WO2008003044A2 - Method and apparatus for human behavioral monitoring - Google Patents

Abstract

The present invention provides a human paradigm, namely, human behavioral pattern monitor (hBPM), that is analogous to an animal paradigm, and a method of use thereof, for validating rodent animal models of psychiatric diseases. The present invention further provides a translational approach to utilize measures characterizing rodent locomotor behavior for application in human locomotor behavior using hBPM in neuropsychiatry disorders, such as bipolar disorder. Furthermore, the present invention provides that the hBPM can be used to characterize hyperactivity, differentiate diagnostic groups, or study habituation, and further provides a potential measure of drug efficacy, and assessment of the effects of medications on cognition.

Description

HUMAN BEHAVIORAL PATTERN MONITOR AND METHOD OF USE THEREOF

STATEMENT OF GOVERNMENT TNTEREST

|00l| This invention was made with a government support under NIH Grant/Contract MH071916-02. As such, the U.S. Government may have certain rights in this invention.

I002J CROSS-REFERENCE OF RELATED APPLICATION

This PCT application claims priority from U.S. Provisional Application No. 60/806,060, filed on June 28, 2006, the entire contents of which is incorporated by reference herein.

FIELD OF THE INVENTION

(003| The present invention relates to a translational approach to utilize measures characterizing rodent locomotor behavior for application to human locomotive behavior. More particularly, the present invention relates to an animal and human paradigm, namely, behavioral pattern monitor (BPM), and method of use thereof, for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, and assessment of cognition on mediations.

BACKGROUND OF THE INVENTION

|004| Translational models of human behavior are an important link to better understand the effect of psychotherapeutic drugs. Several translational models have been developed to better understand the neural circuitry underlying schizophrenia. However, similar attempts for bipolar disorder are missing. Locomotor hyperactivity is a commonly measured behavior in rodents and is often used to quantify the effects of novel drugs. Hyperactivity is also a common characteristic of bipolar patients; nevertheless, a strictly quantitative approach to examine hyperactivity in humans has been missing. 1005] Bipolar Disorder (BD) is a highly recurrent and chronic psychiatric disorder that is characterized by states of mania and affects approximately 1% of the population. BD is a national health issue as it shortens life expectancy, and causes functional impairment and disruption to social, occupational, and family life (Post et al 2003). The underlying neural circuit abnormalities associated with BD are not well understood. During the manic episodes of BD, patients exhibit impulsive behavior, hypersexuality, pressured speech and flight of ideas, and motor hyperactivity, which are symptoms that result from the inability to inhibit behavior and thought (Goodwin & Jamison 1990). Inhibition in the sensory, motor, and cognitive domain is critical for adaptive functioning in a constantly changing environment. Inhibition can be probed with different experimental approaches, which makes this construct amenable to studies in humans and animals. Studying inhibitory deficits using a cross-species translational approach has been helpful in uncovering neurological substrates and genetic bases of other related psychiatric illness, e.g. schizophrenia (Swerdlow 1996). Few studies (Franks et al 1983; Gooding & Tallent 2001 ; Murphy et al 1999), however, have examined inhibitory deficits in BD. Recently, inhibitory deficits in bipolar manic patients has been identified. Nevertheless, several unanswered questions remain such as whether inhibitory deficits in bipolar manic patients are related to the state of mania or reflect a general trait of the illness, and whether neural circuit abnormalities relating to inhibitory deficits can be identified using animal models of mania.

|006| The Dopamine Transporter: Dysregulation of the dopamine (DA) system is thought to contribute to several psychiatric disorders including mania in BD. Hence, DA agonist-induced locomotor hyperactivity has long been used to model the mania phenotype in animals (Lyon 1991). Marked hyperactivity can be triggered in humans or rodents by DA psychostimulants such as amphetamine, which act via the DA transporter (DAT), suggesting that this DA mechanism may be involved in these behaviors. Indeed, amphetamine can induce relapse in remitted manic patients (Goodwin & Jamison 1990). Accordingly, several studies have focused on the DAT as a possible candidate gene in linkage studies in patients with BD (Crowe & Vieland 1998; Fujiwara et al 1997; Greenwood et al 2001 ; Kelsoe et al 1996). Nevertheless, some conflicting results have also been reported (Georgieva et al 2002; Heiden et al 2000). |007| Sensorimotor Inhibition: When mammals are exposed to a sudden loud stimulus, a startle response is elicited. This ubiquitous reflexive response to intense stimuli (acoustic or tactile) is subject to inhibition when a low intensity prepulse precedes a startle stimulus resulting in a reduced startle response. Figure 1 illustrates a robust phenomenon, called prepulse inhibition (PPI). PPI is considered to be a measure of sensorimotor gating. It has demonstrated that PPI is disrupted in certain neuropsychiatry disorders that are characterized by an inability to filter or "gate" sensory (and, theoretically, cognitive) information, such as schizophrenia. Perry et al. (2001a) reports that manic BD patients exhibit robust deficits in PPI, and that similar deficits are evident in DAT knockout mice (Ralph et al 2001a). Geyer et al (2001) reports that DA agonists such as amphetamine disrupt PPl in rats, ft has also reported that PPI can be disrupted in mice by administration of DA agonists such as amphetamine or by deletion of the DAT gene (Ralph et al 2001a; Ralph et al 200I b). These effects can be reversed by antipsychotics having selective antagonist effects at DA D2 but not Dl receptors (Brody et al 2003a; Ralph et al 1999).

[008) Hyperactivity in BD: Increased motor activity is a cardinal feature of a manic episode, described in the DSM-IV as an "increase in goal-directed activity ... or psychomotor agitation". Manic patients often report that they have more energy than usual and are observed to be physically restless, fidgeting, and changing posture and position frequently. Medications used to treat mania such as lithium, divalproex sodium, and atypical antipsychotics significantly decrease motor hyperactivity in mania (Berk et al 1999; Bowden 2000; Swann et al 2002). However, few studies have elaborated on the underlying neural mechanisms that mediate the hyperactivity of mania. Consistent with the central role of DA transmission in the expression of mania, it has been shown repeatedly that DA agonists induce hyperactive states and are therefore considered a model for mania (Lyon 1991).

|009] An enormous literature addresses the motor-activating effects of DA agonists Ln many species (Swerdlow et al 1986; UhI et al 2002). There have been a variety of approaches to developing rodent models of mania, involving baseline locomotor activity, psychostimulant-induced hyperactivity, sleep deprivation, or modification of the Na, K-ATPase pump. Models of BD have also focused on progressive or cycling changes in behavior, often induced by sensitization to DA psychostimulants, amygdala kindling, or biphasic responses to the DA agonist quinpirole (Einat 2000). The effects of pharmacologically and genetically induced states of hyperdopaminergia have been focused by several researchers in part because of the large literature indicating that lithium can attenuate hyperactivity associated with either ***e or amphetamine (Antelman et al 1998; Einat 2000; Flemenbaum 1977; Lyon 1991). One major advantage of using psychostimulant models in animals is that stimulants can precipitate mania in humans (Goodwin & Jamison 1990). It has also reported that knockdown mice exhibit hyperactivity in a novel environment and that valproate reduces this hyperactivity in the DAT knockdown mice (Ralph- Williams et al., 2003b).

[00101 Studies of motor hyperactivity in BD patients have typically used self-report and observer-rated symptom scales. Symptom ratings may not be the most sensitive means of measuring motor activity, as clinician observations and self-reports of physical activity are typically unreliable (Sims et al 1999). Therefore, it is important to develop and validate a more objective and accurate measure of activity. The use of an accelerometer to record physical activity has been informative in medical populations (Bussmann et al 1998; Schasfoort et al 2003; Sims et al 1999; Sisto et al 1998) including psychiatric patients (Teicher 1995; Teicher et al 1986). Recent advances in ambulatory monitoring of motion (e.g. LifeShirt System, Vivometrics 2002), have made it possible to collect physical activity data via a 2-axis centrally mounted accelerometer. This approach has advantages over the more commonly used methods of wrist or ankle-mounted accelerometers, where changes in posture in the absence of motion of the arm or leg are not captured.

[0011] Sequential patterns and perseveration in humans and animals: Studies of nonlinear systems have revealed remarkable new insights into how to characterize and study the manner in which biological and physical systems evolve over time (Braiman et al 1995; Eckmann & Ruelle 1985; May 1987; Rossler & Rossler 1994). Among these insights is the finding that a random-appearing series of events across time can be generated by simple underlying rule-based systems (Feigenbaum et al 1982; Mayer- Kress & Kaneko 1989). For example, the random-appearing sequential flight pattern of an albatross is actually characterized by a lawfiil pattern that depends on the earth's geometry and the bird's fundamental drive to forage for food (Stanley et al 1996). |OO12| The mathematical characteristics of this type of sequential pattern occur in a wide range of systems (Shlesinger et al 1987), including heartbeat variability (Peng et al 1993), DNA coding sequences (Ossadnik et al 1994), and, even at the "macro" level, information flow in large companies (Mantegna & Stanley 1997) and abstract neuronal models (Mandell & SeIz 1997). This striking relationship of simple underlying systems leading to random-appearing phenomena has important and well documented implications for the study of mathematical, theoretical, and physical systems (Ruelle 1994). The methods and techniques that have been developed in these nonlinear approaches are now being applied to many biological and neurobiological systems (Glass & Kaplan 1993; Mandell 1983).

[0013) A rat Behavioral Pattern Monitor (rBPM) was designed almost 25 years ago. When a rat explores a novel or familiar environment, one obtains a large number of consecutive observations that describe not only the amount of the animal's behavior, but also the sequential patterns of the behavior. The rat BPM, which consists of a 30.5 x 61 cm rectangular box equipped with a grid of photobeams, a rearing touch plate, and 10 holes along the walls and floor, was designed to measure sequences of movements and investigatory rearings and holepokes, as well as the amount of locomotor activity (Geyer et al 1986).

[00l-)| Perseveration in BD: Perseverative behavior refers to repetitive and potentially maladaptive sequences of behavioral responses (Crider 1997). Patients who have excessive perseverative behavior have a reduction in their observed response repertoire because the repetitious behavior occurs with increased probability, without the formerly appropriate stimulus present. The perseverative activity intrudes into the formation and/or the execution of the ongoing behavior and may be inappropriate to the current context and thus is different from "goal directed and intentional forms of repetition"

(Crider 1997, p. 63). Perseverative activity can extend from the execution of simple responses to complex problem-solving behaviors and can manifest as motor behaviors or as a "persistent repetition of words, ideas, or subjects so that, once a patient begins a particular subject or uses a particular word, he continually returns to it in the process of speaking" (Andreasen 1979, p. 1320). Thus, the degree of perseverative activity varies depending upon the specific task (Freeman & Gathercole 1966). 10015] Perseverations are a hallmark feature of patients with frontal-cortex pathology (Goldberg & Costa 1986; Luria 1980; Milner 1963; Stuss & Benson 1984). In fact, patients with right frontal lobe damage, exhibit a high degree of perseverative behavior as well as characteristic symptoms of mania, e.g. grandiosity, impulsivity, and hypersexuality (Joseph 1999). Perseverative behavior is also frequently observed in psychiatric disorders (Balder & Goldberg 1987), especially schizophrenia and BD. Although perseverative errors are reported in schizophrenia patients independent of their symptom state, perseverative errors in BD patients are usually associated with the manic state (Murphy et al 1999) and appear to abate in fully recovered patients (Quraishi & Frangou 2002). Perseverative behavior in BD patients has also been observed in behavior during a two-choice button-pressing task used to elicit sequences of responses over time (Lyon & Kemp in press). These authors concluded that the stereotyped or perseverative behavior noted represent a time-related symptom of mania, not observed in other affect-disordered states.

|0016] Effects of Psychopharmacological Treatment: Antimanic medications such as lithium and divalproex sodium reduce manic symptoms in BD patients within three (McElroy et al 1996) to seven (Bowden et al 1997; Vasudev et al 2000) days of treatment initiation. (Note that atypical antipsychotic drugs have antimanic effects, but use the term "antimanic" to refer to the class of agents represented by lithium and anticonvulsants such as divalproex sodium). Surprisingly little is known about how these medications exert antimanic effects and whether the improvement is related to restored inhibitory functioning. There is some suggestion that these medications are successful in reducing impulse control problems in non-bipolar patients (Donovan et al 2000; Olvera 2002; Pallanti et al 2002), supporting the general notion that antimanic agents may exert their effects via attenuating inhibitory deficits.

|00i7| Recently, there has been increased use of atypical antipsychotic medication as treatments for mania (Ghaemi 2000). The combination of antimanic and atypical antipsychotic agents may be more effective in reducing manic symptoms than treatment with an antimanic medication alone. Increased efficacy has been demonstrated across the range of commonly used atypical antipsychotic medications, including risperidone (Sachs et al 2002; Yatham et al 2003), quetiapine (Sajatovic et al 2001), olanzapine, and clozapine (Guille et al 2000). Similar to lithium, divalproex sodium reduces hyperactivity, racing thoughts, and other key symptoms of mania within days of treatment (Motohashi 1999; Token & Grundy 1999). Moreover, animal studies have shown that both typical and atypical antipsychotic agents attenuate inhibitory failures as measured by PPI. Thus, the antimanic property of these medications may also be due to the fact that they attenuate inhibitory deficits in humans.

|0018] It has been reported that a current need is not only to design new animal models, but to design and conduct new experimental studies in clinical populations using paradigms and measures that are suitable to implement in analogous or homologous fashion in animals (Geyer & Markou 2002). The validation of a putative animal model can be no better than the quality of the parallel experimental data from the relevant human clinical population. Accordingly, there is a need to develop human paradigms that mimic the rodent behavior pattern monitor (BPM) as closely as possible, and to design studies of motor activity and perseverative motor patterns in patients to provide an empirical reference for the validation of potential animal models. Such human paradigms can further be used for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, and assessment of cognition on mediations.

SUMMARY OF THE INVENTION

|00l9] The present invention provides a translational approach to develop an explicit human analog of all rodent behavioral test paradigms, exploratory motor activity in a novel environment, i.e., the "open Field" test. The present invention further provides that the analyses taken from the paradigm yield information about how a person responds to novelty and problem solving. The human paradigm, namely, human behavioral pattern monitor (hBPM), of the present invention provides an ability to directly compare findings from rodent models of a psychiatric disease with those obtained from psychiatrically ill human subjects. The human paradigm, e.g., hBPM, of the present invention can further be used for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, and assessment of cognition on medications and/or in a real world problem solving environment.

|0020l The present invention provides a hBPM that mimics the rodent open field as closely as possible. In one of the preferred embodiments, human subjects are told they will see a study coordinator in a short period of time, and are directed into a novel room. The room is 10 by 13 feet and equipped with ten exploratory objects and one table, two open book shelves on opposing walls, a cabinet, and no chairs. The exploratory objects are: 1. feather mask, 2. stuffed doll, 3. slinky, 4. yo-yo, 5. small sculpture, 6. musical instrument, 7. kaleidoscope, 8. soft toy, 9. balls, 10. toy animals. The objects are evenly dispersed in the room on the shelves, table, and on top of the cabinet. All ten objects are selected because they are uncommon, colorful and interactive and represent a variety of shapes, colors and textures and invite close visual and tactile inspection. During the 15-minute experimental session, subjects are asked to enter into the room and wait for the examiner. The subject is instructed to make his/her self comfortable. No additional instructions are provided. An examiner is stationed outside the door of the room to redirect the subject if they open the door or have questions. At the end of the 15-minute period, the experimenter directs the subject back to the laboratory.

f002lj The present invention provides two sources of data in the hBPM The subject wears an ambulatory monitoring device that continuously collects movement data with the use of an accelerometer. The room is also equipped with a hidden digital camera that captures the spatial location of the subject's position, in the form of x and y coordinates at a high sampling rate. The present invention provides that the accelerometry data and the spatial location data are subjected to further novel non-liner dynamic analyses. In one preferred embodiment, the present invention provides human subjects that run through the "open field" test paradigm, and physiologic and spatial location data are collected.

|0022) In one of the preferred embodiments, the present invention provides a translational approach and method to utilize the measures characterizing rodent locomotor behavior for application in human locomotor behavior. More specifically, the present invention provides methods to measure the locomotor behavior symptom in humans quantitatively. Locomotor hyperactivity is a commonly measured behavior in rodents and is used to quantify effects of new drug candidates. It is known that hyperactivity is a common characteristic of bipolar disease in humans. The present invention provides two main measures to quantify human locomotor behavior: overall levels of activity as measured by 3-dimensional acceleration and dynamical entropy, defined as the degree of predictability of sequences of human accelerations. These measures are used to define different types of behavior of human exploring a novel environment. Furthermore, the present invention provides classification procedures of using measures that quantify acceleration and sequences of movements to classify them into different patterns of behavior (sitting, standing, walking etc.).

[0023] The present invention provides that the levels of locomotor activity are not correlated with predictability of locomotor activity, a result that replicates a similar finding in animals However, the present invention provides that unique combinations of locomotor activity levels and predictability of locomotor sequences characterize different behaviors. Therefore, the present invention provides a first approach to quantify human behavior similar to methods used to quantify animal locomotor behavior. By using similar measures, the present invention further provides a translational model of drug actions such as antimanic agents, which can be used for future drug development.

(00241 In yet another preferred embodiment, the present invention provides studies of Bipolar Disorder (BD) and/or bipolar mania in both clinical human populations and related animal models. The present invention also provides the use of human measures of motor activity and perseverative patterns in a manner that is parallel the corresponding measures of dopaminergic activation in pharmacologic and genetic animal models. The present invention further provides studies of both antimanic and atypical antipsychotic treatments in animal models, using measures of PPI of startle, locomotor hyperactivity, and perseverative patterns of exploratory behavior. The evaluation of the effects of pharmacologic treatment modalities using parallel experimental measures in manic BD patients, on hospital admission and during treatment, will enable firm assessments of the predictive validity of potential animal models.

BRIEF DESCRIPTION OF THE DRAWINGS

|0025| Figure 1 illustrates a robust phenomenon, called prepulse inhibition (PPI).

[0026| Figure 2 shows a photograph illustrating the layout of the open field, as well as an example of how a human subject is spatially tracked. [0027] Figure 3 shows a diagram illustrating the translate information that can be gained from basic experimental models of hyperdopaminergia, e.g. acute amphetamine administration and DAT knockdown or knockout mice, as it relates to mania.

10028] Figure 4 indicates that BD patients with psychotic mania, tested within 96 hours of acute psychiatric hospitalization, had significant deficits in PPI that were comparable to those of acutely hospitalized schizophrenia patients.

|0029] Figure 5 illustrates the acceleration data and the local dynamical entropy calculated.

|0030] Figure 6 illustrates that entropy differentiates perseverative from non- perseverative movements.

[00311 Figure 7 shows that two atypical antipsychotics: clozapine (3 mg/kg) and quetiapine (2.5 mg/kg) reduce the PPI deficit in male DAT KO mice.

[O032| Figure 8 shows that an acute administration of fluoxetine (15 mg/kg) unmasks a deficit in PPl in -/- DAT KD that otherwise exhibited normal PPl.

[00331 Figures 9A and 9B show that 2 mEq/kg LiCI reduced the PPI-disruptive effects of 10 mg/kg amphetamine in both 129 (A) and C57(B) mice.

|0034] Figure 10 illustrates measures of sequential organization of behavior, i.e. the geometrical patterns of movements and the predictability of movement sequences, and levels of locomotor activity in vehicle-treated C57BL/6J, 129S6, and 129X1 mice.

[0035] Figure 11 provides a graphical example of activity data using the LifeShirt®.

[0036| Figure 12 illustrates specific areas in the mBPM. There are 4 areas defined per enclosure: (1) 4 corner areas (dark gray), (2) 2 short-wall and (3) 2 long-wall areas (gray), and (4) the center (white). Locations of the holes are indicated by circle. An entry into a new area is counted as the basic measure of locomotion.

|0037] Figure 13 illustrates stimulant effects on rat locomotor patterns in the Behavioral Pattern Monitor. The effects of saline (a), amphetamine (b), phencyclidine (c) and scopolamine (d) on rat behavioral organization, a) The rat treated with saline explored little of the environment, making two or three excursions around the chamber, exhibiting short meandering movement in one or two areas then moving on. b) The amphetamine-treated rat however made numerous circuits of the chamber, crossing from one area to another in relatively straight lines, crossing the center as often as being close to the chamber walls, leading to a great variability of movement. The rat also exhibited more than one area of focused activity ['home corner' (Geyer 1982) or 'home base' (Eilam and Golani 1989)]. c) The phencyclidine-treated rat exhibited sweeping movement patterns, from one corner to another, often circling from a home base that covered the right side of the chamber, with repetitive movements, d) The scopolamine- treated rat displayed increased long and straight movements particularly close to the chamber walls, deviating very little from this path. The level of activity was as great as amphetamine but with scopolamine the rat did not spend time focusing on any areas in particular, nor did it cross the center very often, displaying very repetitive movements. Data are provided for measures of locomotor activity (transitions), locomotor pattern (spatial d) and exploration (holepokes).

[0038] Figure 14 illustrates effects of amphetamine on mouse locomotor patterns in the Behavioral Pattern Monitor. The effects of saline (a) and amphetamine (b) on the behavioral organization of a representative mouse are presented, a) Mice treated with saline exhibit exploration throughout the chamber, but only perform a limited number of excursions around the chamber. This animal spent most of the time in the bottom right hand side of the chamber (the "home corner"), b) Mice treated with amphetamine however, exhibit a large number of excursions around the chamber, covering the chamber floor many times in a variety of paths. They also display several areas where their behavior is concentrated, suggesting several home bases as opposed to the one home corner observed in the saline administered mouse. While the x-y plots represented here are genuine movements, they also reflect a limitation of the number of photobeams used to identify a subject's position. Data are also provided for measures of locomotor activity (transitions), locomotor pattern (spatial d), and exploration (holepokes). Mice treated with amphetamine display a hyperactive phenotype, with lower spatial d and lower exploratory behavior when compared to control animals.

J0039J Figure 15 illustrates locomotor patterns of dopamine transporter knockdown and wild type littermate mice in the Behavioral Pattern Monitor. Representative locomotor patterns of dopamine transporter wildtype (WT; a) and knockdown (KD; b) mice are shown, a) WT mice spend most of their time near the chamber walls and while some movement is made to explore the center, activity is concentrated in the left wall, where the mouse circles back and forth, b) In contrast to the time spent in the home corner by the WT mouse, this KD mouse displayed numerous areas of interest and exhibited more varied paths of activity. Locomotor activity (transitions), pattern (spatial d), and exploration (holepokes) data are provided, with the KD mice displaying greater activity, exploratory behavior, and straighter line movements (lower spatial d) when compared to their WT littermates.

|0040] Figure 16 illustrates effects of the selective dopamine transporter uptake inhibitor GBR 12909 on mouse locomotor patterns in the Behavioral Pattern Monitor. The representative locomotor patterns are shown for mice treated with saline (a) or GBR 12909 (b). a) Mice treated with saline display very limited activity, making only one or two excursions around the chamber with limited exploration into the center of the chamber, b) Mice treated with GBR 12909 display far greater levels of activity, with numerous areas of focused activity and greater variety of paths taken. Data are also presented for locomotor activity (transitions), pattern (spatial d), and exploratory behavior (holepokes) with mice treated with GBR 12909 displaying hyperactivity, increased exploratory behavior, and straighter line movements (lower spatial d) than mice treated with saline.

10041] Figure 17 illustrates patterns of human subjects in the human Behavioral Pattern Monitor. The layout of the room as observed through a fisheye lens is outlined in red. The locomotor pattern of a representative healthy subject (a), or manic BD (b) and schizophrenia (c) patients are shown in black. The location of the subject's upper torso (specifically, the LifeShirt® vest) in x- and y-coordinates was recorded by tracking software (Clever Systems, Inc) as the subject examined the room and the objects located therein. An accelerometer embedded in a wearable ambulatory monitoring device (Lifeshirt®) also recorded levels of motor activity in digital units for each subject, a) The healthy comparison subject walked around the room once, investigated the window, which is covered, examined some objects placed on the bookshelves farthest from the door, and finally moved to the desk, spending the remainder of time examining that area and the objects found there, b) The manic BD patient conducted numerous excursions around the room, often concentrating movement at specific locations such as the window and bookshelves farthest from the door and the small filing cabinet. Apart from the obvious quantity of movements that differentiate this subject from the healthy comparison subject, the manic BD subject also clearly failed to exhibit a preference for one location, spending time in numerous areas. This subject also displayed longer tracks of movement from one area of the room to another, with a large variability in the paths chosen, c) The schizophrenia patient displayed a virtual lack of exploratory behavior. This subject remained at the desk for the duration of the session. Some objects were investigated on the desk, but all exploration was specific and within a limited area. The quantitative data are shown for these representative subjects' acceleration, transitions, spatial d, and exploratory behavior (object interactions).

|0042| Figure 18 illustrates an observed walking and entropy-derived probability. The close correspondence between walking behavior as observed by video ratings (gray line) and entropy-derived probability of walking (black line) for one healthy human subject across a 15-minute session in the human BPM. The entropy-derived probability of walking corresponded exactly to the observed walking at each time-frame.

[0043] Figure 19 illustrates the "open field" studies with Bipolar Mania and ADHD patients and sequences of (x, y) analyses.

|0044| Figure 20 illustrates that normal comparison and Bipolar Mania subjects, but not ADHD subjects showed habituation of locomotor activity.

[00451 Figure 21 illustrates an increased motor activity and straighter movement patterns of Bipolar Mania subjects. Left (a): individuals with Bipolar Mania but not ADHD showed increased motor activity relative to comparison subjects; Right (b): Bipolar Mania subjects exhibited straighter movement patterns than both ADHD and normal comparison subjects.

100461 Figure 22 illustrates a pattern of a time spent in the "open filed" in Bipolar Mania, ADHD and normal comparison subjects. Left (a) illustrates that Bipolar Mania subjects spent more time in the center of the room; Right (b) illustrates that normal and ADHD individuals spent more time along the short wall in the room. 100471 Figure 23 shows example patterns of comparison subject (a), Bipolar Manic subject (b), and ADHD subject (c) in the "open field".

(0048] Figure 24 illustrates the Human Behavioral Pattern Monitor (hBPM), a novel room furnished with ten engaging stimuli and no chairs. Subjects are directed inside with no instruction except to wait for the experimenter.

10049] Figure 25 illustrates a use of hBPM for measuring behavioral patterns for Manic Bipolar Disorder (BD) and Schizophrenia (SCZ) patients. (A) denotes how many times the subjects interacted with any object; (B) gives the amount of time the subjects were walking (in seconds); and (C) gives the quantity of time spent with all objects. * Denotes p<0.05 when compared to normal comparison (NC) subjects, # Denotes p<0.05 when compared to SCZ patients.

[0050] Figure 26 illustrates the LifeShirt®-Clinical System (VivoMetrics, 2002), a wearable ambulatory monitoring device that records physiologic functions. Motor activity is quantified with a centrally mounted accelerometer embedded in the device.

|0051) Figure 27 illustrates motor activity in session one for manic BD patients, SCZ patients, and NC subjects. ^♦* manic BD > nonpatients, p < 0.01 ; ## manic BD > schizophrenia, p O.01 ; # manic BD > schizophrenia, p < 0.05.

|0052] Figure 28 illustrates motor activity across all three sessions for manic BD patients and NC subjects. ** manic BD > nonpatients, p < 0.01 .

DETAILED DESCRIPTION OF THE INVENTION

|0053| The present invention provides a behavioral pattern monitor for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medications, comprising a test subject locomotor space, means for recording information about test subject's locomotor information within the space, and information relating at least one control subject to a locomotor pattern for comparative neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on mediations. In preferred embodiments, the behavioral pattern monitor is specific to rodent behavior and may be used for validating animal models of psychiatric diseases. (0054] The present invention also provides that the behavioral pattern monitor can be specific to human behavior. In preferred embodiments, the space is a room furnished with a plurality of engaging stimuli, and said means for recording information about the test subject's locomotor information is an overhead video camera. In preferred embodiments, the human subject's locomotor information comprises the number of interactions with said stimuli, the time spent in locomotion, the time spent interacting with each stimulus, and the pattern of interacting with each stimulus. In preferred embodiments, the pattern of interacting includes distance pattern of activity, geometric or spatial pattern of activity, and time spent in different parts of the room. In preferred embodiments, the engaging stimuli comprises exploratory objects selected from a mask, a doll, a toy, a sculpture, a musical instrument, a kaleidoscope, a ball, a stuffed animal, and combinations thereof.

|0055| The present invention provides a model that can be used to diagnose or assess a patient having bipolar disorder-manic episode, schizophrenia, Attention Deficit Hyperactivity Disorder, or other neuropsychiatric disorders. Furthermore, the locomotor information can comprise measuring locomotor behavior to characterize hyperactivity, differentiate diagnostic groups, or study habituation.

[0056] Therefore, the present invention provides a method of determining a behavioral pattern of a human subject comprising: a) providing a behavioral pattern monitor for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medications, comprising a room for test subject locomotor space, a means for recording test subject locomotion while within the space, and information relating at least one control subject to a locomotor pattern for comparative neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medication said behavioral pattern monitor; b) guiding said human subject inside said room, c) recording information about said human subject's locomotor behavior for at least 15 minutes, and d) comparing information about human subject's locomotor pattern to the information relating at least one control subject to determine the human subject's relative behavioral pattern.

|0057i I" preferred embodiments, the method provides that the the space is a room furnished with a plurality of engaging stimuli, and said means for recording information about test subject's locomotor information is an overhead video camera. In preferred embodiments, the human subject's locomotor information comprises the number of interactions with said stimuli, the time spent in locomotion, the time spent interacting with each stimuli, and the pattern of interacting with each stimuli. In preferred embodiments, the pattern of interacting includes distance pattern of activity, geometric or spatial pattern of activity, and time spent in different parts of the room. In preferred embodiments, the engaging stimuli comprises exploratory objects selected from a mask, a doll, a toy, a sculpture, a musical instrument, a kaleidoscope, a ball, a stuffed animal, and combinations thereof.

[0058) The present invention provides a method that can be used to diagnose or assess a patient having mania bipolar disorder, schizophrenia, Attention Deficit Hyperactivity Disorder, or other neuropsychiatric disorders. Furthermore, the locomotor information can comprise measuring locomotor behavior to characterize hyperactivity, differentiate diagnostic groups, or study habituation.

|0059| Inhibition, or the ability to withhold or attenuate an action or a thought, is of central importance in the regulation of behavior. Inhibitory deficits are key features of the mania of Bipolar Disorder (BD) and provide a behavioral target for translational research. The present invention provides a hBPM which asseses multiple domains of inhibition in manic BD patients and in animal models based on pharmacological challenges and gene engineering technology. The assessment of inhibitory failures in manic BD patients will enable the validation of animal and human behavioral pattern monitors that can predict novel treatments of BD and help identify the neural substrates underlying BD.

10060] Mania is the defining feature of BD, a highly recurrent and severe psychiatric disorder. Recent evidence linking a subgroup of BD patients to an alteration in the genetic sequence in the vicinity of the dopamine transporter (DAT) supports that the manic state may involve a dysregulation of dopaminergic systems. These systems are critical for the control of inhibitory functions. Moreover, several antimanic agents, which may "normalize" cortical and subcortical hyperactivity also modulate directly or indirectly the dopaminergic system. Nevertheless, there is a striking paucity of preclinical models related to BD, which has hindered examination of neural circuit abnormalities contributing to BD. The present invention provides translational research involving cross-species measures that reflect abnormalities in dopaminergic systems to further the understanding of BD. Specifically, the present invention provides parallel studies of inhibitory deficits in manic BD patients and in mice in which the dopamine (DA) transporter (DAT) has been manipulated either pharmacologically (i.e. amphetamine) or genetically (i.e. DAT knockdown and knockout mice).

|006l I To address the state versus trait nature of inhibitory deficits in BD, manic BD patients are studied during highly symptomatic and remitted states. To examine treatment effects, manic BD patients and hyperdopaminergic mice are tested before and during treatment with antimanic and/or atypical antipsychotic drugs. Comparing these results across species provides that mutant mice lacking the normal complement of dopamine transporters serve as a model of the inhibitory deficits in BD and that DAT- deficient mice provide an animal model with predictive validity for the identification of antimanic agents.

|0062| Behaviorally, the unifying construct is inhibition, which cannot be characterized adequately by any one measure. Therefore, the present invention assesses inhibitory deficits in three domains; 1. sensorimotor inhibition, i.e. stimulus-induced attenuation of motor action, is assessed using prepulse inhibition (PPI) of the startle response; 2. motor hyperactivity, i.e. inability to inhibit ongoing actions, upon exposure to a relatively novel environment is assessed by species-appropriate ambulatory monitoring devices that characterize motor activity with a high degree of spatial and temporal resolution; and 3. perseveration, i.e. an inability to inhibit prepotent or ongoing responses that results in a decline in behavioral variety, is measured in humans and mice using non-linear analyses of spatial and temporal patterns of motor responses.

|0063] At a physiological level, the invention's central construct is hyperdopaminergia, i.e. the increased availability of DA as a modulatory transmitter in prefrontal cortex and subcortical areas, possibly related to the observed genetic association between BD and DAT. The present invention provides two established treatments: (1) the direct modulation of the hyperdopaminergic state via DA D2 receptor antagonism as represented by atypical antipsychotic agents, and (2) indirect modulation of this state via antimanic agents such as lithium and valproate.

|0064] The present invention combines these pharmacological manipulations in both manic BD patients and the animal models of hyperdopaminergia with behavioral measures that are closely matched across species to elucidate the pathways that underlie mania. One of the objects of the present invention is to develop and validate parallel animal and human models as predictive tools for evaluating new treatment interventions in bipolar mania and to identify key target variables, i.e. prepulse inhibition, hyperactivity, perseveration, which can be used to track treatment efficacy.

[00651 The present invention provides a test model that mania is highly associated with impaired inhibitory functioning and that impaired inhibitory functioning is associated with hyperdopaminergic states. Manic BD patients are assessed on three inhibitory measures during highly symptomatic and remitted states. The present invention further provides an assessment performance on the three inhibitory measures in patients treated with medications that are direct DA antagonists versus medications that affect DA transmission indirectly. The present invention provides an explicit human analog of the most classic of all rodent behavioral test paradigms, exploratory motor activity in a novel environment, i.e. the "open field" test, namely, Human Behavioral Pattern Monitor (hBPM).

|0066| In one preferred embodiment, the present invention provides an assessment of the inhibitory functioning of manic BD patients using the hBPM. The present invention further provides that manic BD patients exhibit significantly greater deficits on measures of inhibition (less PPl, more hyperactivity, and more perseveration) than normal comparison (NC) subjects. The present invention also provides an assessment of the relationship between inhibitory functioning and manic symptoms in BD patients using the hBPM. In one of the preferred embodiments, manic BD patients are tested at three intervals: 1) within 72 hours (Session 1) of acute hospitalization; 2) 7 days later (Session 2); and 3) 28 days after Session 1 (Session 3). The present invention provides that manic BD patients show improvement in inhibitory function (increased PPI, decreased hyperactivity, and decreased perseveration) across the three test sessions compared to the NC subjects. The present invention further provides that, in manic BD patients, decreased inhibitory deficits is related to decreased symptoms as measured by Young Mania Rating Scale Scores over the three test sessions.

|0067) In yet another preferred embodiment, the present invention provides a test to whether lithium, anticonvulsants, and atypical antipsychotic medications differentially improve inhibitory functioning. The present invention provides that manic BD patients treated with atypical antipsychotic medication (alone or in combination with lithium or an anticonvulsant) demonstrate significantly greater rate of change (improvement) than patients treated with lithium or an anticonvulsant on measures of inhibitory function (increased PPL, decreased hyperactivity, and less perseveration) over the 3 test sessions. The present invention further provides that, at Session 3, manic BD patients treated with an atypical antipsychotic, lithium, or an anticonvulsant alone exhibit more inhibitory deficits than patients treated with a combination of an atypical antipsychotic and an antimanic medication.

100681 The present invention further provides a potential validity of two mouse models related to the mania phenotype characteristic of bipolar disorder. Both of the models are based on the deficient PPI, locomotor hyperactivity, and perseverative patterns of activity associated with increases in synaptic DA produced by: (1) acute dopaminergic activation produced by the administration of amphetamine; or (2) chronic dopaminergic activation in the DAT-deficient mice. For each of the models, validity is assessed specifically by assessing the ability of the model to detect the effects of drug regimens used in the treatment of mania and/or bipolar disorders (e.g. acute and chronic valproate or lithium, and acute atypical antipsychotics).

|0069| In one preferred embodiment, the present invention provide an assessment of the ability of antimanic drug treatments to prevent the disruptions in three domains of behavioral inhibition produced by acute hyperdopaminergia (amphetamine) using the mouse model. The present invention provides that acute and/or subchronic treatments of lithium, valproate, and atypical antipsychotic medications prevent the amphetamine- induced decreases in PPI, increases in motor activity, and increases in perseverative locomotion in the mouse model.

|0070) In yet another preferred embodiment, the present invention provides an assessment of the ability of antimanic treatments to reverse the disruptions in three domains of behavioral inhibition produced by chronic hyperdopaminergia in DAT- deficient mice. The present invention provides that subchronic treatment with antimanic and atypical antipsychotic medications reverses the reductions in PPI (DAT - /- KO), increases in motor activity, and increases in perseverative locomotion in DAT- deficient (DAT -/- KO and DAT -/- KD) mice. Acute pharmacological treatments also reverses some of the less dramatic phenotypes in DAT-deficient mice. (007l| In yet another preferred embodiment, the present invention provides an assessment of the ability of antidepressant treatments to precipitate mania-like disruptions in behavioral inhibition in DAT +/- knockout or DAT knockdown mice. The present invention provides that subchronic (and acute) treatment with antidepressant medications induces disruptions in the three domains of inhibition DAT- deficient mice that exhibit minimal or no phenotypic abnormalities at baseline (DAT +/- KO and, for PPI, DAT -/- KD) mice.

[0072| The present invention further provides a translational integration in implementing PPI as a cross-species paradigm of inhibition to the study of BD. The present invention provides a similar cross-fertilization of paradigms involving motor activity that have been developed extensively in rodents and are applied to innovative studies in humans. In one preferred embodiment, the present invention provides the use of the LifeShirt® in a novel paradigm to assess motor activity and perseverative patterns of behavior in SD patients. The present invention provides that the combination of inhibitory deficits (altered PPI, hyperactivity, and perseveration) are a composite endophenotype that is unique to BD patients and can be used to assemble and validate animal models of mania in BD. Before the present invention, there are no empirical studies of quantifiable measures of motor activity and response perseveration in BD that can be linked directly to parallel measures in animals. The present invention fills this gap by generating longitudinal profiles of inhibitory dysfunctions in highly symptomatic manic BD patients, using measures derived from animal paradigms. These profiles can be used in animal studies to explore a prominent neurotransmitter target implicated in mania, namely hyperdopaminergia.

(0073| The present invention provided herewith can help to identify endophenotypic markers of BD that could aid in future studies of the genetics of this disorder. The present invention further provides that using an operational measure of sensorimotor inhibition offers an innovative approach to studying BD because: 1) there are existing models for understanding the neural substrates of central gating processes; 2) impaired sensorimotor inhibition has been shown to be associated with, and perhaps causally related to, disturbances in thinking, while normal gating is thought to be relevant to adaptive functioning; and 3) PPI is also reliably measured in rodent populations, making it an excellent tool for translational research. The present invention yet provides that antimanic treatments attenuate motor hyperactivity by either direct or indirect influences on DA neurotransmission.

(0074| The present invention further provides a mouse behavioraJ pattern monitor (mBPM) completely analogous to the rat behavioral pattern monitor (rBPM). The mBPM system confirms that sequences of locomotor behavior are not randomly associated events but comprise highly organized sequences of behavior. The organization of these sequences is complex because unconditioned locomotor behavior is contingent on many factors. Thus, in addition to applying a priori definitions for measures of different concepts such as novelty seeking, approach-avoidance, exploration, establishing a home base, and habituation, a set of measures quantifying the sequential organization of behavior is developed. This approach is based on the general concept that the sequential characteristics of behavior, i.e. how behavior unfolds over time, are not adequately measured by overall levels of activity, measures of exploratory behavior, or distribution of activity in the enclosure. The present invention of these measures confirms that measures of sequential patterns add significantly to the understanding of the effects of and differences between various psychostimulant drugs, including dopaminergic agents, and these measures enable to quantify perseverative patterns of behavior without requiring tedious and time- consuming ratings.

[0075| The present invention further provides that both amphetamine-treated and DAT KO mice show an abnormal sequential pattern of locomotor activity that is characterized by rapid running around the perimeter of the test chamber. This form of hyperactivity is referred as being "perseverative" because the same pattern of movement is repeated again and again. The degree to which animals exhibit such straight-path patterns of locomotion is quantified using the non-linear measures of sequential patterns that was developed originally using data from the rat BPM. For example, the present invention provides that a measure of dimensionality derived from fractal geometry - spatial d - which was first developed for studies in rats (Paulus & Geyer 1991) and subsequently extended to studies of mice and humans, is dramatically altered in DAT KO and KD mice.

|0076] In both rats and mice, the present invention provides that drugs or genetic factors - independent of differences in the amount of locomotor activity - alter spatial d. That is, the pattern of the behavior is orthogonal to the amount of the behavior. This "perseverative" locomotor activity may reflect a failure of motor inhibition, insofar as it results when an animal fails to pause when encountering a corner or other object in the environment, as rodents normally do. Indeed, if another animal is in the same chamber, a DAT KO mouse simply runs or jumps over it rather than exhibits the normal social interactions characteristic of such an encounter between mice. Thus, it appears that DAT KO mice, like mice treated with amphetamine, cannot inhibit their motor activity in a normal manner.

|0O77] Therefore, the present invention provides rodent and human behavioral pattern monitors, e.g., rBPM, mBPM, and hBPM), and use of these BPMs thereof. In one preferred embodiment, the present invention provides rodent and human studies in the BPM, providing a reverse-translational approach to Bipolar disorder, and other psychological disorders. In yet another preferred embodiment, the present invention provides that hBPM differentiates activity patterns of BD and ADH subjects. In yet another preferred embodiment, the present invention provides hBPM differentiates patients with acute psychotic Mania from Schizophrenia. In yet another preferred embodiment, the present invention provides that hBPM can be used to quantification of motor hyperactivity in Mania.

|0078] These and many other variations and embodiments of the invention are apparent to one of skill in the art upon a review of the description and examples.

EXAMPLES

ANIMAL STUDIES

[0079] Amphetamine Disrupts Prepulse Inhibition in Mice: Several studies were published for demonstrating that amphetamine disrupts PPI in mice, as those had been shown previously in rats (Geyer et al 2001; Mansbach et al 1988). The first report of this effect of amphetamine was published in 1996 (Dulawa & Geyer 1996) and such effect is subsequently reversed by pretreatment with the D2-like DA antagonist raclopride (Brody et al 20113a). Using genetically modified mice, it was shown that the PPI-disruptive effects of amphetamine were maintained in mice lacking Dl, D3, or D4 DA receptors, but were absent in mice lacking the D2 subtype of DA receptors (Ralph et al 1999; Ralph-Williams et al., 2003a). [00801 PPI Deficit in DAT-Deficient Mice: In recent years, dopamine transporter (DAT) mutant mouse lines have been generated that may prove useful in behavioral studies related to disease states involving dysregulation of dopaminergic systems, including BD. A DAT "knockout" (KO) mouse has been created that completely lacks the gene coding for the DAT (Giros et al 1996). It was found that the DAT KO mice exhibit marked deficits in PPI of startle and (Ralph et al., 2001 ) in addition to the profound locomotor hyperactivity seen in these mice.

|008l] Antipsychotics Reverse PPI Deficit in DAT Knockout Mice: In a report of PPl deficit in DAT KO mice, the D2 antagonist and typical antipsychotic raclopride but not the Dl antagonist SCH23390 reduced the deficit (Ralph et al 2001a). More recently, it was found that two atypical antipsychotics - clozapine and quetiapine - also reduce the PPI deficit in male DAT KO mice, as shown in Figure 7 with 2.5 mg/kg quetiapine. The effect of 3 mg/kg clozapine was virtually identical (Ong 2003).

100821 The DAT Knockdown Mouse: Although the full DAT KO mice exhibit a chronic hyperdopaminergic tone, they also have profound physiological alterations

(Gainetdinov et al 1998; Giros et al 1996) which may confound interpretation of results. Recently, a second kind of DAT mutant mouse has bee created. Unlike the full

DAT KO, these "knockdown" (KD) mice have an approximate 90% loss of DAT

(Zhuang et al 2001). The reduction in DAT expression is due to the insertion of an extra 4-kb DNA sequence into the second exon in the 5 '-untranslated region of DAT, resulting in a reduction in gene expression efficient) while keeping the coding sequence intact. These mice also have chronic hyperdopaminergia (e.g., a 70% increase in extracellular striatal DA), but they do not show gross physical changes. Behaviorally, the DAT KD mice are hyperactive in a novel open field and show impaired response inhibition in a number of paradigms (Zhuang et al 2001). Thus, the DAT KD mutant mice may prove useful as an animal model for the manic state associated with BD.

100831 I" the DAT KO mice, the dramatic deficit in PPI is only observed reliably in the null mutant -/- mice. While a partial deficit in PPI in the +/- DAT KO mice was sometimes observed, the +/- mice are typically normal in PPI. Similarly, the DAT KD mice, in which roughly 10% of the transporters remain, typically exhibit minimal changes in PPI (Ralph et al 2001a). It is common to find a slight decrease in PPI in the -/- DAT KD mice, but this difference has been significant only occasionally and only in large groups of mice. Thus, partial deletions of the DAT, in either the +/- DAT KO or the -/- DAT KD mice, produce an animal that appears to be near the threshold for demonstrating a mania-like decrease in PPI.

100841 With regard to locomotor hyperactivity, the +/- DAT KO mice appear to be normal, while the -/- DAT KD mice are clearly hyperactive. The possibility that DAT- deficient mice may be useful as a model of vulnerability to mania was tested, by examining the ability of antidepressant medications to precipitate a deficit in PPI or increases in motor activity and perseveration only in the +/- DAT KO or the -/- DAT KD mice and not in wildtype mice. The results suggested that even an acute administration of 15 mg/kg fluoxetine may unmask a deficit in PPI in -/- DAT KD that otherwise exhibited normal PPI, as shown in Figure 8. Although the effect o this acute treatment with fluoxetine was small, the ANOVA revealed a significant genotype-by- drug interaction (F=5.68, df=l,37; p<.025).

[0085| Effects of Antimanic Agents on Drug-Induced Deficits in PPI: To develop murine models to help discover new treatments for BD and/or mania, several studies of various antimanic agents in mice treated with drugs that disrupt PPl acutely were conducted. Specifically, both amphetamine and the NMDA antagonist ketamine disrupted PPI in mice. Because the putative mood-stabilizer lamotrigine blocked the perceptual disturbances produced by ketamine in normal volunteers (Anand et al 2000), the interaction of lamotrigine and either ketamine or amphetamine on PPI in both 129SvPasIco (129) and C57BL/6J (C57) mice were also tested (Brody et al 2003b). Acute lamotrigine prevented the ketamine- but not the amphetamine-induced disruption of PPI in 129 mice, and generally increased PPI in C57 mice.

(0086) The effects of chronic administration of lamotrigine in this paradigm were also determined. Similarly, it was found that an acute administration of 50 mg/kg carbamazepine prevented the ketamine effect on PPI and reduced the amphetamine effect on PPI in 129 mice. Studies with more protracted administrations of such anticonvulsant drugs used in the treatment of BD were also performed. The data indicated that acute treatment with lithium can reduce the PPI deficit produced by amphetamine in both 129 and C57 mice. As seen in Figures 9A and 9B, 2 mEq/kg

LiCl reduced the PPI-disruptive effects of 10 mg/kg amphetamine. [0087] Non-linear Measures of Perseveration in Mice: Based on the extensive experience pioneering the use of non-linear measures to characterize sequential patterns of movements in rodents, and perseverative patterns in particular, and as detailed in the article (Pauius et al., 1999), this work was extended from rats to mice using a Video- tracking system to monitor unconditioned locomotor behavior in a novel environment. Figure 10 illustrates the patterns obtained. The movement patterns were analyzed analogously to the scaling pattern that was advanced previously based on studies of rats (Pauius & Geyer 1991) and which was derived from similar observations in physical and mathematical systems (Mandelbrot 1982).

10088] Briefly, sequences of movements observed with varying spatio-temporal resolutions may not yield a simple linear relationship between the (measured) distance traveled and the observational resolution, The resolution is determined by the number of micro-events that are considered for the calculation of the distance traveled. For example, straight movements along a wall of the enclosure or traversing the center are characterized by the fact that the distance traveled doubles if the number of micro- events used to calculate the distance is doubled. Therefore, there is a linear relationship between the number of micro-events and the distance traveled. However, meandering or circumscribed movements are not characterized by this simple linear relationship.

(0089| Here, the distance traveled was less than double if the number of micro-events used to calculate the distance was doubled. Thus, the measured distance traveled as reported frequently in the literature is not an absolute metric, but depends upon the resolution used to obtain the measure and the interaction between the resolution and the pattern of behavior exhibited by the animal. The spatial scaling exponent, d, quantifies the relationship between the number of micro-events, k, used to calculate the distance, L, and the change in resolution. The average spatial scaling exponent, d, typically varies between 1 and 2 for straight and highly circumscribed movements, respectively

(see Fig 2 in Pauius et al 1999). The dynamical entropy measure, h, is described in detail in the publication. Briefly, this measure quantifies the degree of uncertainty of predicting the next movement based on the sequence of preceding movements and is based on similar measures assessing the emergence of uncertainty in nonlinear dynamical systems (Eckmann & Ruelle 1985). [00901 The dynamical entropy is expressed in bits/step and is related directly to the number of different movement possibilities per step. Thus, the more different paths are observed, the larger the value of h (see Fig 3 in Paulus et al 1999). The dynamical entropy approaches zero if the animal exhibits a highly repetitive path pattern. Conversely,

indicates that the animal moves from any point in the chamber in all different directions with equal probability. Therefore, this measure quantifies the variety of different locomotor patterns. A principal component analysis extracted a two factors accounting for 75.8% of the variance and demonstrated that the amount of motor activity was independent of the perseverative patterning of the activity (see Table 3, Paulus et al, 1999). Thus, at least two independent factors related to the amount of locomotor activity and to the geometrical and dynamical path patterns govern locomotor behavior of mice in a novel environment.

[0091] Amphetamine-induced Hyperactivity in Mice: The behavioral profile induced by acute administration of amphetamine in different strains of mice was established. As in rats, amphetamine increased levels of locomotor activity and the altered the patterns of locomotor movements. Furthermore, the effects of different doses of amphetamine differed significantly across strains. These results underscore the important contributions of genetic factors to the behavioral profile induced by stimulants.

[0092| Different strains of mice were used to determine whether measures of sequential organization of behavior, i.e. the geometrical patterns of movements and the predictability of movement sequences, are independent of levels of locomotor activity. It was confirmed that robust strain differenced in motor phenotypes in the vehicle- treated C57BL/6J, 129S6, and 129X1 mice (Ralph et al., 2001b). C57BL/6J mice continued to explore the enclosure during the entire test session. The 129X1 mice initially explored the enclosure, briefly moving into the center, while the 129S6 mice sampled only a small area of the test environment, rarely entering the center.

[0093| These strains of mice were also tested after administration of amphetamine. The amount of locomotor activity and the spatial pattern of motor sequences were altered differentially by amphetamine in C57BL/6J and 129X1 mice, but not in 129S6 mice. Of particular relevance is the combination of hyperactivity and perseverative locomotion seen in the C57BL/6J mice at higher doses of amphetamine, which is similar to the pattern of activity seen in the DAT knockout mice (see Fig. 5 in Ralph et al 2001a). Given that amphetamine releases presynaptic DA, these findings are consistent with the role of DA in the modulation of motor pattern in mice (Ralph et al 2001b). The significant differences in both locomotor activity and patterns across strains observed here support that some, but not all, dimensions of the behavioral phenotype are strain-dependent. Such observations support the possibility that these behaviors are in part genetically determined and may provide endophenotypes relevant to BD.

10094] Hyperactivity in DAT-Deficient Mice: The studies of the DAT KO mouse confirmed and extended previous observations of dramatic hyperactivity when these mutant mice were stimulated or placed in a novel environment. Although their activity levels were normal if undisturbed in their homecages, in a novel chamber, the locomotor activity of DAT KO -/- mice was characterized by repetitive, perseverative straight movements in the periphery of the enclosure. In contrast to both DAT +/+ and heterozygous +/- mice, these animals did not sample the entire enclosure; rather, the

DAT KO -/- showed a restricted repertoire of locomotor behavior (Ralph et al., 2001).

This restriction of the behavioral repertoire is similar to the behavioral patterns that are observed in rats treated with the direct D1/D2 agonist apomorphine or C57BL/6 mice treated with amphetamine (Figure 10). Thus, stimulation of the DA system in mice appears to produce a restricted locomotor phenotype characterized by repetitive or perseverative straight sequences of movements.

[0095) To identify which of the DA receptor subtypes was modulating the locomotor behavior in the DAT -/- mice, the DA mutant mice were treated with the D2-like receptor antagonist raclopride and the Dl receptor antagonist SCH23390 (Ralph et al., 2001a). Raclopride attenuated the locomotor hyperactivity of DAT -/ mice, but the drug did not significantly alter the perseverative movement patterns. Interestingly, raclopride produced more perseverative patterns in the DAT heterozygous +/- mice compared to vehicle-treated +/- mice, but not in DAT +/+ or -/- mice. Thus, blocking the D2 receptor in a mouse with mild hyperdopaminergia, which may result in a relative increase of Dl occupancy, increased perseverative locomotor pattern .

|0096| This finding is consistent with the effect of the Dl antagonist SCH23390 in reducing the perseverative locomotor patterns in the hyperdopaminergic DAT KO -/- mouse. Therefore, with increased dopaminergic tone, occupancy of Dl receptors may be important in regulating the perseverative aspect of locomotor behavior in mice. These findings support the likelihood that the different domains of disinhibition, though affected in common by pharmacological or genetic manipulations of the DAT, may involve some different neural circuits beyond the DA scopes, suggesting that antimanic and antipsychotic treatments may have differential influences on the three domains of inhibition in both human and animal studies.

100971 Valproate Reduces Hyperactivity in DAT Knockdown Mice: The hyperdopaminergic state of the DAT KD mice is consistent with a manic state in humans with BD. It has been suggested that the manic episodes in BD are consistent with a temporarily dysregulated DA system (Diehl & Gersho 1992; Emilien et al 1999). If indeed a dysregulated DA system underlies some of the key symptoms of mania, pharmacological agents that successfully treat manic symptoms would attenuate the hyperactivity displayed by the DAT KD mutant mice. It is confirmed that DAT KD mice were hyperactive in an open field compared to WT controls ((Zhuang et al., 2001, Ralph-Williams et al 2003b). In addition, the DAT KD mice exhibited more perseverative patterns of locomotor behavior than did the WT controls. Specifically, DAT KD mice exhibited significantly straighter sequences of motor patterns (lower spatial d values). When the DAT KD mice were treated with 100 mg/kg valproate, a dose which had no effect to WT mice, their hyperactivity was reduced significantly. Furthermore, while drug treatment had no effect on spatial d in the +/+ mice, valproate attenuated the perseverative patterns of motor behavior (diminished the predominance of straight sequences of locomotor activity as evidenced by increased spatial d) seen in the DAT mice.

[00981 Thus, when the DAT KD mice were treated with the clinically effective antimanic drug valproate, both their hyperactivity and their perseverative motor behavior were significantly attenuated. These data indicate that the DAT KD mice may emerge as a useful tool in investigating some disorders characterized with dysregulated DA systems, including potential drugs that may be useful in treating these disorders. While the work monitored motor activity in a video tracker, recent experiments have confirmed this phenotype in the mouse version of the BPM. Indeed, a pilot study indicated that the robust hyperactivity of the DAT KD mice in the BPM is reduced by pretreatment with 2 mEq/kg lithium chloride, with significant drug by genotype interactions in transitions, distance, corner entries, long wall entries, and counts.

|0099| Converging Evidence For PPI, Motor Activity, And Perseveration As Targets for Translational Models of Mania in BD: The results of the preliminary experiments highly suggest the utility of the three domains as a model of bipolar mania. Successful completion of the following experiments would enable us to utilize completely parallel experiments in humans and animals to: (1) determine treatment efficacy of novel antimanic agents in humans based on animal data; (2) identify mechanisms underlying bipolar mania using the animal model; and (3) assembling a neural systems model of bipolar mania by integrating animal and human experimental results:

looioo] The potential validity of two mouse models related to the mania phenotype characteristic of BD were evaluated. Both of the models are based on the deficient PPI, motor hyperactivity, and perseverative patterns of locomotion associated with increases in synaptic DA produced by: (1) acute dopaminergic activation produced by amphetamine; or (2) chronic dopaminergic activation in the DAT deficient mice. The ability of the two models were assessed to detect the effects of drug regimens used in the treatment of mania in BD patients (e.g. acute and chronic valproate, lithium, and acute atypical antipsychotics).

1001011 a) C57BL/6 Mice: The C57BL/6 mice were selected after consideration of a number of complex factors. However other inbred as well as out-bred strains of mice may well be warranted. The C57BL/6 mouse is the most extensively studied largely because it is the most common background strain on which gene knockouts are developed. Congenic lines (i.e. back-crossed at least 10 generations) of DA Dl, D2, D3, and D4 receptor KO mice were established, and DAT KO are on a mixed C57/129 background that most closely resembles C57s in its behavioral profile. Studies of PPI at baseline and after treatments with amphetamine or NMDA antagonists in all these DA-related KO mice on the C57 background were successfully done (see cited papers). Hence, the dose regimens and experimental parameters appropriate for the proposed studies were known.

(001021 The effects of antimanic medications on PPI deficits produced by amphetamine and ketamine have used both 129SvEv and C57BL/6J mice in the previous studies. Most importantly, the ability of acute lithium treatment to prevent the PPI disruptive effects of amphetamine has now been observed in both strains. Although the current line of DAT KD mice is on a mixed 129-strain background, the backcrossing the DAT KD mice to C57 is available. The current DAT +/+ KD mice are excellent for studies of PPI, but - like most 129 strains of mice - do not exhibit adequate levels of motor activity even during the dark cycle to reliably detect suppressive effects of treatments on locomotor behavior. (Unlike the +/+ mice, the DAT KD -/- mice are, of course, hyperactive). Also, C57 mice treated with amphetamine exhibit dose-related increases in motor activity and perseverative locomotion, while 129-related strains show less reliable effects of amphetamine.

1001031 Therefore, both male and female C57BL/6J mice were selected for the studies. For practical reasons, all the studies using DAT-deficient mice utilize both males and females. In addition, differential sensitivities of male and female C57BL/6 mice to both the administration of amphetamine and the deletion or disruption of the DAT gene were observed. Although often fairly subtle, these sex differences have been noted for all three domains of inhibitory function and may be important and informative to examine further. Thus, the pharmacological studies of the effects of amphetamine as a model of acute hyperdopaminergia include both sexes in order to optimize doses of amphetamine for the appropriate behavioral effects and to enable more complete comparisons with the parallel studies of DAT-deficient mice as a model of chronic hyperdopaminergia.

GENERAL PROCEDURES FOR MICE STUDIES

100104] Animal Subjects: In general, mice were allowed at least a 5-day period for acclimation to the animal room before behavioral testing and are handled every day. Male and female 157BL/6J (Jackson Laboratories, Bar Harbor, Maine) mice were housed in single-sex groups of no more than 4. They were caged in clear plastic cages in a climate-controlled animal colony with a reversed day/nig t cycle (lights on at 8:00 pm, off at 8:00 am). This room was dedicated for use only by the assigned laboratory. AU testing occurred between 9:00 am and 6:00 pm. Food (Harlan Teklad, Madison, WI) and water were freely available throughout the experiments, except during behavioral testing. Although the laboratory worked with both rat- and mice, the two species were never mixed in the same rooms or the same equipment. |00i05| Genotyping: Genotyping of both DAT KD and DAT KO mice consisted of the phenol-chloroform extraction method and PCR cycling methodology as published by the originators of the mice (Giros et al., 1996; Zhuang et al., 2001). One (1) cm tail lengths were obtained from each mouse at weaning and digested in proteinase K and buffer. Phenol-chloroform extraction were used to obtain DNA from each mouse. PCR were perform using Amersham PCR beads and 3 primers (1 volume of primer for the WT DNA, 1 volume for the mutant DNA, and 2 volumes of primer common to both strands). PCR analysis were analyzed on polyacrylamide gels.

10100) Drugs: Injected drugs were delivered s.c. or i.p. at a volume of 5 ml/kg. Drugs were dissolved in isotonic saline o other appropriate vehicle just prior to use.

|0l0l| Startle Apparatus, Mice: Startle reactivity, habituation, and PPI were measured using San Diego Instruments startle chambers, as described in Geyer & Dulawa (2003) and Ralph et al. (2001). Eight (8) mouse chambers were used simultaneously in a separate sound-attenuated room. Each box consisted of a clear nonrestrictive Plexiglas cylinder resting on a platform inside a ventilated chamber. A high frequency speaker produced both a continuous background noise of 65 dB and the various acoustic stimuli. Vibrations of the Plexiglas cylinder caused by the whole-body startle response of the mouse were transduced into analog signals by a piezoelectric unit attached to the platform, digitized, and stored. Sound levels referred to the A weighting scale (SPL) and were measured as previously described (Geyer & Swerdlow, 1998). The multiple chambers were calibrated weekly using a dynamic calibration system to ensure equivalent and consistent sensitivities.

fθlθ2| Prepulse Inhibition Test Session, Mice: The same test session described below for human studies were the primary session used in mice. In some experiments, stimulus parameters and session designs were varied to address specific questions or to optimize paradigms for simultaneous assessment of multiple variables. Whenever feasible, the primary session were followed by a brief additional test in which the intensity of the prepulse stimuli (rather than the ISI) is varied (69, 73, and 77 dB) as used in the publication (Ralph et al., 2001a). Analyses matched those used for humans. As needed, a variety of specific session designs were used, including cross-modal tests with lights as prepulse and tactile startling stimuli to address hearing loss, or different startle intensities to assess startle thresholds. |0l03| Mouse Behavioral Pattern Monitor (mBPM): Although the previous motor activity studies of mice utilized the VideoTracker system, the present studies use the more powerful mBPM, which monitors additional behaviors and enables testing in the dark. The mBPM system includes ten chambers, each enclosed within a ventilated external box. A well-developed software used for the rat BPM system (Geyer et al 1986)was adapted to accommodate the increased number of photobeams used for the mBPM. The system was housed in a dedicated and sound-attenuated room. Each chamber consists of a 30.5 x 61 cm x 38 cm Plexiglas hole board that is equipped with 3 floor holes in the front, middle, and rear part of the floor and 8 wall holes (3 along either side of the long walls, and two holes in the front and back walls). The location of the mouse was obtained from a grid of 12 x 24 photobeams 1 cm above the floor (height is adjustable). Each hole is also equipped with a photobeam. Rearing is detected by an array of 16 photobeams placed 2.5 cm above the floor an, aligned with the long axis of the chamber. The status of the photobeams is transmitted with a frequency of 18 Hz to a PC-compatible computer. It is confirmed that this sampling frequency is adequate to capture even the very rapid movements of, for example, DAT KO and KD mice. A change in the status of photobeams triggers the storage of the information in a binary data file together with the duration of the photobeam status. Subsequently, the raw data files are transformed into (x,y,t, event) ASCII data files comprised of the (x,y) location of the mouse in the mBPM chamber with a resolution of 1.25 cm, the duration of each event (t), and whether a hole poke or a rearing occurred (event).

|0i04] mBPM Test Sessions: For a typical experiment, the number of male or female mice were determined based on estimated effect sizes and power calculations using a full factorial ANOVA model with β>0.8 and α< 0.05. Previous studies using the primary measures have shown that, on average, 8-12 animals per group provide the projected power. The animals are brought to the laboratory 1 h before testing.

101051 mBPM Measures: The primary measures include: total entries, duration of time in the center, rearings hole pokes, spatial d, and dynamical entropy h. When appropriate, additional measures include: number and durations of hole pokes, number of entries into the center, and duration of time in the comers. |0l06) Statistical Analyses: In general, tests for homogeneity of variance across groups were first performed on the data. If the scores did not violate the assumption of homogeneity of variance, parametric statistics were performed, e.g., Analysis of Variance (ANOVA). Greenhouse-Geisser corrections were used for within-subjects statistics to account for violations of sphericity. Following significant differences, specific comparisons were made using the appropriate post hoc analysis (i.e., LSD, Dunnett's, Tukey's, Newman-Keuls, or Scheffe tests). The 5% rejection region was used for statistical comparisons. If data were highly skewed or kurtotic, the data were transformed using one of the appropriate transformation techniques. All statistics were analyzed using the Statistical Package for the Social Sciences (SPSS 1995).

Example 1

Mice Model (mBPM): Assess the Ability of Antimanic Treatments to Prevent the

Disruptions in Three Domains of Behavioral Inhibition Produced by Acute

Hyperdopamineraia (Amphetamine)

|0i07| Acute and/or subchronic treatments with antimanic and atypical antipsychotic medications prevent the amphetamine-induced decreases in PPI, increases in motor activity, and increases in perseverative locomotion.

|0i08) Acute Amphetamine Model. PPI: Studies of male and female mice were conducted separately using C57BL/6J mice shipped from Jackson Laboratories and adapted to the reversed light cycle for at least one week. Testing nocturnal animals during the dark phase of a reversed light cycle reduces variability in studies of either PPI or locomotor activity. Mice were handled routinely for 3-4 days prior to any behavioral testing. This procedure minimizes stress-related effects of handling and injections on subsequent test days and thereby reduces experimental variance.

[0i09| Groups for each treatment condition typically contained 10 mice each, based on our extensive experience with the effects of amphetamine on these measures in C57BL/6J mice. In each experiment, two doses of the pretreatment drug and one dose of amphetamine were used. Amphetamine sulfate was administered intraperitoneal Iy (i.p.) at a dose of 7.5 mg/kg for male mice and 10 mg/kg for female mice (calculated as the salt), based on the prior dose-response studies in C57BL/6J mice (Brody et al 2003b; Dulawa & Geyer 1996; Ralph et al 2001b; Ralph et al 1999). As in rats (Gold et al 1989), amphetamine was a within-subjects factor and pretreatment a between- subjects factor in a baselinematched, cross-over factorial design. Thus, a typical experiment assessing the interaction between a pretreatment drug and amphetamine on PPI use 30 mice, 10 for each level of the pretreatment factor.

[Oi 10) After adaptation to the vivarium and handling, mice are tested in a brief baseline session that were used to create groups that were matched for both startle magnitude and level of PPI. Each mouse was then tested twice at an interval of one week after a fixed pretreatment and either vehicle or amphetamine in a counter-balanced order. The pretreatment were given at an appropriate time (typically 30-60 min) prior to testing and the vehicle or amphetamine treatment were given 10 min prior to testing (Brody et al., 2003). Thus, each mouse receives only one amphetamine treatment. Note that neither sensitization nor tolerance nor carryover effects of amphetamine were typically found in tests of PPI in rodents (Dirks et al 2001; Druhan et al., 1998).

loin i Acute Valproate: The first treatment was an acute administration of sodium valproate, a sodium channel blocker, inhibitor of GABA transaminase, and inhibitor of phosphoinositol turnover (50 or 100 mg/kg, free base, i.p. 30 min pretest). The 100 mg/kg dose of valproate was sufficient to reverse the locomotor hyperactivity phenotype in DAT -/- KD mice without affecting locomotor activity in the +/+ mice (Ralph-Williams et al 2003b). This dose has similarly been shown to block the effects of acute administrations of amphetamine on motor measures of distractibility in rats (Agmo et al 1997). Higher acute doses of valproate (150 - 400 mg/kg, unpublished; Ralph- Williams et al 2003b) have significant behavioral effects in control mice. The data provided that valproate reduced the PPI-disruptive effects of amphetamine. Confirmation of this result would require a pretreatment-by-treatment interaction in a mixed design ANOVA (see General Methods). As in all the human and animal studies of PPI, multiple prepulse conditions were examined to assess the behavior at different levels and thereby aided the detection of possible floor and ceiling effects, but interactions with the repeated measures factor of prepulse condition were unknown. Post-hoc tests determined which doses of valproate contributed to the predicted reversal of the amphetamine effect.

[0112) Chronic Valproate: The studies were expanded with valproate by feeding animals food containing valproate, as developed by Dr. Husseini Manji at NIMH. With Dr. Manji's consultation, it was confirmed that the expected blood concentrations of valproate were achieved in the testing wild-type C57 mice. The study of chronic valproate is important to better mimic the clinical use of the drug and to establish a more steady-state blood level of the drug prior to the amphetamine challenge. The amphetamine results showed that 100 mg/kg valproate was not effective in 129SvPaslco mice and did not reverse the PPI deficit seen in DAT -/- KO mice (unpublished).

10113) The experimental design were similar to that used above, with valproate pretreatment being a between subjects factor and amphetamine treatment a within subjects factor in a counter-balanced order cross-over design. Separate experiments were conducted on male and female mice. As with the acute valproate manipulation, two different concentrations of valproate in the food were used to produce two different stable blood levels. Custom valproate chow were manufactured by Bio-Serv (Frenchtown, NJ); control chow were identical to the valproate chow except for the addition of valproate. Preliminary studies were used to specify the parameters for these treatments and ensure that the treatments do not compromise the health or relevant behavior of the treated mice.

[0H4] A study by Gould et al. found that 2 days of treatment with 10 g/kg sodium valproate chow followed by 9 days of 20 g/kg sodium valproate chow decreased beta- catenin protein levels in the frontal cortex of rats (Gould et al 2003). Chronic chow administration of lithium but not chronic chow administration of carbamazepine also resulted in measurable decreases in beta-catenin protein levels. Since Gould et al arrived at the valproate dose by testing different doses, and the concentration and duration of drug produced noticeable changes at the molecular level, it is provided that an identical dosing regimen were effective in the mouse since the ratio of consumed, metabolized chow to the weight of the rodent should be comparable between mice and rats. The purpose of the half dose for two days is to reduce side effects and to mimic the clinical administration of valproate (Gould et al 2003).

I0li5| After baseline matching (test-retest reliability is high even with a month between successive tests in humans, rats, and mice), mice were placed on either the control or valproate diet for 9 days (as adjusted via pilot studies) prior to the first test session with either vehicle or 10 mg/kg amphetamine sulfate (dose to be adjusted based on Aim 4 studies). One week later, mice were re-tested after the alternative treatment (amphetamine or vehicle). Blood levels of valproate were determined for each mouse during the week between tests using samples obtained from orbital eye-bleeds. 5-10 ul samples from mice were obtained. The valproate assays were conducted on a 3 ul sample. Blood levels of individual mice were used in additional regression analyses to assess the relationship between effective dose and behavioral effect. Thus, amphetamine treatment is continued to be used as a categorical factor, but pretreatment condition is replaced by individual blood levels as a predictor variable.

(01161 In the case of valproate, the blood level assays were sufficiently sensitive. Hence, orbital eye-bleeds, rather than sacrifice were used to determine blood levels of valproate. The mice on the same diet were kept for an additional week after the end of the PPI experiment. The animals in the locomotor activity paradigm, with or without amphetamine, were tested. The specific design and procedures are specified below.

J0117J Acute Lithium: The designs of the acute and chronic lithium studies parallel those described for valproate. The data indicated that 85 mg/kg (2 mEq/kg) lithium chloride (LiCl) was effective in reducing the PPI-disruptive effects of 10 mg/kg amphetamine in male mice from both the S 129SvPasIco and C57BL/6J strains. This dose was based on the literature (Gould et al 2001) and confirmed to yield a serum lithium concentration that was within the therapeutic range (Goodwin & Geddes 2003). It was confirmed using doses of 1 and 2 mEq/kg i.p. LiCl in both male and female mice using the standard paradigm and doses of amphetamine proposed for these more systematic studies of C57 mice. As above, a pretreatment-by-treatment interaction in a 3X2 factorial ANOVA was provided in which pretreatment was a between subjects factor and treatment is a repeated measure.

I0U8J It remains possible that the acute administration of a bolus injection of LiCl could alter salt balances in the animal and influence physiological processes such as blood pressure that might affect PPI. There is no evidence that alterations in blood pressure affect startle or PPI, but any such influences were controlled by testing an appropriate isomolar dose of NaCI in additional animals.

|0ii9] Chronic Lithium: Using a design similar to that described for chronic valproate, the effects of amphetamine on PPI in mice treated chronically with lithium in the food were also assessed. As described above for valproate, preliminary studies were conducted using different concentrations of LiCl in the food (as has been used successfully in rats) to identify concentrations that yield appropriate serum levels of lithium (0.5 mmol - 1.5 mmol). It is provided that 10 g LiCl per kg of food for 2 days followed by 20 g/kg were appropriate based on previous study of rats (Gould et al 2003). A dose of 1.5 mEq/kg administered s.c. in mice followed by a dose of 2.3 mEq/kg daily resulted in a significant loss of weight of light mice but not heavy mice, and was attributed to the anorexic effects of lithium in rodents and humans (Wood & Morton 2003). Lithium administration resulted in no significant change in weight in wild-type mice after 3.7 weeks of administration (Wood & Morton 2003). LiCl administration at this dose did not affect mortality in wild-type KO mice over 2 months of administration, which suggests that a one month dose should be safe (Wood & Morton 2003). Since humans require up to 2 weeks of lithium administration to reach steady-state levels, and 2 weeks is a safe interval in mice, 2 weeks is the interval of choice in mice.

{0120} After baseline matching and sufficient time on the diet, mice were treated with either vehicle or 10 mg/kg amphetamine sulfate. One week later, mice were retested after the alternative treatment (amphetamine or vehicle). Mice were then maintained on their specific diet and sacrificed without anesthesia within a few days of their last PPl test for trunk blood collection and serum lithium determinations. Thus, the design is a counter-balanced order, factorial design with 3 levels of the pretreatment factor (diet; between subjects) and 2 levels of the treatment factor (amphetamine; within subjects). The pretreatment by treatment interaction were the same as in the above studies. As above, regression analyses were also used, with the serum levels of individual mice serving as a predictor variable.

|0l2l] Although the preliminary studies indicated that acute administrations of lithium were effective in reducing the PPI-disruptive effects of amphetamine, these effects were not large and were sometimes limited to only part of the test session. Thus, the rapidly changing serum levels associated with acute injections of lithium may have complicated the study. It is necessary to confirm that chronic lithium is effective in this paradigm independently of the effects of acute lithium. Moreover, unlike with valproate (above), no pilot studies in the mBPM are done in this instance because the mice must be sacrificed in order to verify their serum lithium levels in close temporal proximity to the behavioral tests.

[O122| It is provided that both valproate and lithium, administered chronically, are effective in, reducing the PPI disruptive effects of amphetamine. Additional studies with other antimanic or mood-stabilizing agents are undertaken, additional drugs that are used to treat mania were first focused, although chronic treatment with some mood stabilizers that exhibit prophylactic influences on manic episodes could also prevent the disruption of PPI produced by amphetamine. In this regard, the acute amphetamine challenge might be conceptualized as a qualitatively different model than the genetically determined alteration in dopaminergic function represented by the DAT- deficient lines of mutant mice.

10123] Antimanic compounds of interest may include, but not limited to: carbamazepine, olanzapine, or quetiapine. Additional mood stabilizers may include, but not limited to: lamotrigine; topiramate; and gabapentin. One of the most important of additional drugs would be acute and/or subchronic carbamazepine. Carbamazepine is a tricyclic anticonvulsant drug used in the treatment of mania. Acutely, carbamazepine are administered at a dose of 50 mg/kg (to be confirmed in pilot dose- finding studies) and tested as in the prototypical experiment above (i.e. 2-by-3 design with amphetamine). The subchronic treatment matches that used previously to block the induction of cFOS by 2 mg/kg methamphetamine, namely one week on a chow containing 0.25% carbamazepine and a second week on a 0.5% chow (Lee et al 2000). Pilot studies and blood assays are conducted to confirm the effectiveness of this regimen and its effects on motor activity by itself.

[01241 Acute Amphetamine Model, Motor Activity: Based on the dose-dependent increases in locomotor activity and perseverative patterns seen in the C57BL/6J mice, the ability of antimanic agents to reverse these behaviors in mice was examined. In each case, treatment regimens that do not significantly reduce locomotor activity by themselves was utilized. As in the PPI experiments, studies of male and female mice were conducted separately using C57BL/6J mice adapted to the reversed light cycle for at least one week and handled repeatedly. Testing during the dark phase of the light cycle is extremely important for ethologically relevant studies of exploratory locomotor activity in rodents. Groups for each treatment condition typically contain 10 mice each, based on the extensive experience with the effects of amphetamine on locomotor activity in C57BL/6J mice. In each experiment, two doses of the pretreatment drug and one dose of amphetamine were used. Amphetamine sulfate were administered at a dose of 4.0 mg/kg for male mice and 5.0 mg/kg for female mice (calculated as the salt), based on the prior dose-response studies in C57BL/6J mice (Ralph et al 2001 b; Kadner, Geyer, in preparation). These doses produce robust locomotor activation and perseverative patterns without being close to the threshold for the focused stereotypies that can compete for expression with locomotor activity (Kadner, Masten, Geyer, in preparation).

|O1251 As discussed above, amphetamine is a within-subjects factor and pretreatment is a between-subjects factor in a cross-over factorial design. Thus, a typical experiment assessing the interaction between a pretreatment drug and amphetamine use 30 mice, 10 for each level of the pretreatment factor. Prior to any treatments, mice were tested in a 30-min baseline session, which constituted their first exposure to the test chamber. This baseline session is important to characterize the phenotype in the studies of genetically modified mice and is used here to maximize the comparability of the pharmacological and knockout models. In addition, it is found that some prior familiarization with the test increases the comparability of the two subsequent experimental test sessions used for the cross-over design. Such a design has proven to be an effective and efficient utilization of precious animals, which is particularly important when using mutant mice. Each mouse wad then tested twice at an interval of two weeks after a fixed pretreatment and either vehicle or amphetamine in a counterbalanced order. The pretreatment was given at an appropriate time (typically 30-60 min) prior to testing and the vehicle or amphetamine treatment was given 10 min prior to testing (see Brody et al 2003b). Locomotor and exploratory behavior in the mouse Behavioral Pattern Monitor (mBPM) chambers were monitored for 2 h to cover the bulk of the locomotor response to amphetamine in C57BL/6J mice.

(01261 Acute Valproate: The same doses of sodium valproate used in the PPI studies: 50 and 100 mg/kg i.p., were tested. The higher dose is below threshold for altering locomotor activity by itself in C57BL/6J mice, while being effective in reducing the locomotor hyperactivity phenotype seen in the DAT KD mice (Ralph-Williams et al 2003b). Thus, a prototypical experiment involve a 3 (vehicle, 50, or 100 mg/kg valproate pretreatment) by 2 (vehicle or amphetamine treatment) factorial design with groups of 10 mice tested for 2 h in the mBPM beginning 30 min after pretreatment and 10 min after treatment. The data were analyzed by ANOVA for each of the primary measures (entries, distance traveled, holepokes, rearings, and spatial d. it is provided that valproate reduces the hyperactivity produced by amphetamine that is confirmed by a significant pretreatment-by-treatment interaction.

[0127] Relative to the studies of PPI, the potentially complex dose-response function for the effects of amphetamine on motor activity may complicate the interpretation of the results of these initial experiments. This dose function in both male and female C57 mice was characterized, and the test doses were chosen to be on the ascending portion of the function with respect to locomotor activation. Nevertheless, depending upon the nature of the observed interactions between the pretreatment and amphetamine, additional studies using additional doses of amphetamine may well be required to confirm and extend these experiments.

1O128| Chronic Valproate: The dietary valproate regimen matched that used in the PPI studies above. As discussed above, two different blood levels of valproate were examined, with testing presumably being conducted at weeks 2 to 4 after starting the mice on the appropriate chow. The corresponding pretreatment-by-treatment interactions in both ANOVA and regression (using blood valproate levels in each mouse as predictor variables) analyses matched those described for the PPI experiments.

|0l29| If the above PPI studies are extended in time to include locomotor activity tests after the completion of the PPl experiments, the specifics of these studies may be adjusted accordingly. For example, if the lower dose of chronic valproate is effective in reducing the activating effects of amphetamine in both sexes in the above experiments, only one sex and only the lower dose of dietary valproate would be tested here. This test is still required to confirm that the same duration of treatment used in the PPI studies was similarly effective in the locomotor activity paradigm. It is also important to confirm that the same interaction between valproate and amphetamine was obtained in mice that had not been treated previously with amphetamine, in the unlikely event that some sensitization might have influenced the results in the previous studies. Note that little if any sensitization is expected in mice given only one prior dose of amphetamine in a different environmental context and at a considerable time before the subsequent testing.

[01301 Acute Lithium: Doses of 1 or 2 mEq/kg LiCl one hour before testing have been shown to reduce the locomotor-activating effects of 3 mg/kg amphetamine in C57 mice (Gould et al 2001). Higher doses of LiCl reduced activity by themselves, but especially the low dose only altered the amphetamine response. Similarly, it is found that 2 mEq/kg LiCl reduces the PPI-disruptive effects of amphetamine. Same two doses of LiCl (1 and 2 mEq/kg or 42.5 and 85 mg/kg i.p.) were used for these experiments, with a separate 3-by-2 design for each sex as in the valproate studies detailed above.

[0i3l| Chronic Lithium: These studies determine whether the locomotor hyperactivity and perseveration produced by acute administration of amphetamine (3 & 4 mg/kg for males and females) is reduced in mice maintained for 2-4 weeks on a diet containing lithium. The studies indicated that the concentration of lithium in the chow needed to establish appropriate serum levels in mice. The associated pretreatment by treatment interactions matched those listed above.

101321 Atypical Antipsychotic Studies: Not specified above were parallel studies of atypical antipsychotic drugs. Parallel studies of both clozapine and risperidone were conducted. Clozapine were used because it is considered to be the most unusual of the second-generation or atypical antipsychotics in its clinical effects and because it is the compound with which we have the most experience. Risperidone were used because it is currently the most commonly prescribed antipsychotic for the treatment of manic patients on our inpatient service. Each compound poses some complications for each behavioral paradigm.

101331 At least acutely, both compounds are likely to produce some reduction in motor activity in wildtype mice. The exploratory behavior tests were designed to ensure that decreases in activity by drugs such as antipsychotics (testing in relatively novel and dark environments during the animal's active dark cycle) can be detected. While the standard procedure of habituating the animals was used to the chamber and then administering amphetamine, this procedure failed to distinguish non-specific decreases in activity from decreases in the response to amphetamine (Geyer 1990; Geyer & Segal

1991). 10134] Pilot studies were conducted to identify acute doses that have minimal effects in control animals. With clozapine, the highest acute dose is likely 3.0 mg/kg. With regard to PPI testing, risperidone increases PPI in control mice (Olivier et al 2001; Ouagazzal et al 2001), complicating the interpretation of the increases in PPI in amphetamine-treated mice. To ensure that at least the lower dose does not increase PPI in control mice, doses of 0.3 and 1.0 risperidone were used. It is found that up to 3 mg/kg, clozapine has no such effect in C57BL/6J or DAT KO +/+ mice, although it does reduce startle magnitudes to some degree. Hence, clozapine was used in doses of 1 and 3 mg/kg acutely. In cases where the acute antipsychotic treatment fails to prevent the effects of amphetamine or leads to uninterpretable results, chronic treatment regimens were explored. In the extensive similar studies in rats, chronic treatments have not been required to block the effects of dopaminergic agonists on PPI (Geyer et al 2001 ; Mansbach et al 1988).

Example 2

Mice Model (mBPM): Assess the Ability of Antimanic Treatments to Reverse the

Disruptions in Three Domains of Behavioral Inhibition Produced by Chronic

Hyperdopam inergia

101351 It is provided that subchronic treatment with antimanic and atypical antipsychotic medications reverses the reductions in PPI (DAT -/- KO), increases in motor activity, and increases in perseverative locomotion seen in DAT-deficient (DAT -/- KO and DAT -/- KD) mice. Acute pharmacological treatments may also reverse some of the less dramatic phenotypes in DAT-deficient mice.

|0l36l Dopamine Transporter Knockdown (DAT KD) Model, Motor Activity: The potential validity of the hyperactivity seen in the DAT KD mice as a behavior having sensitivity to antimanic agents were tested using the same treatments listed above. It is demonstrated that 100 mg/kg valproate attenuates the hyperactivity and perseverative behavior characteristic of the DAT KD mice, while having no discernable effect in the wildtype mice or in C57BL/6J mice. This chronic hyperdopaminergic phenotype was tested with the same treatments and measures described above for use with amphetamine administration. Unless further studies reveal conditions under which the DAT KD mice show deficits in PPI, only the locomotor activity studies were done in these mice. For studies of mutant mice, only the offspring of heterozygous matings were used. Genotype was generated, and study cohorts of 200+ mutant mice derived from simultaneous breeding of 20-25 +/- pairs. It is likely that only the +/+ and -/- offspring were used in these drug studies, as it is seldom seen robust phenotypes in the KD +/- mice (though any such phenotype would be pursued avidly). Thus, a given cohort of mutants for testing likely involve 50 +/+ and 50 -/- mice. Both sexes were used with sex being included as a factor in the ANOVA. Meanwhile, the +/- offspring are utilized in the breeding to generate another cohort for further studies.

[O137| DAT KD - Acute Pretreatments: Using the doses of valproate and LiCl described above, similar studies were conducted in the mBPM on DAT KD +/+ and -/- mice. In each case, it is provided that the antimanic agent reduces both the hyperactivity and the perseverative pattern of locomotion seen in the DAT KD -/- mice, as confirmed by a pretreatment by genotype interaction. The results of the above studies were used to adjust pretreatment regimens. Thus, pretreatments that have significant (or even trend) effects in control C57 mice were not used in these studies. If both doses of a given pretreatment (e.g. 50 and 100 mg/kg valproate) were deemed of value, the design were a 3 (pretreatment) by 2 (sex) by 2 (genotype) between-subjects factorial design, with at least 8 mice per cell (96+ mice split into 12 groups). In the absence of interactions with sex, the group sizes become about 16 per cell. Such interactions occurred at least at a trend level in some but not the majority of studies. All mice were first tested in a 30-min baseline characterization session, which were used to create groups matched for overall level of locomotor activity prior to treatment. Two weeks later, mice were tested in a 60-min test session after the appropriate pretreatment.

|0l38l Given the investment in generating and genotyping each cohort of mutant mice, each cohort was typically used in more than one experiment. With a minimum of two weeks between treatments and exposure to the mBPM chambers, it is found that carryover effects from one experiment to another were typically minimal. Nevertheless, groups were counter-balanced according to drug history and assess possible carryover effects by including history as a grouping factor. Any indications of an interaction involving prior test history prompted confirmatory tests in a subsequent experiment. 101391 A fundamental concern with any constitutively mutated animal was that compensatory changes during development may contribute to any observed phenotypes. In the use of the DAT-deficient mice as possible models of genetic abnormalities (though not likely to be identical to what one might expect in human populations), it is considered that the DAT mutants may be models of compensation. Nevertheless, the comparison of the effects of amphetamine with the abnormalities in DAT mutants began to assess this issue. Such concerns were addressed using temporally controllable alterations of the DAT gene. A doxycycline-sensitive DAT mutant was in development with a normal complement of DAT until given doxycycline. Use of such mice, testing the mutation on different genetic backgrounds would be possibilities in future studies.

10140) DAT KD - Chronic Pretreatments: Using the chronic pretreatment regimens for valproate and LiCl described above, similar studies were conducted in the mBPM on DAT KD +/+ and -I- mice. Depending upon the size of the cohort available for study, either one or two concentrations of valproate or lithium in the food were examined in a given experiment. Anticipating some variance in blood or serum levels between individuals, groups of 10-12 were initialed for these studies, which could limit any individual study to only one dose of the pretreatment. The prioritization of which chronic regimens to utilize were based on the results of the above studies.

|0l4i I Given that -/- KD mice consume about 20% more food than their +/+ littermates (Pecina et al., 2003), some adjustment of the concentration in the food might be required. Indeed, preliminary evidence indicated a higher serum level of lithium in DAT-KO -/- mice than in +/+ controls fed the same diet. Hence, preliminary studies using mice that had already been tested in one of the acute experiments were undertaken to determine the optimal dosing regimens to obtain comparable serum levels in +/+ and -/- KD mice.

[0142J Dopamine Transporter Knockout (DAT KO) Model, PPl: Unlike the DAT KD -/- mice, the DAT KO - /- mice exhibit a robust deficit in PPI that mimics that seen in the manic BD patients (though the deficit is more robust in the mice). Hence, these studies provided that antimanic treatments reverse ("rescue") the PPI deficit in -I- DAT KO mice. It is further provided that both clozapine and quetiapine (3 and 2.5 mg/kg, respectively), atypical antipsychotics, were effective in reducing the deficit in PPI in male DAT KO -/- mice.

{0143] DAT KO - Acute Pretreatments, PPI: Using the doses of valproate and LiCl described above, the sensitivity of the PPI deficit in DAT KO -/- mice to reversal by antimanic agents was assessed. For each antimanic treatment, it will reduce the PPI deficit seen in the DAT KD -/- mice, as confirmed by a pretreatment by genotype interaction. It is provided that no such interaction on measures of startle reactivity. The offspring of heterozygous matings were genotyped by PCR and tested in a baseline startle test to develop groups matched for startle and PPl prior to drug testing. One week later, +/+ and -/- mice were tested after vehicle or one or two doses of the antimanic agent (depending upon the size of the cohort). If necessary, mice were tested again at weekly intervals after the alternative pretreatment so that pretreatment became a within subject factor. Analytical designs matched those used above.

10144] DAT KO - Chronic Pretreatments, PPI: The effects of chronic valproate and lithium on the PPI deficits in DAT KO -/- mice were assessed, as described in the above studies. Adjustments in the concentrations of drugs in the food were warranted for the DAT KO -/- mice. Mice were tested on the diet weekly, as repeated testing of

PPI was a standard and very effective strategy in mice, rats, and humans. The dietary treatment was continued for up to 4 weeks. Serum lithium levels were assessed using eye-bleeds every two weeks; blood valproate were assessed in each mouse from trunk blood samples taken at sacrifice after the last test.

|0l45] Dopamine Transporter Knockout (DAT KO) Model, Motor Activity: The effects of antimanic agents on the locomotor hyperactivity and perseverative patterns of locomotion exhibited by DAT KO -/- mice were tested in the mBPM. In contrast to the DAT KD studies, +/- as well as +/+ and -/- mice were used in these studies, because some gene-dosage effects of the DAT deletion were seen in measures of the perseverative patterns of locomotion (Ralph et al 2001a). From each cohort, approximately half of the +/- mice were used in the experiments rather than in the breeding for the next cohorts. Although the hyperactivity phenotype and the perseverative pattern phenotype were not be differentiated, it is provided that in both rats and mice that these two categories of behavior were independent and can be affected differentially. For example, both Dl and D2 DA receptor antagonists reduce the hyperactivity phenotype in the DAT KO -/- mice, but only the Dl antagonist attenuated the perseveration phenotype. In contrast, D2 but not D 1 receptor antagonism reversed the PPI phenotype in these mice. Thus, the three dimensions of inhibitory function can be individually altered pharmacologically even in a mutant mouse that exhibits all three abnormal phenotypes.

[0l46| DAT KO - Acute Pretreatments. Motor Activity: The DAT KO studies match the treatments, designs, and predictions above for the DAT KD mice, except that all three genotypes were used. As discussed above, it is provided that the antimanic agent reduced both the hyperactivity and the perseverative pattern of locomotion seen in the DAT KO -/- (and to some extent the +/-) mice, as confirmed by a pretreatment by genotype interaction.

(01471 DAT KO - Chronic Pretreatments. Motor Activity: The studies of chronic antimanic treatments on the motor activity of DAT KO mice match the treatments, designs, and predictions above for the DAT KD mice, except that all three genotypes were used.

|0l48i Atypical Antipsychotic Studies: As discussed above, clozapine and risperidone were also tested using similar designs. It is provided that there is no need for chronic administrations of atypical antipsychotics to prevent the effects of amphetamine in above studies, but chronic treatments were required to reverse the preexisting phenotypic abnormalities in the DAT-deficient mice. As with the antimanic agents, preliminary studies using administration of the antipsychotics in the food were conducted to select the appropriate dose regimens.

Example 3

Mouse Model (mBPM): Assess the Ability of Antidepressant Treatments to Precipitate Mania-Like Disruptions in Behavioral Inhibition in DAT +/- Knockout or DAT

Knockdown Mice

(01491 It is provided that subchronic (and perhaps acute) treatment with antidepressant medications induces disruptions in the three domains of inhibition DAT-deficient mice that exhibit minimal or no phenotypic abnormalities at baseline (DAT +/- KO and, for PPI, DAT -/- KD) mice. If both antimanics (e.g. lithium and valproate) and atypical antipsychotics (e.g. risperidone and quetiapine) are effective in reversing inhibitory deficits in the DAT-deficient mouse models, the animal model would have some degree of predictive validity for identifying either class of drugs, but would not have discriminant validity in distinguishing between antimanics and antipsychotics. Note that such a lack of discriminant validity is not much different from the clinical realities in the current use of these medications. One approach to distinguishing the relevance of the putative animal model to treatments of BD rather than treatments of schizophrenia is to assess the effects of antidepressant treatments in the model.

|O15O] Clinically, antidepressants when used alone have been shown to precipitate an episode of mania in a BD patient (Goodwin & Jamison, 1990). In contrast, schizophrenia patients are not uncommonly treated with both an antipsychotic and an antidepressant. It is provided that the DAT +/- KO mice and the DAT -/- KD mice have a genetic predisposition or partial loading toward mania-like behavior, which is sufficient to produce some hyperactivity (at least in the case of the DAT KD mice), even though they fail to exhibit a robust phenotype of behavioral disinhibition. Accordingly, whether an acute treatment with an antidepressant unmask a disinhibition phenotype was tested. Specifically, it is provided that DAT +/- KO mice and DAT -/- KD mice that exhibit normal PPI in the absence of treatment, exhibited deficits in PPI after treatment with fluoxetine or imipramine.

|0i5l] Acute Antidepressants. DAT KD and KO, PPI: Doses of 10 and 20 mg/kg fluoxetine were administered to +/+, +/-, and -/- DAT KD mice prior to tests of PPI. Both males and females were used, with sex included as a factor in the ANOVA. As above, mice were baseline-tested for determination of groups matched for baseline startle and PPI and then tested with each of the 3 pretreatments at weekly intervals. If carryover effects or order interactions were noted, the study was repeated using a between-subjects design. The serotonin selective reuptake inhibitor (SSRI) fluoxetine was first focused on because it is found that a serotonin2A antagonist (M 100907) attenuates the PPI, hyperactivity, and perseverative phenotypes in DAT KO -/- mice (Barr et al 2003). It is provided that the -/- (and perhaps the +/-) knockdown mice exhibited normal PPI after vehicle pretreatment and reduced PPI after fluoxetine. A pretreatment by genotype interaction and appropriate post-hoc comparisons would confirm this finding. 101521 A similar study using acute administrations of the tricyclic antidepressant imipramine were then conducted. As with fluoxetine, it is provided that the antidepressant unmasked a tendency for deficient PPl in the DAT deficient mice in which the loss of transporter sites was incomplete and insufficient to produce a robust phenotype in the absence of the antidepressant.

[0153) DAT KQ: Doses of 10 and 20 mg/kg fluoxetine were administered to +/+ and +/- DAT KO mice, both males and females. In this case, the -/- mice were not tested because their PPI was already disrupted to the point that it would be unlikely to observe any further disruption after pharmacological treatment. If acute antidepressant treatments do not alter PPI in DAT KD -/- or DAT KO +/- mice, fluoxetine or imipramine were administered chronically using established procedures (Santarelli et al., 2003) and the mice were tested weekly for up to 4 weeks. Thus, if chronic but not acute antidepressant treatments were required to unmask the mania-like phenotype, as they are required for clinical efficacy in depression and for other behavioral effects in mice (Santarelli et al., 2003), this chronic regimen produced a deficit in PPI.

101541 Acute and Chronic Antidepressants. DAT KD and KO. Motor Activity: Similar studies were conducted to assess the effects of antidepressants on motor activity and perseverative patterns of locomotion. Again, it is provided that the antidepressants unmasked a disinhibition phenotype in DAT-deficient mice that are relatively normal without pharmacological treatment. Hence, DAT KD +/+ mice were compared with DAT KD +/- mice after vehicle or an antidepressant treatment (acute or chronic). Similarly, DAT KO +/+ mice were compared with DAT KO +/- mice with and without the antidepressant treatments. The -/- mice were not used in either case, as they already express robust phenotypes.

[01551 The completion of these parallel human and animal studies establish the profile of inhibitory deficits in manic BD patients both on hospital admission, when patients are highly symptomatic, and during treatment with antimanic and/or antipsychotic drugs. The results of these studies in humans provide the essential information needed to assess the validity of the murine models of hyperdopaminergia. The human studies provide clear evidence regarding the effects of various clinical treatments on the profile of behavioral measures of disinhibition in BD patients. Because patients to treatment conditions were randomly assigned, further studies are needed to refine and clarify the results of these naturalistic clinical experiments.

[0!56| Nevertheless, the information gathered herewith enables important evaluations of both mouse models of hyperdopaminergia and other animal models in which relevant behavioral measures are examined. It is provided some examples that illustrate the potential value of the translational research strategy. Based upon the initial studies in DAT-deficient mice, it is provided that divalproex sodium attenuates hyperactivity and response perseveration in manic BD patients, without altering the deficit in PPI. Note that acute valproate reduces hyperactivity and perseverative locomotion in DAT KD mice, but does not appear to reduce the PPI deficit in DAT KO mice. Lithium might also normalize PPI in manic BD patients, as it reduces drug- induced deficits in mice.

(0157] An animal model (which at baseline exhibited deficient PPI, hyperactivity, and perseveration) in which hyperactivity and perseveration were attenuated by both valproate and lithium and in which lithium but not valproate reversed PPl deficits, can receive considerable empirical validation. An animal model that exhibited such a high degree of predictive validity is an invaluable tool for examinations of the neurobiological substrates involved in the expression and/or etiology of BD. Identifying differential effects among the measures of inhibition in manic BD patients facilitates model development and helps to better specify the specific disorder(s) and treatments being modeled. Such discriminations among the three domains of inhibition are likely to be observed in both human and animal experiments. Recall that DA D2 antagonists reduce the hyperactivity and PPI deficit, but not the perseverative abnormalities, in DAT KO mice (Ralph et al., 2001). In contrast, a DA Dl antagonist, which does not share with D2 antagonists the ability to block the PPI-disruptive effects of amphetamine (Ralph- Williams et al., 2002), reduces both the hyperactivity and the perseverative locomotion in the DAT KO mice (Ralph et al., 2001). Hence, although manic BD patients exhibit abnormalities in all three of the domains of inhibition, as do amphetamine-treated or DAT deficient mice, all the antimanic and atypical antipsychotic treatments have uniform effects on all these measures.

101581 Similarly, some treatments have therapeutic effects that can be indexed by one or another of the measures of inhibition differentially after acute versus more chronic treatments. Such information provides further criteria by which to evaluate the specific results that can be derived from the animal models. Further, the profile of deficits across the domains of inhibition at admission may represent clinical responses to particular pharmacological treatments. Some of the measures are rapidly responsive to treatments in a manner that represents the treatment efficacy, or lack thereof, over a longer term. Moreover, one or another of the measures of disinhibition is non- responsive to treatments and independent of clinical state. Such a measure has utility as a trait marker or endophenotype of BD.

HUMAN STUDIES

|0l59) Studies that demonstrate: I) that manic BD patients have sensorimotor inhibition deficits that may be responsive to treatment; 2) depressed and non-manic BD patients may not have these marked PPI deficits; and 3) that continuous motor activity can be reliably monitored, were completely. To study of perseverative behavior as an indicator of inhibitory deficits, both traditional and alternate methods of measuring perseverative patterns patients were used and applied these analytic techniques to help understand the nature of inhibitory deficits.

[0160] PPI deficits in manic BD patients: It has been widely reported that patients with clinical features of inhibitory deficits have PPI deficits (Braff et al 2001; Swerdlow et al 2001). Relatively little has been published on PPI in manic BD patients. As shown here and in a recent paper (Perry et al 2001 a), it was the first to demonstrate that BD patients with psychotic mania, tested within 96 hours of acute psychiatric hospitalization, had significant deficits in PPI that were comparable to those of acutely hospitalized schizophrenia patients (Fig. 4).

[01611 A remaining question, however, is whether dysregulation in the dopaminergic and related circuitry during acute manic states is restored when the patient's manic symptoms have been stabilized. To examine the stability of sensorimotor gating deficits, a study was conducted to test manic BD patients on their admission to a psychiatric hospital and again approximately 2 weeks later, when their symptoms are at least partially stabilized, 9 manic BD patients were tested. The results suggested that manic BD patients demonstrate PPI deficits at baseline, consistent with the previously published results, and that these deficits decrease during the second test session, paralleling the level of improvement in their symptoms. It is important to note that it has been shown repeatedly that PPI performance is stable both in NC subjects and in clinically stable schizophrenia patients over as long as 2 months (Braff 1978; Cadenhead et al 1999; Ludewig & Vollenweider 2002). The findings demonstrated that adequate PPI data from manic BD patients can be obtained to complete the study on PPI as well as other inhibitory measures.

|0l62] Normal PPI in depressed and non-manic BD patients: Although there is evidence to suggest that impairment in sensorimotor gating is evident during manic states, PPI deficits do not appear to be associated solely with disruptions in mood or general symptom states. For example, PPI in acutely hospitalized patients with Major Depressive Disorder (MDD) without psychosis to schizophrenia patients and NC subjects were compared (Perry et al in press). The MDD patients had PPI levels that were not significantly different from NC subjects and significantly higher than the schizophrenia patients. Additionally, PPI data on 4 subjects with BD, depressed type were collected. These patients were tested within 72 hours of psychiatric hospitalization, when they were in a highly symptomatic state. The initial analysis of the data revealed that the BD depressed subjects had an average PPI of 75% at the 120- ms ISl, which was similar to the PPI levels exhibited by normal comparison sample at the same ISI (Fig. 4). Albeit preliminary, the result further supported that in affective disordered patients, PPI deficits may be specific to mania and psychosis and does not constitute a general feature of acute mood symptoms.

|O163] Monitoring continuous motor activity and perseverations: Preliminary data were collected to determine: (1) whether high temporal resolution motor activity data in humans can be obtained; and (2) whether measures derived from nonlinear dynamical systems theory to quantify perseverative patterns in human motor activity can be used. In particular, sequential pattern information was extracted to quantify the degree of perseveration using an accelerometer tracking device (LifeShirt®). The decision to measure continuous motor activity as well as perseverative movement was based upon the studies in genetically altered rodents. Thus, the animal work had led us to develop the human paradigm.

|0164] A healthy human volunteer, who had extensive experience observing manic bipolar patients, was instructed to generate different "types" of whole body motion while wearing a LifeShirt®. Five different movement patterns were simulated: sitting very still, sitting relaxed with some movement, sitting but fidgeting in the chair, walking in the room, and walking and rocking, respectively. These movement patterns were selected based on prior observations of manic patients. Accelerometry data were sampled for a total of 28 minutes at a 10 Hz sampling rate and were stored numerically in digital units. Inspection of the acceleration distribution revealed a highly skewed density towards few high-acceleration bouts.

|0l65] All accelerations were logarithmically transformed prior to the analysis. To examine the degree of perseveration, the dynamical entropy was calculated, which quantified the degree to which sequences of observation, which were initially very similar evolve differently across time. The calculation of the dynamical entropy was based on determining the minimal length of a subsequence that renders this sequence unique in a data sequence, e.g. the subsequence "01" but not the subsequence "11" is unique in the sequence "1 1 10110", i.e. there was no other "01" subsequence but there were three "1 1" subsequences (in the first, second, and fifth position of this data sequence). The logarithmic distribution of accelerations was divided into four equal sized bins. The sequence of accelerations was then transformed into sequences of bin numbers. For example, a small acceleration value followed by a large acceleration value would correspond to a sequence { 1, 4} . Using a method developed by Grassberger (Grassberger 1989), the unique sub-sequence lengths of the accelerations were obtained. The unique subsequence length was determined for each sequence starting at the i* data point and the local dynamical entropy was computed via log (number of data points)/[unique subsequence length].

(01661 The data provided that average acceleration increased with increasingly active movement patterns (Fig. 5). Naturally, the overall measures of activity reflect the combination of fidgeting, standing/walking, and pacing/ pocking. In contrast, the dynamical entropy measure began to discriminate these different patterns of activity while exhibiting the expected inverted U-shaped response curve as a function of increasing activity (Fig. 6). Specifically, both very low active movements and highly repetitive movements were more predictable than movement patterns in the mid-level range of activity. Therefore, the data not only showed that measures of perseveration can be applied to the LifeShirt® data, but also, that perseveration followed the well established relationship with levels of activity. The inverted U-shaped response curve of the dynamical entropy as a measure of perseveration is completely analogous to the inverted U-shaped dose responses previously reported using both dynamical entropy and the spatial scaling exponent, d, in both rat (Paulus & Geyer 1.91) and mouse (Paulus et al 1999a) experiments.

|O167) Alternate methods of studying perseverative behavior: Several alternative methods of indexing perseverative behavior were published to examine perseverations as indicators of inhibitory dysfunction (Minassian et al 2003; Paulus et al 1999b; Perry & Braff 1998; Perry et al 1998). For example, a two-choice prediction task was used to characterize decision-making behavior in 15 manic BD patients in the acute phase of their hospitalization (Minassian et al in press). The results demonstrated that manic BD patients are characterized by a decision-making strategy that is consistent with a decreased ability to inhibit alternative responses. Minassian & Perry (2003) recently completed a study on perseverative patterns of eye movements during the visual scanning of complex objects, using the non-linear dynamic approach that has been developed to examine sequential movement patterns in rodents. They found that schizophrenia patients with highly predictable and highly circumscribed, restricted scanning, as measured by a derivative of the spatial */ exponent used in the rodent work, had a higher number of verbal perseverations (r = -.42, p = .02). These studies provide alternate methods of indexing perseverations and underscore the usefulness of multiple means of assessing perseverative behavior in an effort to better understand the nature of inhibitory deficits.

|0168J Overall Design & Subject Selection: A cross-sectional study on differences in inhibitory functions between manic BD patients and Normal Comparison Subjects (NCS) as assessed by PPI, levels of motor activity, and perseveration was performed, as indicated in Example 1 below, and a longitudinal follow-up study on the effects of treatment on these functions in manic BD patients was also conducted, as indicated in Example 2 & 3 below. In particular, Examples 2 & 3 were structured to complement the animal experiments by addressing how medications affect inhibitory failures in manic BD patients. The BD patients include, but are not limited to, 1) subjects meet diagnostic criteria for Bipolar Affective Disorder (BD), most recent episode manic as determined by the Structured Clinical Interview for DSM-IV (SCID); 2) have young Mania Rating Scale (YMRS) score that is equal to or greater than 20; 3) have no other active Axis I disorder, and 4) have ability to give informed consent as evaluated prior to the study. Normal comparison subjects (NCSs) include, but not limited to, individuals that meet the following criteria: 1) no lifetime history of an Axis I or Axis II diagnosis according to the SCID interviews, and 2) no first-degree relative with a history of psychotic illness. All subjects are between 18 and 55 years of age.

|0l69| Subjects were excluded from the study if they have a neurological disease or damage, or if they have systemic medical illness that may compromise cognitive functioning (e.g., liver disease, etc), history of head injury with a documented loss of consciousness, or DSM-IV defined alcohol or substance abuse or dependence within the last 30 days. Subjects who are under the influence of illegal substances on admission or at the time of Session 3 as determined by urine toxicology screens were terminated from the study. Normal comparison subjects were excluded from the study if they report significant psychopathology, are currently taking a psychoactive substance, or have a first-degree relative with a psychotic illness. Women, minority, and people from different ethnic groups could be involved in the study.

|O17O) Procedures: Subjects were assessed using the SCID, and manic BD patients were administered the YMRS. Subjects were asked to give a sample of urine for toxicology analysis to ensure the absence of active substance use. Subjects were screened for hearing impairment using a Grason Stadler Audiometer.

[0171] Session 1 : Manic BD patients were tested within 72 hours of their admission to the hospital for the following measurements and/or assessments:

10172] Activity Measurement: Both NCS and manic BD patients were fitted with the LifeShirt® ambulatory monitoring device and a brief (10-min) calibration procedure were conducted. These calibration data can be used as covariates to enable us to carry out between and within-subject comparisons. The goal of the activity measurement is to mimic the mouse exposure to a novel environment as closely as possible. Thus, subjects were told that they would see a study coordinator in a short period of time and were directed into the study room, for 15 minutes. This room is equipped with minimal furniture and no chairs. Although physiologic data were collected during the entire time the subject was wearing the LifeShirt®, only the physiologic data from the 15-min waiting period were examined. At the end of the 15-min period, the experimenter would direct the subject back to the laboratory.

|0J73| Sensorimotor inhibition Measurement: Subjects were seated comfortably in a lounge chair in a sound attenuated testing room and assessed for PPI. This session had been developed to be completely parallel for both humans and animals. The wearing of the LifeShirt® for the entire experimental period was necessary so that the subject does not associate the LifeShirt® solely with the 15-min waiting period. At the end of the PPl session, the experimenter would assist in removing the LifeShirt®.

|O174] Follow-up sessions: All subjects underwent Session 2 of testing 7 days after Session 1, and Session 3 of testing 28 days after Session 1. As such, every effort were made so that subjects were re-tested on the same day of the week as previously tested.

The name and dose of the BD subject's medication were recorded. The YMRS were administered to BD subjects. All subjects again underwent the identical experimental procedure detailed above. At Session 3, 60% of the BD patients and NCS that were tested at Session 2 were tested. Several methods, such as contacts with patient at discharge, and contacts patients' friends and family members, were used to maximize the follow up rate in manic BD patients.

GENERAL PROCEDURES FOR HUMAN STUDIES

(01751 Structured Clinical Interview for DSM-IV (SCID): All potential subjects were assessed with the SCID in order to determine eligibility for the study. The SCID (First et al 1994) is a clinical interview where information is obtained from the subject for the purposes of determining an Axis I or II diagnosis. The examiner may also obtain supplementary information from the subject's medical chart, professionals, and family and friends. The SCID may be used in its entirety to establish a DSM-IV diagnosis. Alternatively, select modules from the interview may be used to confirm a suspected diagnosis. The SCID has been widely used in research to characterize samples in terms of diagnosis (Spitzer et al 1992). The authors of the SC]D-III-R, predecessor to the most recent version of the SCID, assert: "For research studies in which a clinical diagnostic evaluation is necessary, a SCID in the hands of a well-trained interviewer is an efficient way of systematically making DSM-IIl-R diagnoses" (Spitzer et al 1992 p.

628). The SCID was subjected to a multi-site test-retest reliability study with 592 subjects interviewed and 25 raters (Williams et al 1992). Interrater agreement was considered good at a kappa coefficient of 0.61. When compared to other diagnostic interviews, the SCID showed similar test-retest reliability. It has previously established a 98% agreement for determining Axis I diagnoses using the SCID (Perry et al 2001b).

|0l761 Young Mania Rating Scale (YMRS): Manic BD patients were assessed with the YMRS at all three test sessions. Those subjects with a score of 20 or above at Session I were included in the study. The YMRS (Young et al 1978) is an interview- based scale consisting of eleven items rated on five defined grades of severity from "symptom not present" to "symptom extremely severe". The items are designed to address symptoms associated with mania i.e. elevated mood, increased motor activity, hypersexuality, decreased need for sleep, irritability, and increased speech. The scale also includes items targeting language disturbance, unusual thought content, disruptive behavior, appearance, and insight. Possible scores on this scale range from 0 to 60. Subjects' scores on all eleven items are recorded. The reliability and validity of YMRS were published with the original scale (Young et al 1978). The scale demonstrated high interrater reliability for both the individual item ratings (r =.66 -.92) and for the total score (r =.93). The YMRS total score was also highly correlated with clinicians' independent global ratings of illness as well as other mania rating scales. Per convention, YMRS scores of 20 are reflective of moderate mania (Tohen et al 2002).

(0177) Prepulse Inhibition Test Session, Humans: To assess PPI in humans, pairs of small cup electrodes (Ag/AgCI) were placed below and lateral to the right eye over the orbicularis oculi muscle, with a reference electrode on the right mastoid. The subject was then fitted with headphones. Sound levels were measured using continuous tones and a calibrated Quest Sound Level Meter on the A scale, coupled to the headphones by an artificial ear. Subjects were seated upright in a comfortable lounge chair and asked to focus on a small square on the wall. The PPI session consisted of 82115-dB 40 ms bursts of white noise with a 70-dB continuous white noise background. After a 5-min acclimation period, the session began with 1 block of 5 pulse-alone trials. Following this block were 2 blocks of 36 trials each, each containing 12 pulse-alone and 24 prepulse-pulse trials in a pseudorandom order. The 20 ms 86-dB prepulse stimuli preceded the noise burst by 30, 60, or 120 ms. Within each of the 2 prepulse blocks were 6 "hidden" no-stimulus trials where no stimulus was delivered but data were recorded. Therefore there were 4 prepulse-pulse trial types (30 ms, 60 ms, 120 ms, no- stimulus), with 6 of each trial type in each of the 2 blocks. The session ended with a block of 5 pulse-alone trials. Inter-trial intervals averaged 16 s, with a range of 8 to 22 s. Voluntary and spontaneous blinks were excluded from analysis by blindly rated morphological criteria. The session duration was 30 min.

[01781 PPI is defined as the percent reduction in startle magnitude in the presence of the prepulse compared to the magnitude in the absence of the prepulse [100 - (100 x magnitude on prepulse trial/magnitude on corresponding pulse-alone trial)]. Thus, a large percent score indicates a high degree of PPl, while a smaller percent score indicates less PPl. PPI difference scores (magnitude on pulse-alone trials minus magnitude on prepulse trials) were also analyzed since percent and difference scores could differ and contribute complementary information. Percent PPI was calculated separately for each of the 3 ISI (30, 60, 120 ms) conditions.

10179] Locomotor Activity and Motor Control: The LifeShirt® System: The LifeShirt® System (Vivometrics, 2002) is an ambulatory, multi-sensor, continuous monitoring system for collecting, analyzing, and reporting health data. The LifeShirt®

System is able to collect reliable objective physiologic data through various sensors, including respiratory inductive plethysmography bands, which measure pulmonary function, electrical activity of the myocardium via a 3-lead EKG, and activity/posture via a two-axis accelerometer. The sensor array of the LifeShirt® System is embedded in a sleeveless undergarment, made of washable Lycra material that fits snugly and can be worn comfortably for extended periods by individuals of varying girth.

10180] For measurement of body posture (angle deviation from horizontal) a two-axis accelerometer is placed onto the shirt over the sternum. The rectified and integrated accelerometer signal is used to detect periods of physical activity and rest. An on board PDA continuously encrypts and stores the patient's activity and posture physiologic data on a compact flash memory card. VivoLogic™, a proprietary PC -based software, decrypts and processes the recorded data, and provides the viewing of- high-resolution waveforms and trends over time. Summary reports are generated that present processed data in concise, graphical, and numeric formats. Data are then exported in ASCII format for analysis in other software programs. Accelerometry data are sampled at 10 Hz and stored numerically in digital units. From these data, changes in posture, average activity levels, periods of peak activity and total activity levels for specified time frames were derived.

10181 j Figure 1 1 provides a graphical example of activity data using the LifeShirt®. Note that changes in type of activity were reflected by deflections in the motion data. The continuous high-frequency sampling of activity data also allowed us to derive nonlinear dynamic variables related to motor activity. Two measures to quantify levels of activity and perseveration were obtained. First, levels of activity were obtained as the average acceleration during a given time bin. This measure (shown in the figure) differentiated different types of behavior in terms of their overall activity level. Second, the dynamical entropy, h, were obtained from the sequence of accelerations over time, for a given time bin. To calculate h, all acceleration values were first logarithmically transformed. Then, each acceleration measure wad transformed into a bin number based on equally spaced bins derived from the distribution of accelerations. For example, a typical distribution of accelerations ranged from .01 to 15 local acceleration units.

[0182| After the log transform, given 4 bins, one obtained the following ranges for the different bins: 1 = {0.01, 0.06}, 2= {0.06, 0.38}, 3= {0.38, 2.41 }, 4= {2.41, 15}. Therefore, the sequence of acceleration values: 0.2, 1.1, 0.01, 2.6} would translate into the bin sequence: {2, 3, 1, 4}. This sequence was subsequently analyzed using a technique described in detail in (Paulus, 1997). Specifically, local dynamical entropies were defined for the subsequences based on the subsequence occurrence probability, pi, and the subsequence length, h, as hi = log(pi) / 1|. This measure was analogous to a 'local spread of information,' or the rate at which a particular subsequence becomes unique. A technique analogous to nearest-neighbor methods has been suggested to calculate efficiently the average dynamical entropy and can be easily extended to calculate these local dynamical entropies (Schumann & Grassberger 1996). This method determines the uniqueness of a subsequence and calculates the local dynamical entropy via h| = log(N)/l i; where N corresponds to length of the entire sequence and I i is length of the subsequences that identifies the subsequences that identifies the subsequence as unique. Unique is defined as: there is no other subsequence in the entire sequence with that particular sequence of bins. 10183} Power analyses: Based upon previous studies of PPI in normal subjects and patients with schizophrenia (Adler 982; Clementz et al 1998; Light et al 2000), the average effect size for PPI differenced between psychiatric patients and normal comparison subjects is d > 0.8. For a 2-tailed test and α = 0.05, when testing the difference between two means, for power = .80, 30 subjects were needed in each group. Taking into account attrition rates previously encountered in the longitudinal studies, a total of 258 BD subjects and 60 nomal comparison subjects were recruited in order to have at least 30 subjects complete the study all the way through to Session 3.

Example 4

Human BPM: Assess the Inhibitory Functioning of Manic BD Patients

|0184) Manic BD patients showed altered inhibitory functioning as measured by prepulse inhibition (PPI). To determine whether manic BD patients show inhibitory deficits in all three domains (PPI, hyperactivity, perseveration), manic BD patients to a group of NCS on these three measures of inhibitory function at admission (Session 1) were examined. The results indicated that manic BD patients exhibit significantly greater deficits on measures of inhibition (less PPI, more hyperactivity, and more perseveration) than NCS.

(0185) The primary dependent measure for the assessment of PPI are percent PPI in each of 3 prepulse conditions (30, 60, and 120 ms ISI). Multiple conditions were included to ensure that the behavior at different levels was measured in order to detect floor or ceiling effects. Hence, group- or treatment-by-prepulse interactions were not expected. Changes in PPI are most readily interpreted in the context of no changes in startle. Startle reactivity would also be assessed, however, with difference as well as percent PPI scores being used to help interpret the possible contribution of changes in startle to changes in PPl. The primary dependent measure for motor activity was derived from the standard measure of average acceleration from the LifeShirt® device. To quantify the degree of perseveration, the average dynamical entropy was utilized as a function of exposure time. Higher entropy corresponds to increasingly unpredictable sequences of alterations and therefore decreasingly perseverative movement patterns.

[0l86| A multivariate ANOVA with group (manic BD patients vs. NCS) as the independent variable were used for analysis. To quantify the magnitude of the findings, effect sizes were computed (Rosenthal et al 2000). As manic BD patients were medicated with a variety of antimanic agents, an analysis of covariance with medication status as a covariate was also conducted.

Example 5

Human BPM: Assess the Relationship Between Inhibitory Functioning and Manic

Symptom in BD Patients

|0l87) It has been well established that manic BD patients show rapid and dramatic changes in symptomatic state (McElroy et al 1996). To determine whether the inhibitory dysfunction in these patients is related to clinical state, these patients were examined multiple times. Thus, manic BD patients were tested at three intervals: 1) within 72 hours of hospitalization (Session 1); 2) 7 days later (Session 2); and 3) 28 days later (Session 3). The data provided that manic BD patients showed significant improvement in inhibitory function (increased PPF, decreased hyperactivity, and decreased perseveration) across the three test sessions compared to NCS; and in manic BD patients, decreased inhibitory deficits were related to decreased symptoms as measured by YMRS scores over the three test sessions.

101881 A mixed group-by-session (2x3) ANOVA were used and a group-by-session interaction would confirm. Post-hoc comparisons were conducted to reveal group differences at Session 1, which were absent by Session 3. The shape of the distributions in the scatterplots were first examined, and then the appropriate correlation coefficients (Spearman's or Pearson's r) between the inhibitory measures and YMRS scores were computed.

|0l89] Although a majority of the patients were discharged by Session 3, less than 5% of BD patients were expected to require extended hospitalization due to the severity of their symptoms, and this group of BD patients was examined separately. As the aim of re-testing BD patients at Session 3 was to examine whether inhibitory deficits resolve as symptoms stabilize, BD subjects with YMRS scores equal to or greater than 20, indicating continuing mania, were also examined separately. Furthermore, detailed analyses of the individual measures were also conducted, yielding information about the heterogeneity of inhibitory deficits and how the measures were differentially impacted by medication. Such information is particularly important in evaluating the predictive validity of potential animal models.

Example 6

Human BPM: Test Whether Lithium, Anticonvulsants, and Atypical Antipsychotic Medications Differentially Improve Inhibitory Functioning

[01901 One important way to assess the predictive validity of the animal model is to examine how antimanic medications differentially affect inhibitory functioning. It is reported that both anticonvulsants (valproate, lamotrigine, and carbamazepine) and atypical antipsychotics (quetiapine and clozapine) reverse PPI deficits in either pharmacologically treated mice or in DAT KO or KD mice and, to some extent, reduce levels of locomotor activity and perseveration in the DAT-deficient mice. Thus, manic BD patients were divided into four groups based on the medications prescribed by their treating psychiatrist: treatment with lithium alone; treatment with an anticonvulsant (e.g. divalproex sodium, carbamazepine, lamotrigine) alone; treatment with an atypical antipsychotic medication alone (e.g. risperidone, olanzapine, quetiapine); or treatment with a combination of lithium or an anticonvulsant plus an atypical antipsychotic medication. These groups did not include all possible treatment combinations, but were the most common and therefore largest groups at NBMU.

IO191| The data provided that manic BD patients treated with an atypical antipsychotic medication (alone or in combination with lithium or an anticonvulsant) demonstrated a significantly greater rate of change (improvement) than patients treated only with lithium or an anticonvulsant on measures of inhibitory function (increased PPI, decreased hyperactivity, and less perseveration) over the three test sessions. This is based upon the conceptual model illustrated in which the effects of the antipsychotics were attributed to direct and immediate blockade of dopamine (DA) receptors. In contrast, the effects of lithium or anticonvulsant agents alone were due to a longer-term reduction of hyperdopaminergia by virtue of a functional reorganization involving circuitry that was either upstream or downstream of the DA synapse. Parallel results were made in comparisons of acute antipsychotic versus subchronic or chronic antimanic effects in the animal models of hyperdopaminergia. |0192| At Session 3, manic BD patients treated with an atypical antipsychotic, lithium, or an anticonvulsant alone exhibited more inhibitory deficits than patients treated with a combination of an atypical antipsychotic and an antimanic medication. As patients treated with combination therapies were likely to be more symptomatic, Session I data were included as a covariate.

101931 A mixed 3x3 group (antipsychotic, lithium alone, or anticonvulsant alone) by session ANOVA were used, and would be confirmed by a group by session interaction. Also, a one-way ANOVA was used for comparing patients on monotherapy versus patients on combined treatment with both an atypical antipsychotic and an antimanic at Session 3.

10194] The above studies addressed medications as classes and, with the exception of lithium, did not distinguish between individual medications within a class. Given that large numbers of subjects were collected in these groups, the effects of specific medications on inhibitory measures in a post-hoc fashion were also determined. For example, lithium to divalproex sodium was compared, as well as comparing atypical antipsychotic agents to one another (e.g. risperidone versus olanzapine versus quetiapine). Additionally, a substantial number of subjects who: 1) were taking medications prior to admission but at sub-therapeutic doses; and 2) are hospitalized, refuse to take medications, but are willing to participate, was collected. Exploratory examination of these subgroups allowed to better understand the impact of pharmacological treatment on inhibitory deficits.

Example 7

Human BPM Differentiates Activity Patterns of Bipolar Manic (BD) and Attention Deficit Hyperactivity Disorder (ADHD) Subjects

|0195l As discussed above, locomotor activity measures are used to assess effects of therapeutics in animals. Hyperactivity occurs in various psychiatric disorders including BD and ADHD. Surprisingly, few studies have examined whether measurement of locomotor activity in humans could be useful biomaker. This study used video- tracking to measure locomotor activity to measure distinguishable patterns of human behavior. |0196| Forty-three (43) subjects with BD or ADHD and a comparison group signed informed consent and were exposed for 15 minutes to a standardized room equipped with a ceiling-mounted video camera, allowing to track movements. The video-stream was processed to obtain (x.y) locations with a temporal resolution of 0.1 second and a spatial resolution of approximately 2 cm. These events were processed analogously to rodent data to assess amounts of activity (transitions between corners, walls, and center) and spatial patterns of activity (spatial scaling exponent, d).

|0l97i In this study, 13 subjects with BD, 14 subjects with ADHD, and 16 comparison subjects signed informed consent and participated. Each subject was exposed to an unfamiliar room for 15 minutes. During this time period a video camera mounted on the ceiling recorded the individual movements. Image processing was conducted to obtain the center of mass coordinate, Sequences of (x.y) coordinates were subjected to several analyses, including distance traveled as a measure of activity, the spatial scaling exponent, d, to quantify geometric patterns of activity, and time spent in different parts of the room, to assess exploratory patterns (Figure 19).

|0l98i The following table indicates that BD, ADHD, and normal comparison groups differed significantly on (1) levels of overall activity, (2) patterns of motor activity, and (3) exploratory patterns, particularly during the first 5 minutes in the unfamiliar environment.

[01991 These studies also showed that comparison and BD subjects, but not subjects with ADHD, showed habituation of locomotor activity over the length of the session (Figure 20). Spatial movement patterns distinguished BD and ADHD individuals from comparison subjects and from each other. The data provided that individuals with BD but not ADHD showed increased motor activity relative to comparison subjects (Figure 21A), while BD subjects exhibited straighter movement patterns than both ADHD and comparison subjects (Figure 21B). The data also provided that BD subjects spent more time in the center of the room (Figure 22A) while ADHD individuals spent more time along the short wall (Figure 22B). AH these data indicate that BD subjects engaged predominantly in distance-covering movements, while ADHD subjects exhibited an increased number of circumscribed or localized movements. Example patterns of locomotor activity of normal comparison, BD, and ADHD subjects are shown in Figure 23.

[0200] Therefore, the results show that one can use locomoter behavior in humans to characterize hyperactivity, differentiate diagnostic groups, and study habituation. It is concluded that individuals with BD and ADHD show differentiable patterns of activity when assessed in an objective behavioral paradigm: hyperactivity was observed for BD subjects, but not for ADHD subjects; the geometric characteristics of movement patterns generated by BD subjects but not those ADHD was altered; exploration of different parts of the novel environment differed across group. Taken together, BD individuals generate a distinct pattern of hyperactivity, which can be differentiated from other diagnostic group. The patterns observed here are reminiscent of those observed in Dopamine Transporter mutant mice. Such differentiated patterns can be developed into a biomarker, providing guidance for future diagnosis and treatment for BD and other psychological disorders.

Example 8

Exploratory Behavior Differentiates Patients with Acute Psychotic Mania from

Schizophrenia

(02011 As discussed above, human BPM, adapted from the classic animal open field test, is developed to study exploratory behavior. In this study, exploratory behavior in acutely psychotic BD, Schizophrenia (SCZ), and normal comparison (NC) subjects were examined, engaging stimuli in a novel environment. The following table indicates the number, gender, age, and years of education of the subjects involved in the study.

[0202] In these studies, subjects were exposed to a novel room furnished with 10 engaging toys without a chair in order to better encourage exploration (Figure 24). Subjects were asked to wait alone in the room while their behavior was recorded for fifteen minutes using a video camera embedded in the ceiling, in which video footage of the session was scored frame-by-frame for the following: total number of interactions with objects, total number of time spent walking, and total number of time spent interacting with objects. Manic BD and SCZ patients were tested within 72 hours of admission to an acute inpatient facility. The results obtained using a rating scale for the hBPM were presented in Figure 25 A-C. Figure 25 A denotes how many times the subjects interacted with any object, Figure 25B gives the amount of time the subjects were walking (in seconds), and Figure 25C provides the quantity of time spent with all objects.

|0203] The results provided that BD patients showed markedly more object exploration than both SCZ and NC subjects. Number of object interactions in BD was highest in the first 5 minutes and decreased there after, while in SCZ and NC groups object interaction was low, but consistent throughout the session. The results also provided that BD patients spent significantly more time walking around the room compared to

NC subjects, but no significance was found between BD and SCZ patients. Significant between-group effects were found for number of object interactions [F(2,47)=24.784, p<0.01], average time spent with objects [F(2,47)=l 2.023, p<0.01], and average time spent walking [F(2,47)=6.405, p<0.005].

10204] These studies conclude that acutely psychotic BD patients demonstrate a unique pattern of exploratory behavior characterized by initially high exploration which may habituate over time. Manic BD patients are also characterized by distinctive patterns of distance-covering movements in the human BPM. These data, combined with mathematical quantification of locomotor activity (See Example 9 below) and exploratory patterns, may provide a signature that can differentiate diagnostic groups. The human BPM captures motor hyperactivity in manic BD patients, and may be a useful paradigm for translational studies comparing locomotor activity in humans and in mouse models of mania. Example 9

Human Behavioral Pattern Monitor (hBPM): Quantification of Motor Hyperactivity in

Mania

|0205] As discussed above, motor hyperactivity is a cardinal feature of the mania of BD. Most studies use self-report and observer-rated scales to assess hyperactivity, but these can be biased and unreliable. Using hBPM, a human analogue of the classic rodent "open field" or the rodent BPM, one can objectively quantify motor activity among different diagnostic groups in this novel exploratory environment, using a continuous ambulatory monitoring device.

|0206] The following Table provides demographic information of the testing subjects.

Group n Age Gender Yrs Education

Manic BD 21 32.5 (14.0) 13 M, 8 F 12.9 (1 •8)

Schizophrenia (SCZ) 14 38.9 (1 1.9) 9M, 5 F 13.0 (1 9)

Nonpatient (NC) 18 29.6 (10.9) 9 M, 9 F 14.7 (1 7)

[0207J In these studies, manic BD and SCZ patients were tested within 72 hours of admission to a psychiatric hospital. Manic BD patients were retested one week after their first session (n=16), and again two weeks after their second session (n=8). NC subjects were retested at same time intervals as manic BD patients.

|0208] As indicated in Figure 24, hBPM refers to a novel room furnished with ten engaging stimuli and no chairs. Subjects are directed inside with no instruction except to wait for the experimenter. Figure 26 shows a wearable ambulatory monitoring device, namely, the LifeShirt®-Clinical System (VivoMetrics, 2002). Motor activity is quantified with a centrally mounted accelerometer embedded in the device. Using hBPM and LifeShirt®-Clinical system, motor activity in Session One for manic BD patient, SCZ patients, and NC subjects was presented in Figure 27, while motor activity across all three sessions for manic BD patients and NC subjects were presented in Figure 28. The following Table shows Pearson r correlation coefficients between motor activity and YMRS Total Scores for manic BD patient.

102091 These studies indicate that use of the hBPM represents an original attempt to objectively capture motor hyperactivity in manic BD patients. The data provide that quantitative assessment of hyperactivity is only modestly correlated with observer-rated symptoms of mania, highlighting the need for objective assessment. The data also provide that motor activity in the hBPM does not show significant short-term or long- term habituation. Once again, because the manic BD patients are also characterized by increased exploration of novel objects and distinctive patterns of distance-covering movement in hBPM, hBPM may be a useful paradigm for translational studies comparing locomotor activity in humans and in mouse models for mania.

Example 10

A Reverse-Translational Approach to Bipolar Disorder: Rodent and Human Studies in the Behavioral Pattern Monitor

(02101 The hBPM has been used to assess and characterize behaviors in bipolar disorder. Mania is the defining feature of Bipolar Disorder (BD). There has been limited progress in understanding the neurobiological underpinnings of BD mania and developing novel therapeutics, in part due to a paucity of relevant animal models with translational potential. Hyperactivity is a cardinal symptom of mania, traditionally measured in humans using observer-rated scales. Multivariate assessment of unconditioned locomotor behavior using the rat Behavioral Pattern Monitor (BPM) has shown that hyperactivity includes complex multifaceted behaviors. The BPM has been used to demonstrate differential effects of drugs on locomotor activity and exploratory behavior in rats. Studies of genetically engineered mice in a mouse BPM have confirmed its utility as a cross-species tool. In a "reverse-translational" approach to this work, the human BPM has been developed to characterize motor activity in BD patients. Increased activity, object interactions, and altered locomotor patterns provide multidimensional phenotypes to model in the rodent BPM. This unique approach to modeling BD provides an opportunity to identify the neurobiology underlying BD mania and test novel antimanic agents. |02li] Modeling BD mania: Bipolar disorder (BD) is a severe psychiatric disorder that affects approximately 2-7% of the general population (Friedman et al. 2006). Mania is the defining feature of BD although patients can also manifest depressive symptoms. During the manic episodes of BD, patients exhibit impulsive behavior, hypersexuality, pressured speech, flight of ideas, and motor hyperactivity, which have been conceptualized as inhibitory failures of behavior and thought (Goodwin and Jamison 1990). These symptoms profoundly impair and disrupt social, occupational, and family life in individuals with BD (Post et al. 2003). Despite the prevalence of this disorder, surprisingly few studies (Franks et al. 1983, Gooding and Tallent 2001, Murphy et al. 1999), have attempted to develop adequate translational models to elucidate the neurobiology underlying BD.

]02l2] One approach to bridge the gap between animal research and clinical research in BD is to develop translational models that extend human studies to animal paradigms that examine analogous constructs. Alternatively, one can use a "reverse translational" approach where existing measurements of activity in animals can be adapted to study activity in patients with BD. Here, the development of the human Behavioral Pattern Monitor (BPM), an analog of the rodent BPM and a method with which to sensitively quantify the characteristics of human hyperactive and exploratory behavior are discussed. The requirements that must be met for translatable applicability are particularly addressed, and a novel behavioral paradigm in humans that is designed to enhance the translational potential of research on BD is introduced.

|02l3l Multivariate assessment of Locomotor Behavior: Motor hyperactivity, which manifests as agitation, is a cardinal feature of the mania of BD and is typically quantified using observer-rated and self-report scales. Such scales may not be sensitive to potentially subtle alterations in activity levels and activity patterns. Moreover, they may not be informative about qualitative aspects of hyperactivity such as instances of stereotypy, nor differentiate between large-scale hyperactive movements (e.g. pacing around a room) and smaller-scale hyperactive behavior (e.g. fidgeting and restlessness). The current situation regarding the assessment of hyperactivity in BD individuals is not unlike the state of the art in assessing animal motor behavior forty years ago (Lat 1965). 10214] Since that time, several research groups (Eilam and Golani 1989, Geyer et al. 1982, 1986, Szechtman et al. 1985) have developed sophisticated approaches to quantity locomotor and exploratory behavior in rodents. The results from these approaches have shown that assessment of motor activity is complex (Eilam and Golani 1989, File and Wardill 1975, Geyer 1990, Geyer and Paulus 1996, Geyer et al. 1986, Paulus and Geyer 1996, Szechtman et al. 1985) but important for understanding a wide range of phenomena ranging from how drugs affect behavior to the phenotype of genetic mutant mice. It is provided that multivariate assessments of both the amounts and sequential patterns of motor actions in humans can potentially provide far greater knowledge on the characteristics of locomotor behavior as well as the effects of treatment compared to existing methods.

10215) The Complex Conceptual Basis of Locomotor Behavior: The assessment of unconditioned locomotor and exploratory behavior has become one of the most widely used behavioral paradigms in rodents to determine the effects of various experimental manipulations, ranging from genetic changes, e.g. knockout mice, to pharmacological challenges, e.g. amphetamine-induced locomotor activity (Crawley 1999). This wide range of applications is based on the fact that unconditioned motor activity probes a variety of behaviors, can be recorded automatically, and can quickly generate an effect profile (Geyer 1990). A variety of different concepts have been applied to the interpretation of aspects of unconditioned motor behavior of rodents in an open field, including arousal, novelty seeking, diversive and inspective exploration, anxiety, stereotypy, and perseveration (Eilam and Golani 1989, Geyer 1990, Geyer et al. 1986, Golani et al. 1993, Lat 1965, Paulus and Geyer 1991, Sanberg et al. 1985, Szechtman et al. 1985).

|O2161 In an extensive review of the open field test, Walsh and Cummins suggest the open field elicits affective components of behavior such as fear and excitability (Walsh and Cummins 1976). Numerous investigators have recognized the necessity for analyses of multivariate profiles and/or spatio-temporal patterns of motor activity and proposed different approaches to quantify the various components of open field behavior (Drai and Golani 2001, Eilam and Shefer 1997, Ossenkopp et al. 1996, Robbins 1979). Some of these approaches were based on observer ratings, while others have attempted to automate the entire measurement process. As detailed below, the measurement approaches developed in studies in rats are now being applied to phenotypic assessments of mice.

[O217| Over the past three decades, two strategies have been focused on in order to address several shortcomings of the traditional approaches to the characterization of unconditioned motor activity and exploratory behavior. First, multivariate assessment techniques have been developed (Ossenkopp et al. 1996). Second, a range of novel quantitative measures based on nonlinear dynamical systems methods and fractal geometry to assess important aspects of the hierarchical and sequential organization of behavior has been developed. Here, these strategies are discussed first to the characterization and differentiation of the behavioral profiles produced in rodents by the administration of various psychostimulant drugs. Psychostimulants provide a relevant focus here because many animal models related to BD and especially to mania rely on the behavioral effects of these drugs. Then, the application of these same strategies and measures to the study of exploratory motor activity in hospitalized patients with BD and other psychiatric disorders are discussed.

|02l8| Assessing Multivariate Behavioral Profiles: Although drugs such as amphetamine, phencyclidine, caffeine, nicotine, and MDMA have been characterized primarily as psychostimulants based upon how they affect locomotor activity, they have diverse mechanisms of actions and differential effects in a variety of behavioral paradigms. Because this classification represents an oversimplification, many investigators have developed additional tests and measures to help differentiate the behavioral profiles of these and other psychoactive drugs. As a consequence, the original Open Field was expanded to include computer-monitored activity chambers based on photobeams or video-tracking as well as measures of exploratory behaviors such as rearings or holepokes in holeboard chambers (Berlyne 1966, File and Wardill 1975, Geyer 1990, Geyer and Paulus 1996, Makanjuola et al. 1977, Paulus and Geyer 1996).

[0219| Among others, a computer-monitored activity chamber, a rat Behavioral Pattern Monitor (rBPM) was developed. The rBPM is a 30.5 by 61 cm chamber equipped with rearing touchplates on the walls and 10 holes in the floors and walls that serve as discrete stimuli for rodents to investigate. This system collects information about the locomotor movements and investigatory responses (rearings and holepokes) of rats at high levels of temporal and spatial resolution (Geyer et al. 1986). The multivariate profiles of locomotor and investigatory behaviors provided by the rBPM helped to elucidate the behavioral characteristics and neuropharmacological mechanisms of psychoactive drugs (Adams and Geyer 1982, Flicker and Geyer 1982).

|0220) Multivariate profiles have many advantages over univariate assessments that are limited to measures of the amount of activity. These advantages are most clearly demonstrated in research assessing the effects of stimulant drugs on rodents. Depending upon dose, drugs such as amphetamine, apomorphine, caffeine, 3-4- methylenedioxymethamphetamine (MDMA), nicotine, phencyclidine and scopolamine all induce similar increases in the amount of activity, as measured in the Open Field, photobeam activity chambers, or the BPM (Bushnell 1987, Collins et al. 1979, Fink and Smith 1979, Fitzgerald et al. 1988, Geyer et al. 1986, Gold et al. 1988, Gould et al. 2001 , Krebs-Thompson et al. 1998, Kulkarni and Dandiya 1975, Meliska and Loke 1984, Paulus and Geyer 1992, Sessions et al. 1980).

[02211 Some differential effects have been noted however, when even the simplest of multivariate assessments is used (see following Table for summary). For example, while caffeine also increased rearing behavior in the Open Field and rBPM, as well as holepoking in the holeboard and rBPM (Collins et al. 1979, Geyer et al. 1986, Meliska and Loke 1984, Rao et al. 1999), nicotine had no effect on rearing behavior in the Open Field or rBPM (Geyer et al. 1986, Meliska and Loke 1984). In fact, higher doses of nicotine reduced the amounts of locomotion and holepoking behavior (Marco et al. 2005, Marks et al. 1986), likely due to hypothermic effects.

Table: Differential Effects of Stimulants on Behavioral Organization

denotes a dose-dependent change in effect * denotes a time-dependent change in effect n/a denotes not assessed

[0222] Likewise phencyclidine can raise or lower exploratory levels dependent upon dose (Krebs-Thompson et al. 1998). Moreover, while the dopamine releaser amphetamine stimulates exploratory behavior in the holeboard apparatus and rBPM, the direct dopamine agonist apomorphine inhibits holepoking behavior (Geyer et al. 1986, Makanjuola et al. 1977). Similarly, MDMA, a derivative of amphetamine that preferentially releases presynaptic serotonin rather than dopamine, also reduces holepoking and rearing behavior (Gold et al. 1988). Also, while stereotypy behavior is observed in conjunction with both amphetamine- and apomorphineinduced hyperactivity (Antoniou and Kafetzopoulos 199], Geyer et al. 1986, Gordon and Beck 1984, Rebec and Bashore 1984), higher doses are required for amphetamine-induced stereotypy (Makanjuola et al. 1977).

102231 Additionally, the type of stereotypy induced by these various psychostimulants differs greatly, with amphetamine at low doses simply producing an exaggerated wide range of activities, while apomorphine produced a more restricted behavioral repertoire (Antoniou and Kafetzopoulos 1991, Geyer et al. 1987), and MDMA produced repetitive behaviors that could not be readily scored using the rating scales defined for amphetamine-treated animals (Gold et al. 1988). These differential effects highlight the need for multivariate assessment of activity, where fine discriminations can be made as to the distinctive characteristics of the behavioral profiles and thus the potentially unique effects of the compounds. 10224] Assessing the Organization of Unconditioned Exploratory Behavior: Despite the great advantages of multivariate assessments, they commonly retain the traditional approach of characterizing behavior in terms of pre-defined response categories. Psychomotor stimulant drugs, however, are known to disrupt normal behavioral responses, fragment behavioral sequences, and introduce new elements into the normal repertoire. The situation is complicated even further by the fact that rating scales designed for one drug (e.g. amphetamine) are often inappropriate for another drug, even one having a related chemical structure such as MDMA (Gold et al. 1988). This fundamental problem constrains the ability of traditional measures to support inferences about the relationship of drug effects to the normal behavioral repertoire of the animal. Second, the traditional approaches are insufficient in quantifying the sequential arrangement of behavioral elements. Third, in traditional approaches, the temporal and spatial resolution used to define the measures is chosen arbitrarily. This choice is based frequently on the qualitative separation of temporal and spatial scales. As indicated, there appears to be no distinct separation of temporal scales. Instead, "pauses" and "behavioral actions" are found on all time scales. Moreover, this separation fundamentally neglects the hierarchical nature of behavioral organization.

|0225] Recent studies in rodents have clearly demonstrated the additional utility of assessments of sequential patterns of locomotor activity in pharmacological and neurobiological studies (Drai and Golani 2001 , Eilam and Golani 1988, Geyer and Paulus 1996, Kafkafi et al. 2003), insofar as measures of the organization of the behavior provide further information that complements traditional multivariate profiles. Behavioral organization within this context can be defined as the selection, ordering, and sequencing of behavioral elements in response to external or internal stimuli to form flexible, yet stable, macroscopic patterns of behavior. The assessment of behavioral organization, as in the case with locomotor behavior, can be approached from both hierarchical and sequential points of view. Hierarchically, behavioral elements are thought to form organized behavioral components on successively larger spatial or temporal scales. Accordingly, the evaluation should quantify the scale- invariant properties of the behavioral organization. Sequentially, behavioral elements are thought to be arranged serially into organized behavioral patterns. Thus, the evaluation should quantify the sequence length-independent properties of the behavioral organization. |0226] Two of the measures described below were developed originally for studies of rat locomotor activity and are now being extended for studies of humans. These two measures, the spatial scaling exponent, d, and the dynamical entropy, h, quantify the hierarchical and sequential properties of behavioral organization, respectively. These measures have several advantages over traditional rating scales and other scale- dependent assessments of motor behavior. Specifically, this approach does not depend on response categories that are defined a priori, is resolution and sequence-length independent, can be extended to include time-dependent characteristics, and allows one to obtain detailed statistical evaluations.

10227] Spatial scaling exponent, d, measures the hierarchical and geometric organization of behavior. Specifically, d is based on the principles of fractal geometry and describes the degree to which the path taken within an enclosure by the subject is one-dimensional or two-dimensional. To obtain spatial d, the distance traveled is plotted against the number of micro-events (defined as the smallest change that can be observed) using a double-logarithmic coordinate system, and a line of fit between these two variables is generated (PauJus et al. 1990, Ralph et al. 2001). Spatial d typically varies between 1 (a straight line) and 2 (a filled plane), with values closer to 1 reflecting straight movements and values closer to 2 reflecting highly circumscribed, local movements.

|0228] At both ends of this spectrum, the geometric pattern of movement around the BPM is highly predictable but exhibits either an almost straight-line movement or a highly circumscribed geometrical pattern, respectively. Exemplars of spatial d values for the locomotor paths exhibited by rodents and humans are presented in Figures 13- 17. Whereas spatial d measures the hierarchical and geometric organization of behavior, the dynamical entropy h measures the degree to which behavior is observed along a continuum between complete order and disorder (Paulus et al. 1991). The derivation of h has been described in detail in our previous work (Paulus et al. 1990). Briefly, a given sequence of activity is compared to similar preceding sequences, and this comparison is conducted for varying sequence lengths. Lower values of h (low entropy) suggest highly predictable or ordered sequences of motor activity, while higher values (high entropy) suggest a greater variety, or disorder, in level of motor activity. [0229] Another aspect of the spatial pattern of the animal's movement is described by the spatial CV statistic, which reflects the degree to which an animal makes the same repetitive transitions from one area to another and is described in greater detail elsewhere (Geyer et al. 1986). Analogously, the amount of time spent in each of the regions can also be quantified and used to calculate a CV statistic in the time domain. This temporal CV defines the degree to which the animal remains in one area or distributes its time across multiple areas (high vs. low temporal CV respectively; (Geyer et al. 1986). Collectively, such descriptors as spatial scaling exponent d, dynamical entropy h, spatial CV, and temporal CV assess various aspects of the hierarchical and sequential organization of behavior at a macroscopic level. This approach using non-linear measures has provided a useful complement to the more traditional behavioral profiles based on a priori definitions of categorical events (Paulus and Geyer 1991, Paulus and Geyer 1996).

f0230| In order to determine whether these approaches truly capture different behavioral variance, we conducted factor analyses on 137 control rats (Paulus and

Geyer 1993b). The analyses revealed that the various automated assessments obtained from locomotor behavior in the BPM loaded onto three primary factors: the amount of activity (e.g. total photobeam breaks, transitions from one region of the BPM to a neighboring region, and distance traveled); exploratory behavior (e.g. rearing and holepoking); and sequential behavioral organization (spatial scaling exponent d, dynamical entropy h). Together, these three factors accounted for 77% of the variance in the measures. Subsequent studies have demonstrated that composite variables comprised of the weighted measures for each factor are highly effective in discriminating receptor subtype-specific drug effects in rats (Krebs-Thomson and Geyer 1998)

[023l| Combining Multivariate Profiles and Measures of Behavioral Organization: Using this approach, stimulants can be differentiated into those that do or do not affect exploratory behavior and/or behavioral organization (see Fig 13 comparing saline vs. amphetamine, phencyclidine, and scopolamine locomotor patterns). For example, apomorphine, MDMA, phencyclidine and scopolamine increase spatial CV, reflecting the generation of more repetitive patterns of movements, amphetamine increases the variety of different movements and therefore lowers spatial CV (Geyer et al. 1986, Gold et al. 1988, Lehman-Masten and Geyer, 1991 ). In contrast, neither nicotine nor caffeine had any effect on this measure. Appropriately, opposite effects are observed using the spatial d measure, where apomorphine and scopolamine lower spatial d, suggestive of movements in straighter lines, amphetamine increases this measure at low doses and produces both increases and decreases at high doses, phencyclidine only increases spatial d, while nicotine and caffeine do not affect spatial d (Geyer et al. 1986, Paulus and Geyer 1993a, Lehman-Masten and Geyer, 1991).

10232) Both amphetamine and MDMA increase entropy h, suggesting disordered movement, although higher doses of MDMA lower entropy h (Paulus et al. 1990). The only measure of behavioral organization that is affected similarly by all these psychostimulants is that of temporal CV. Amphetamine, apomorphine, scopolamine, nicotine, phencyclidine and caffeine all lower temporal CV₁ largely due to their common effect of generating hyperactivity that reduces the predominant behavior of the untreated rat to spend time in its home corner (Geyer et al. 1986, Lehman-Masten and Geyer, 1991). Thus despite these compounds all being categorized as psychostimulants, they produce distinctive behavioral profiles that can be readily differentiated using multivariate assessments including analyses of movement patterns.

|0233l This observation is hardly surprising given the different mechanisms of actions of each of these drugs. Neurochemical analysis can differentiate between these drugs and it is reassuring to know that behavioral analysis of spontaneous locomotion and exploration can also differentiate between them. Apomorphine and scopolamine share several characteristics, despite the former being an agonist at Dl and D2 dopamine receptors while the latter is a non-selective antagonist of the muscarinic acetylcholine receptors.

102341 While nicotine and caffeine share some characteristics, they differ in some aspects as well as their mechanism of action. Nicotine is the prototypical Iigand of the nicotinic acetylcholine receptors, while caffeine may act via the GABAergic system, which can indirectly affect dopamine and norepinephrine functions. The myriad effects of amphetamine are a result of increased release of dopamine, serotonin, and norepinephrine, which occur via amphetamine-induced blockade of the respective transporters. Hence it is observed that other drug-induced manipulations of these neurotransmitter systems can also cause hyperactivity and altered exploratory behavior and locomotor patterns. With respect to modeling BD mania in animals, the literature is focused largely on the dopaminergic systems activated by drugs such as amphetamine. However, the degree to which hyperactive behavioral profiles engendered by drugs, such as cholinergic, glutamatergic, or noradrenergic drugs, as well as dopaminergic drugs, may be examined to mimic the hyperactive states associated with mania in BD patients.

(0235] Locomotor Behavior Homologies: From Rat to Mice: The use of simple photobeam activity chambers (Young et al. 2007) or the open field test (Barr et al. 2004, Crusio 2001) has predominated in mouse locomotor research. A video-tracking system was used to assess motor activity in mice. As provided, psychostimulant drugs such as amphetamine produce similar degrees of activation in mice as in rats. Fairly dramatic differences in the patterns of activation produced by amphetamine in different strains of inbred and outbred mice were readily demonstrable (Ralph et al. 2001). Numerous studies have demonstrated the importance of the manipulation of the dopaminergic and serotonergic systems in altering locomotor activity and behavioral organization (Barr et al. 2004, Cabib et al. 2002, Grailhe et al. 1999, Powell et al. 2004). Moreover, a factor analysis on data collected from 84 mice demonstrated that, similarly to rats, transitions and distance traveled both load onto a factor of amount activity, while locomotor pattern measures such as the spatial scaling exponent d and dynamical entropy h load onto a behavioral organization factor (Paulus et al. 1999). The notable lack of exploratory behavior as an independent factor was likely determined by the video-tracking paradigm's inability to record rearing and holepoking behavior.

|0236| It has been suggested that the manic episodes in BD are consistent with a temporarily dysregulated dopamine system (Diehl and Gershon 1992, Emilien et al.

1999), reflecting exaggerated activation of brain dopamine systems. This suggested hyperdopaminergia is consistent with genetic linkage studies connecting dopamine transporter (DAT) abnormalities to BD (Greenwood et al. 2001, Hay den and

Nurnberger 2006), especially as mice with reduced DAT levels exhibit significantly increased extracellular dopamine activity similarly to BD patients (Vawter et al. 2000,

Zhuang et al. 2001). Moreover, amphetamine, which acts at the DAT, has been used widely in animal models of mania (Hayden and Nurnberger 2006). |0237] Hence, studies examining the activity of mice having complete or partial deletions of the gene for the DAT were conducted. Using the video-based open field in DAT knockout mice, it was confirmed and extended previous observations of dramatic hyperactivity when these mutant mice are stimulated or placed in a novel environment (Gainetdinov et al, 2001). Although their activity levels are normal if undisturbed in their homecages, in a novel chamber, the locomotor activity of DAT null mutant mice is characterized by repetitive, perseverative straight movements in the periphery of the enclosure. In contrast to both DAT wildtype (WT) and heterozygous mice, these animals did not sample the entire enclosure; rather, the DAT KO showed a restricted repertoire of locomotor behavior (Ralph et al . 2001 ).

10238] It was also found that DAT knockdown (KD) mice, lacking only 90% of the DAT, also exhibit a hyperactive phenotype characterized by more perseverative patterns of locomotor behavior reflected in lower spatial d values (Ralph- Williams et al. 2003). If indeed a dysregulated dopamine system underlies some of the key symptoms of mania, it may be provided that pharmacological agents that successfully treat manic symptoms would attenuate the hyperactivity displayed by the DAT KD mutant mice. Indeed, when the DAT KD mice were treated with 100 mg/kg valproate, a dose that had no effect on WT mice, their hyperactivity was reduced significantly. Furthermore, while drug treatment had no effect on spatial d in the WT mice, valproate attenuated the perseverative patterns of motor behavior (diminished the predominance of straight sequences of locomotor activity as evidenced by increased spatial d) seen in the DAT KD mice. Thus, when the DAT KD mice were treated with the clinically effective antimanic drug valproate, both their hyperactivity and their perseverative motor behavior were significantly attenuated (Ralph-Williams et al. 2003).

[0239| Encouraged by such findings, a mouse version of the BPM, namely, mBPM, was developed and designed to overcome the lack of exploratory behavior assessment in the video-based open field test (Risbrough et al. 2006). The utility of the mBPM was demonstrated in the description of the different locomotor activity, exploratory behavior, and behavioral organization phenotypes of dopamine receptor KO mice and their responses to MDMA (Risbrough et al. 2006). In another example of cross-species comparability, MDMA increased activity, lowered spatial d, and increased spatial CV in WT littermate mice in a similar pattern of responses to that observed in rats. [0240] The mBPM was also sufficiently sensitive to differentiate between the phenotypes of Dl, D2, and D3 receptor KO mice. Dl KO mice exhibited an exaggerated responsiveness to the MDMA-induced increases in locomotor activity, while D2 KO mice exhibited a reduced amount of MDMA-induced activation (Risbrough et al 2006). Activity levels of D3 KO male mice were unaffected by MDMA while females displayed a reduced expression of MDMA-induced locomotor activation. Interestingly, D3 KO mice did not exhibit the same immediate MDMA induced-increase in perseverative locomotor behavior (spatial CV). While Dl KO mice exhibited straighter locomotor patterns (lower spatial d) than their WT counterparts, the spatial d measures of the Dl KO and WT mice were similarly lowered following MDMA administration (Risbrough et al. 2006). Hence it was suggested that Dl receptors may contribute to the locomotor pattern quality, i.e. linear vs. circumscribed movement (spatial d), while D2 receptors may contribute to perseverative or thigmotactic locomotor effects of MDMA (spatial CV). This study demonstrates the utility and cross-species generalizability of the BPM. Moreover, the mBPM data collected to date does suggest that a factor analysis could be performed similarly to that of rats, which would likely yield the emergence of exploratory behavior as a third independent factor.

[024i| The development of the BPM from rat to mouse is important for two reasons. First the cross-species generalization of the BPM is evident, as discussed above, with supporting evidence of similar locomotor pattern effects of amphetamine in both rats (Fig. 13) and mice (Fig. 14). Secondly, and perhaps more, importantly, the development of the mBPM allows testing to be performed in genetically modified animals. Extending the work with DAT-deficient in the video-based open field, the mBPM is used in an extensive phenotyping of DAT KD mice. As provided, it was confirmed that in addition to their locomotor hyperactivity, they also exhibit increased exploratory behavior as measured by holepokes and reduced spatial d (Fig. 15). In fact when their locomotor pattern is plotted, it is evident that they display a similar profile to that of rats and mice administered low doses of amphetamine (Fig. 13b and Hb) and similarly to mice administered GBR12909 (Fig. 16), a selective DAT reuptake inhibitor. [0242] Moreover multiple 'home bases' are evident consistent with previous reports (Eilam and Golani 1990) that the 'home corner' described as characteristic of untreated rats (Geyer 1982) can elaborate into multiple preferred locations. Most importantly, the behavioral repertoire displayed by the DAT KD mice appeared similar to the behaviors that are often described of BD patients (see below). Thus assessing the 'manic' behavior of other putative models of BD mania in the mBPM may provide further support for these models, such as CLOCK mutants (McClung 2007, Naylor et al. 2000).

[0243| The Human Behavioral Pattern Monitor (hBPM): The richness of the results from locomotor behavior in rodents in terms of elucidating the underlying neural mechanisms has not been paralleled by similar studies in humans. Thus, to date a comparable laboratory-based, multivariate assessment of locomotor activity has not been extended to human populations. Some earlier studies have measured motor activity in psychiatric populations with the use of a wrist or leg accelerometer (Teicher 1995, Teicher et al. 1986, Wolff et al. 1985). The vast majority of studies on hyperactivity, however, have been limited to observer-rated and self-report scales. As discussed above, such scales may not be optimal in detecting potentially subtle alterations in activity levels nor are they informative about qualitative aspects of hyperactivity that may distinguish certain psychiatric populations from others. For example, several diagnostic groups may present with symptoms of hyperactivity but they may be qualitatively distinct and thus reflect different underlying neural circuitry abnormalities.

10244) In response to the need for models of mania and following the work in rodents, the human Behavioral Pattern Monitor (human BPM) was developed, as an analog of the rodent BPM and a method with which to sensitively quantify the characteristics of human hyperactive and exploratory behavior. Unlike other translational paradigms, the hBPM reflects a "reverse-translational" approach using the existing rich animal literature to inform its development. This approach contrasts with other well- established translational paradigms such as prepulse inhibition of the startle response, an index of sensorimotor gating. The assessment of prepulse inhibition in psychiatric patients was first developed in humans using the startle eyeblink response (Braff et al. 1995). It was subsequently extended to rats (Geyer et al. 2001) and then mice (Geyer et al. 2002), using the whole-body startle response. In contrast, the human BPM represents an evolution in the opposite direction, where a paradigm originally developed in rodents is mimicked in humans. The implementation of prepulse inhibition and the hBPM are examples of the bidirectionality of translational science, where human studies inform animal research, and vice-versa.

102451 The hBPM takes place in a 9' by 14' room that the human participant has not been exposed to and therefore is, like the rodent BPM, a novel and unfamiliar environment. As detailed below, similar to the rodent BPM, multiple measures of motor activity can be collected, including spatial d, entropy h, transitions, distance traveled, and others. Along the walls of the room, dispersed evenly on items of furniture, are ten small objects. These objects were chosen using the criteria that they be safe, colorful, tactile, and manipulable and therefore invite human exploration. The objects provide an analog of the exploratory holes in the walls and floor of the rodent BPM chambers. Participants are directed into the room with little instruction or direction and are asked to wait for the experimenter to return. The hBPM session has been fifteen minutes long in the studies to date.

[0246] Data in the hBPM are gathered using three sources of measurement: 1) collection of physiologic data, namely motor activity of the subject's torso, using an accelerometer embedded in an ambulatory monitoring device that the participant wears; 2) x-y coordinates of the subject's spatial location in the BPM, extracted from digital video recording; and 3) experimenter ratings of exploratory activity, obtained by carefully scoring the video recording of the BPM session and measuring events such as interactions with objects. These three sources of measurement capture different qualitative aspects of motor and exploratory behavior, and yet may also be intercorrelated in the case of certain types of behavior, as will be illustrated below. In fact it is provided that similarly to the rat and mouse BPM, three main independent factors will emerge, describing activity (accelerometry, transitions from one region to a neighboring region), exploratory behavior (interaction with objects), and sequential organization of behavior (spatial d and spatial CV).

{02471 In the hBPM, one measure of the amount of activity is quantified with an accelerometer, which is embedded in a wearable ambulatory monitoring device. The LifeShirt® System (Vivometrics 2002) is an ambulatory, multi-sensor, continuous monitoring system that collects objective physiologic data through various sensors, including respiratory inductive plethysmography bands, which measure pulmonary function, electrical activity of the myocardium via a 3-lead EKG, and activity/posture via a two-axis accelerometer. The sensor array of the LifeShirt® System is embedded in a sleeveless undergarment. For measurement of activity level, a two-axis accelerometer is placed onto the shirt over the sternum, and the rectified and integrated accelerometer signal is used to detect periods of physical activity and rest. An onboard PDA continuously encrypts and stores the patient's activity and postural physiologic data on a compact flash memory card. Accelerometry data are sampled at 10 Hz and stored numerically in digital units. Thus, one measure of the amount of motor activity is obtained by averaging acceleration values over the three five-minute intervals of the human BPM session. Exemplars of the acceleration values derived from individual subjects are provided in Figure 17.

[0248] To obtain additional measures of the quantity and patterns of motor activity and exploratory behavior, the room is also equipped with a camera and fish-eye lens system hidden in a ceiling vent. The images from the camera are stored in digital format on a computer in the adjacent room, with a frequency of 30 frames per second. The digital videos of subject's activity in the human BPM are subjected to frame-by-frame analysis with proprietary software (Clever Systems, Inc. 1999), which generates x and y- coordinates of the subject's successive locations. Because the software specifically tracks the blue LifeShirt® vest, the coordinate positions reflect the position of the upper part of the subject's torso. At present, these x-y coordinates are used to plot the path of the subject and to count time spent and transitions between nine arbitrarily defined regions of the hBPM. These regions are analogous to our definition of nine areas of the rodent BPM (Geyer et al. 1986), namely the four corners, four walls, and the center. Delineation of these regions allows to obtain a distribution of amount of time spent in each region as well as to measure the number of transitions, defined identically to the rodent work as movement from one region to an adjacent one.

(0249) As gained further experience with data generated using this system, alternative definitions of regions will no doubt be found to be more relevant to the behavior of humans in this environment than are the regions defined previously for use in rodents.

In any event, transitions between regions and dwell times within specific regions can serve as additional measures to describe different aspects of locomotor activity and to complement the accelerometry data. In addition, the digitized video images enable detailed assessments of the subject's interactions with the 10 objects placed in the room, in analogy to the rodent's investigatory behavior directed toward the 10 holes placed in the walls and floors of the rodent BPM chambers.

|0250| The continuous, high-frequency sampling of motor activity data also allows to calculate dynamical entropy, h, which is comparable to the entropy measure mentioned above in the context of our animal studies. Dynamical entropy quantifies the predictability of a given level of activity based upon preceding patterns of activity. This acceleration-derived entropy measure captures, a unique feature of human locomotor behavior, i.e. how sequences of acceleration events are organized in time. More importantly, it can also be used to derive entropy "signatures" for specific and distinctive patterns of motor behavior. For example, in initial standardization studies, average entropy values were generated for motor behaviors that subjects exhibited in response to audiotaped instructions, e.g. walking, sitting, standing motionless, or exploring an environment. These entropy values were then used to generate mathematical probabilities that new subjects, uninstrυcted in the hBPM, were engaging in those motor behaviors. Figure 18 illustrates that videotape ratings of a subject's walking behavior corresponded precisely with the entropy-derived mathematical "signature" of walking. Given that dynamical entropy h can characterize disordered movement as well as perseverative movement as described above in the animal studies, it provides a potentially informative measure of how human motor behavior is organized across time.

(025i| A measure of sequential organization that can be derived from the hBPM data and is completely analogous to the measurement of the organization of rodent locomotor data is the spatial scaling exponent, d, which, as in the case of the rodent work reviewed above, describes the geometric pattern of a subject's movement in the exploratory environment. Spatial d is derived in a near-identical manner to the rodent

BPM (Paulus et al. 1990, Ralph et al. 2001). The series of x-y-coordinates derived from the digitized video images describes the spatial patterns of the subject's location and is used to calculate spatial d over specified time blocks. [0252] Although the hBPM is in some respects a novel effort to develop a parallel to the animal open field paradigm, laboratory-based exploratory environments for humans have been reported previously. An early study examined the exploratory behavior of infants in a novel environment (Rheingold and Eckerman, 1969). Similarly, (Pierce and Courchesne 2001) quantified exploratory behavior in autistic children by rating videotapes of an eight-minute session where subjects were placed in a room with colorful and interactive objects. Ratings of decreased exploration were correlated with MRl-based measures of altered brain volumes in children with autism, suggesting that this exploratory paradigm was useful in detecting behavioral deficits that are associated with brain dysfunction.

|0253] Therefore, The hBPM is one of the central measures that are used in an investigation of inhibitory deficits in bipolar mania. The original basis for developing the hBPM was to conduct parallel, cross-species studies of inhibitory problems that are features of the mania of BD and to extend the paradigm to other conditions such as schizophrenia, where multivariate assessment of motor behavior can reveal distinctive characteristics of the illness. The importance of this study was to develop and validate rodent models of mania, which has been identified as a need in the literature (Einat 2006). This validation would be in part based upon the potential similarities between the motor activity of manic patients in the hBPM and the corresponding rodent BPM studies of mice that have been genetically or pharmacologically manipulated to create trait or state conditions of hyperdopaminergia. Thus, as noted above, the hBPM is an example of a "reverse-translational" approach to neuroscience research: whereas most paradigms that are eventually applied to both humans and animals are first developed in humans and then modified to be tested in animal models, the hBPM is unique in that it was developed as an analog to a widely used and highly influential animal paradigm, the open field as elaborated into the rodent BPM. Thus far it shows great promise in characterizing the motor behavior of human clinical populations, both in terms of some of the more straightforward measures such as accelerometry and video ratings, as well as the more complex measures of entropy and patterns of sequential movements in space.

[0254] Currently, the hBPM is used in manic BD patients and individuals with schizophrenia who have been hospitalized on an inpatient psychiatric unit for an acute exacerbation of their illness. Figure 17 illustrates representative case examples of hBPM data for the clinical populations as well as the non-patient cohort. The x-y coordinate tracings of the manic BD patient clearly show a very high level of activity in the BPM (Fig. 17b). Both the average acceleration and the number of transitions during the BPM session are substantially higher than those of the schizophrenia patient (Fig. 17c) or the healthy comparison subject (Fig. 17a). In addition, the manic patient exhibits markedly more interactions with the exploratory objects. The relatively low spatial d in the manic patient suggests that this motor behavior is characterized by long, straight movements from one area of the room to the next. This pattern of increased motor activity and increased exploratory behavior in combination with a reduced spatial d is comparable to what have been observed in DAT KD mice (see Fig 15) and mice administered GBRl 2909 (see Fig. 16), suggesting that DAT KD and GBR 12909- treated mice may be intriguing candidates for genetic and pharmacological animal models of mania.

|0255| In contrast, the schizophrenia patient exhibits very low motor activity, little exploration of objects, and a higher spatial d, signifying restricted and localized activity. The striking difference between the manic BD and the schizophrenia patient once again highlights the importance of multivariate assessment of activity, where measurement of multiple parameters may yield distinct "signatures" of locomotor activity that characterize and differentiate these two disorders. From a diagnostic perspective, the hBPM may be able to quantitatively assess an obvious and meaningful difference between two acutely ill populations who, during acute states, are often difficult to distinguish from one another because the behavioral presentation of both patient groups is dominated by psychotic and mood symptoms (Pini et al. 2004).

|0256| The potential objective, sensitive, and multivariate characterization of hyperactivity that is afforded by the hBPM offers many directions for further research. One application would be to conduct pharmacological manipulations in parallel animal and human studies. For example, while the effects of stimulants on rodent motor behavior have been thoroughly characterized in the rodent BPM, studying stimulant- induced hyperactivity in the hBPM may help to further elucidate the behavioral features of an acute hyperdopaminergic state in healthy humans. Similarly, the hBPM may be useful in testing the efficacy of compounds for characterizing disorders that have hyperactivity as a central symptom. Such comparisons could include patients with BD, schizophrenia, schizoaffective disorder, and attention-deficit/hyperactivity disorder (ADHD) as well as developmental illnesses such as autism spectrum and impulse control disorders.

|0257] As part of the current study on bipolar mania, manic subjects were re-tested in the hBPM after several weeks of stabilization on psychotropic medications. It is provided that patients who are treated with a combination of an antipsychotic and mood stabilizer will show faster alleviation of symptoms of hyperactivity than those patients treated with a mood stabilizer alone, since the antipsychotic medications act directly as dopamine antagonists while mood stabilizers probably only indirectly modulate dopamine levels. While it is a naturalistic study in which manic patients are not randomized to medications, a further study is to carry out randomized, controlled investigations of the effects of psychotropic compounds on hyperactivity, and to examine the time course of these effects.

|0258] A possible limitation of the hBPM in longitudinal studies is the potential effect of habituation, insofar as the hBPM ceases to be a totally novel environment with repeated exposures. The same problem is evident in longitudinal studies in rodents. One way to address this issue in a controlled fashion would be to design studies where a group of subjects is first tested in a medicated state and re-tested after withdrawal from medications, subject to an implementation but these subjects hold much promise for informing the field about the efficacy of psychotropic medications.

|0259] In conclusion, the hBPM is an important example of cross-fostering translational research. Given the importance of hyperactivity in many psychiatric disorders in general and in bipolar mania in particular, it is surprising that experimental approaches to measure locomotor behavior empirically in humans have not been more abundant in the literature. It is provided that locomotor behavior in rodents is a complex phenotype that is not sufficiently characterized by quantifying only the amount of behavior. However, measures that quantify its temporal, spatial, and dynamic organization have proven to be valuable tools to differentiate the contributions of different neural transmitter systems on locomotor and exploratory behavior. Similarly, it is provided that multivariate approaches to human locomotor and exploratory behavior will provide powerful insights into the neural bases of these behaviors and may provide new biomarkers as targets for the development of novel antimanic agents.

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Claims

CLAIMSWhat is claimed is:

1. A behavioral pattern monitor for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medications, comprising a test subject locomotor space, means for recording information about test subject's locomotor information within the space, and information relating at least one control subject to a locomotor pattern for comparative neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on mediations.

2. The behavioral pattern monitor of Claim 1 specific to rodent behavior.

3. The behavioral pattern monitor of Claim 1 specific to human behavior.

4. The behavioral pattern monitor of Claim 3, wherein said space is a room furnished with a plurality of engaging stimuli, and said means for recording information about test subject's locomotor information is an overhead video camera.

5. The behavioral pattern monitor of Claim 3, wherein said human subject's locomotor information comprises the number of interactions with said stimuli, the time spent in locomotion, the time spent interacting with each stimulus, and the pattern of interacting with each stimulus.

6. The behavioral pattern monitor of Claim 3, wherein said pattern of interacting includes distance pattern of activity, geometric or spatial pattern of activity, and time spent in different parts of the room.

7. The behavioral pattern monitor of Claim 3, wherein said human subject is a patient having bipolar disorder- rnanic episode, schizophrenia, Attention Deficit Hyperactivity Disorder, or other neuropsychiatric disorders,

8. The behavioral pattern monitor of Claim 3, wherein said information comprises measuring locomotor behavior to characterize hyperactivity, differentiate diagnostic groups, or study habituation.

9. The behavioral pattern monitor of Claim 4, wherein said engaging stimuli comprises exploratory objects selected from a mask, a doll, a toy, a sculpture, a musical instrument, a kaleidoscope, a ball, a stuffed animal, and combinations thereof.

10. A method of determining a behavioral pattern of a human subject comprising:

a) providing a behavioral pattern monitor for neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medications, comprising a room for test subject locomotor space, a means for recording test subject locomotion while within the space, and information relating at least one control subject to a locomotor pattern for comparative neuropsychiatric analyses, drug efficacy measurement, new drug evaluation, or assessment of cognition on medication said behavioral pattern monitor;

b) guiding said human subject inside said room,

c) recording information about said human subject's locomotor behavior for at least 15 minutes, and

d) comparing information about human subject's locomotor pattern to the information relating at least one control subject to determine the human subject's relative behavioral pattern.

1 1. The method of Claim 10, wherein said wherein said room is furnished with a plurality of engaging stimuli, and said means for recording the test subject locomotion is an overhead video camera.

12. The method of Claim 10, wherein said human subject's locomotor information comprises the number of interactions with said stimuli, the time spent in locomotion, the time spent interacting with each stimulus, and the pattern of interacting with each stimulus.

13. The method of Claim 10, wherein said pattern of interacting includes distance pattern of activity, geometric or spatial pattern of activity, and time spent in different parts of the room.

14. The method of Claim 10, wherein said human subject is a patient having bipolar disorder- manic episode, schizophrenia, Attention Deficit Hyperactivity Disorder, or other neuropsychiatric disorders.

15. The method of Claim 6, further comprising measuring locomotor behavior to characterize hyperactivity, differentiate diagnostic groups, or study habituation.

16. A use of said behavioral pattern monitor of Claim 3, comprising validating an animal model for psychiatric diseases.