WO2017087671A1

WO2017087671A1 - Light-regulated gabaa receptors and methods of use thereof

Info

Publication number: WO2017087671A1
Application number: PCT/US2016/062530
Authority: WO
Inventors: Richard H. Kramer; Wan-Chen Lin
Original assignee: The Regents Of The University Of California
Priority date: 2015-11-18
Filing date: 2016-11-17
Publication date: 2017-05-26

Abstract

The present disclosure provides a variant GABA_A receptor having a substitution of a wild-type amino acid with a cysteine. The present disclosure provides a photoregulator comprising: i) a linker; ii) a photoisomerizable moiety; and iii) a GABA_A receptor ligand. The present disclosure provides a light-regulated GABA_A receptor comprising: a) the variant GABA_Areceptor; and b) the photoregulator covalently linked thereto. The present disclosure provides methods of modulating the activity of the light-regulated GABA_A receptor. The present disclosure provides a treatment method comprising administering the variant GABA_A receptor, or a nucleic acid encoding same; and administering the photoregulator. The present disclosure provides a transgenic non-human animal comprising in its genome a transgene encoding the variant GABA_A receptor.

Description

LIGHT-REGULATED GABA_A RECEPTORS AND METHODS OF USE THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

62/257,150, filed November 18, 2015, which application is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. NS090527 awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

[0003] GABA is the main inhibitory neurotransmitter in the brain, acting in counter-point to glutamate, the main excitatory neurotransmitter. The delicate balance between

GABAergic inhibition and glutamatergic excitation is essential for normal sensory processing, motor pattern generation, and cognitive function. Abnormalities in GABA- mediated inhibition have devastating consequences, contributing to pathological pain, movement disorders, epilepsy, schizophrenia, and neurodevelopmental disorders.

[0004] GABA exerts its effects largely through ligand-gated CI^" channels known as GABAA receptors. GABAA receptors are heteropentamers containing two a, two β, and one tertiary subunit. The a-subunit contributes to GABA binding and determines gating kinetics and subcellular localization. There are six a-subunit isoforms expressed differentially during development and across brain regions but the distinct functions of the isoforms remain elusive.

[0005] Pharmacological agents, including agonists, competitive antagonists, and allosteric

modulators, have been the main instruments for elucidating the function of GABAA receptors. However, these efforts are limited by the low spatial and temporal precision of drug application. Moreover, manipulation of GABAA isoforms has been hindered by the lack of subtype-specific drugs for the GABA-binding site, which could be useful for unambiguously correlating receptor activity with function. The are subtype-selective modulators for the allosteric benzodiazepine-binding site, but they have limited specificity and/or low efficacy. Gene knock-out technology provides an alternative strategy for deducing the function of GABAA isoforms but removal of one a-subunit can lead to compensatory changes in the expression of other receptors, transporters, and ion channels.

[0006] There is a need in the art for tools for identifying GABAA isoform-specific agonists and antagonists.

Literature

(Rudolph and Mohler (2014) Annu. Rev. Pharmacol. Toxicol. 54, 483-507; Goldstein et al., (2002) J. Neurophysiol. 88, 3208-3217; Atack et al. (2006) Neuropharmacology 51, 1023-1029; Gorostiza and Isacoff (2008) Science 322:395; Kaufman et al. (1968) Science 162: 1487-1489; Bartels et al. (1971) Proc. Natl. Acad. Sci. U.S.A. 68: 1820- 1823; Fujita et al. (2006) Biochemistry 45:6581-6586; Caamano et al. (2000) Angew. Chem., Int. Ed. Engl. 39:3104-3107; Mayer and Heckel (2006) Angew. Chem., Int. Ed. Engl. 45:4900-4921; Givens et al. (1998) In Methods in Enzymology, Marriott, G., Ed. Academic Press, New York, 291: 1-29; Volgraf et al.(2006) Nature Chem. Biol. 2:47-52; U.S. Patent Publication No. 2007/0128662; Lester et al. J. Gen. Physiol. 75:207-232 (1980); Banghart et al. Nature Neurosci. 7: 1381-1386 (2004); WO 2007/024290.

SUMMARY

[0007] The present disclosure provides a variant GABAA receptor having a substitution of a wild-type amino acid with a cysteine. The present disclosure provides a

photoregulator comprising: i) a linker; ii) a photoisomerizable moiety; and iii) a GABAA receptor ligand. The present disclosure provides a light-regulated GABAA receptor comprising: a) the variant GABAA receptor; and b) the photoregulator covalently linked thereto. The present disclosure provides methods of modulating the activity of the light- regulated GABAA receptor. The present disclosure provides a treatment method comprising administering the variant GABAA receptor, or a nucleic acid encoding same; and administering the photoregulator. The present disclosure provides a transgenic non- human animal comprising in its genome a transgene encoding the variant GABAA receptor.

[0008] The present disclosure provides a light-regulated gamma amino butyric acid

(GAB A) receptor (GABAA receptor), the light-regulated GABAA receptor comprising: a) an alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain; b) a photo switchable group of the formula: linker-azobenzene-R, wherein R is a ligand for the GAB AA receptor, and wherein the photo switchable group is covalently linked to the c steine via the linker. In some cases, R is selected from:

In some cases, the alpha chain is an isoform 1 alpha chain, and the cysteine substitution is a T153C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO: l. In some cases, the alpha chain is an isoform 2 alpha chain, and the cysteine substitution is a Q149C substitution, based on the amino acid numbering of the isoform 2 alpha chain amino acid sequence set forth in SEQ ID NO:3. In some cases, the alpha chain is an isoform 3 alpha chain, and the cysteine substitution is a V174C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO:5. In some cases, the alpha chain is an isoform 4 alpha chain, and the cysteine substitution is a M155C substitution, based on the amino acid numbering of the isoform 4 alpha chain amino acid sequence set forth in SEQ ID NO:7. In some cases, the alpha chain is an isoform 5 alpha chain, and wherein the cysteine substitution is a E156C substitution, based on the amino acid numbering of the isoform 5 alpha chain amino acid sequence set forth in SEQ ID NO:9. In some cases, the alpha chain is an isoform 6 alpha chain, and the cysteine substitution is a M139C substitution, based on the amino acid numbering of the isoform 6 alpha chain amino acid sequence set forth in SEQ ID NO: 11. In some cases, the alpha chain comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8, 10, and 12. In some cases, when the ligand is PAG- IC and when the GABAA receptor comprising an alpha- 1 isomer is exposed to light of a wavelength of from 480 nm to 520 nm, the ligand is in the extended trans isomer configuration and binds to the ligand- binding site of the alpha chain, and inhibits GABA-elicited current mediated by the receptor, and wherein, when the GABAA receptor is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the cis isomer configuration and GABA-elicited current mediated by the receptor is restored. In some cases, when the ligand is PAG-2A, PAG-2B, or PAG-3C, and when the GABAA receptor is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the extended trans isomer configuration and binds to the ligand-binding site of the alpha chain, and inhibits GABA-elicited current mediated by the receptor, and wherein, when the GABAA receptor is exposed to light of a wavelength of from480 nm to 520 nm, the ligand is in the cis isomer configuration and GABA-elicited current mediated by the receptor is restored.

The present disclosure provides an isomerizable light-responsive ligand for a

GABAA receptor, wherein the ligand is of the formula: maleimide-azobenzene-R, wherein R is a li and for the GABAA receptor. In some cases, R is selected from:

PAG-3C. [0011] The present disclosure provides a method of modulating the activity of a GABAA receptor, the method comprising exposing the GABAA receptor to light, wherein the GABAA receptor comprises: a) an alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue at a surface accessible site in the alpha chain; b) a photoswitchable group of the formula: linker- azobenzene-R, wherein R is a ligand for the GABAA receptor, and wherein the photoswitchable group is covalently linked to the cysteine via the linker, wherein the light is of a wavelength in the range of from 360 nm to 400 nm, or wherein the light is of a wavelen th in the range of from 480 nm to 520 nm. In some cases, R is selected from:

o /—\ o /—\ ri

PAG-2C; and

[0012] The present disclosure provides a variant GABAA receptor alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain. In some cases, the alpha chain is an isoform 1 alpha chain, and the cysteine substitution is a T153C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO: l. In some cases, the alpha chain is an isoform 2 alpha chain, and the cysteine substitution is a Q149C substitution, based on the amino acid numbering of the isoform 2 alpha chain amino acid sequence set forth in SEQ ID NO:3. In some cases, the alpha chain is an isoform 3 alpha chain, and the cysteine substitution is a V174C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO:5. In some cases, the alpha chain is an isoform 4 alpha chain, and the cysteine substitution is a M155C substitution, based on the amino acid numbering of the isoform 4 alpha chain amino acid sequence set forth in SEQ ID NO:7. In some cases, the alpha chain is an isoform 5 alpha chain, and the cysteine substitution is an E156C substitution, based on the amino acid numbering of the isoform 5 alpha chain amino acid sequence set forth in SEQ ID NO:9. In some cases, the alpha chain is an isoform 6 alpha chain, and the cysteine substitution is a M139C substitution, based on the amino acid numbering of the isoform 6 alpha chain amino acid sequence set forth in SEQ ID NO: 11.

[0013] The present disclosure provides a GABAA receptor comprising the variant alpha chain as set forth above or elsewhere herein.

[0014] The present disclosure provides a non-human transgenic animal comprising a transgene in the genome of the animal, wherein the transgene comprises a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain.

[0015] The present disclosure provides a treatment method comprising: a) administering to an individual in need thereof a variant GABAA receptor alpha chain as set forth above or elsewhere herein, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain; b) administering to the individual a

photo switchable group of the formula: linker-azobenzene-R, wherein R is a ligand for a GABAA receptor, and wherein the photoswitchable group binds to the variant alpha chain and is covalently linked to a cysteine in the alpha chain via the linker, forming a photoregulated alpha chain, wherein the photoregulated alpha chain associates with two GABAA receptor β chains and either a GABAA receptor γ chain or a GABAA receptor δ chain, thereby generating a light-responsive GABAA receptor; c) exposing the light- responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the ran e of from 480 nm to 520 nm. In some cases, R is selected from:

PAG- IC,

The present disclosure provides a method of modulating the activity of a neuron, the method comprising: a) introducing into the neuron a variant GABAA receptor alpha chain as set forth above or elsewhere herein, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain; b) administering to the individual a photo switchable group of the formula: linker-azobenzene-R, wherein R is a ligand for a GABAA receptor, and wherein the photo switchable group binds to the variant alpha chain and is covalently linked to a cysteine in the alpha chain via the linker, forming a photoregulated alpha chain, wherein the photoregulated alpha chain associates with two GABAA receptor β chains and either a GABAA receptor γ chain or a GABAA receptor δ chain, thereby generating a light-responsive GABAA receptor; c) exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 480 nm to 520 nm, wherein R is a GABAA receptor anta onist. In some cases, R is selected from:

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A-1F depict the operating principle of a light-regulated GABAA receptor

(LiGAB AR) (FIG. 1A); various photo switchable tethered ligands (PTL) (FIG. IB); photo-control of a representative LiGAB AR (FIG. 1C); a comparison of wild-type GABA_A receptor and an al-LiGABAR (FIG. ID); quantification of LiGAB AR photosensitivity for each a-isoform (FIG. IE); and the PTL attachment site for each a- isoform (FIG. IF: al, SEQ ID NO: 19; a2, SEQ ID NO:20; a3, SEQ ID NO:21; a4, SEQ ID NO:22; a5, SEQ ID NO:23; and <x6, SEQ ID NO:24).

[0018] FIG. 2A-2I depict low-resolution mapping of al- and a5-LiGABARs in CAl pyramidal neurons (FIG. 2A-2C) and higher-resolution mapping of al- and a5-LiGABARs along apical dendrites.

[0019] FIG. 3A-3C depict photo-control of inhibitory post-synaptic currents (IPSCs) by a trans antagonist (PAG-1C, conjugated to alT125) (FIG. 3A); photo control of IPSCs by a cis- antagonist (PAG-2A, conjugated to a5E125C) (FIG. 3B); and photo-control of tonic currents by a irans-antagonist (PAG-1C, conjugated to a5E125C).

[0020] FIG. 4A-4C depict the kinetics of LiGAB AR photo-control.

[0021] FIG. 5A-5F depict accessible depth of LiGAB AR photo-control from the surface of the brain.

[0022] FIG. 6A-6D depict in vivo photo-control of visual responses in the mouse cortex.

[0023] FIG. 7A-7H depict characterizations of the al-GABA_A photoswitch-ready mutant

(PhoRM) knock-in mouse.

[0024] FIG. 8A-8E depict in vivo photo-control of visually-evoked responses and gamma

oscillations in the awake al-GABA_A phoRM mouse.

[0025] FIG. 9A-9B depict the design and screening of LiGAB ARs according to embodiments of the present disclosure. [0026] FIG. 10A-10D depict the effect of cysteine mutation or PTL conjugation on receptor functions.

[0027] FIG. 11A-11F depict the effect of PTL treatment on light sensitivity of endogenous ion channels in hippocampal neurons.

[0028] FIG. 12A-12B depict pairing of LiGABAR photoswitching with two-photon GABA uncaging.

[0029] FIG. 13A-13D depict the effect of exogenously expressing a cysteine mutant of GABAA receptors on neuronal excitability or the kinetics of inhibitory postsynaptic currents.

[0030] FIG. 14 depicts a strategy of gene targeting for generating the alT125C knock-in

mouse.

[0031] FIG. 15 depicts the analysis and categorization of data from multi-electrode recordings.

[0032] FIG. 16A-16B provide amino acid sequences of wild-type (rat_Gabral, SEQ ID NO: 13; human_Gabral_vl, SEQ ID NO: l) (FIG. 16A) and mutant (FIG. 16B) GABA_A-receptor al (mutant GABA_A-receptor al, SEQ ID NO:2) .

[0033] FIG. 17A-17B provide amino acid sequences of wild-type (rat_Gabra2, SEQ ID NO: 14; human_Gabra2_vl, SEQ ID NO:3) (FIG. 17A) and mutant (FIG. 17B) GABA_A-receptor a2 (mutant GABA_A-receptor a2, SEQ ID NO:4).

[0034] FIG. 18A-18B provide amino acid sequences of wild-type (rat_Gabra3, SEQ ID NO: 15; human_Gabra3, SEQ ID NO:5) (FIG. 18A) and mutant (FIG. 18B) GABA_A-receptor a3

(mutant GABA_A-receptor a3, SEQ ID NO:6).

[0035] FIG. 19A-19B provide amino acid sequences of wild-type (rat_Gabra4, SEQ ID NO: 16; human_Gabra4_vl, SEQ ID NO:7) (FIG. 19A) and mutant (FIG. 19B) GABA_A-receptor a4 (mutant GABA_A-receptor a4, SEQ ID NO:8).

[0036] FIG. 20A-20B provide amino acid sequences of wild-type (rat_Gabra5, SEQ ID NO: 17; human_Gabra5_vl, SEQ ID NO:9) (FIG. 20A) and mutant (FIG. 20B) GABA_A-receptor a5 (mutant GABA_A-receptor a5, SEQ ID NO: 10).

[0037] FIG. 21A-21B provide amino acid sequences of wild-type (rat_Gabra6, SEQ ID NO: 18; human_Gabra6, SEQ ID NO: 11) (FIG. 21 A) and mutant (FIG. 21B) GABA_A-receptor a6 (mutant GABA_A-receptor a6, SEQ ID NO: 12). DEFINITIONS

[0038] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like. The term "polypeptide" includes polypeptides comprising one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carbohydrate moiety. The term "polypeptides" includes post-translationally modified polypeptides.

[0039] The term "naturally-occurring" as used herein as applied to a polypeptide, a cell, or an organism, refers to a polypeptide, cell, or organism that is found in nature. For example, a polypeptide having an amino acid sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.

[0040] A "host cell," or "a cell," as used herein, denotes an in vivo or in vitro prokaryotic cell, an in vivo or in vitro eukaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured in vitro as a unicellular entity. A cell includes a cell that comprises a subject light-regulated polypeptide. A "host cell" includes cells that can be, or have been, used as recipients for a subject synthetic regulator. A "host cell" includes cells that can be, or have been, used as recipients for an exogenous nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, in some

embodiments a subject host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell. [0041] As used herein the term "isolated" is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0042] The term "conservative amino acid substitution" refers to the interchangeability in

proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic -hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine.

Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0043] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a

desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g. , including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0044] The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, primates, including simians and humans; rodents, including rats and mice; bovines; equines; ovines; felines; canines; and the like. "Mammal" means a member or members of any mammalian species, and includes, by way of example, canines; felines; equines; bovines; ovines; rodentia, etc. and primates, particularly humans. Non-human animal models, particularly mammals, e.g. non-human primates, murines, lagomorpha, etc. may be used for experimental investigations.

[0045] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect such treatment for the disease, condition, or disorder. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0046] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a compound (e.g., an aminopyrimidine compound, as described herein) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0047] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A

pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0048] As used herein, a "pharmaceutical composition" is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a "pharmaceutical composition" is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).

Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular,

subcutaneous, and the like. [0049] By "transgenic animal" is meant a non-human animal, usually a mammal, having a non- endogenous (i.e., heterologous) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to methods well known in the art. A "transgene" is meant to refer to such heterologous nucleic acid, e.g., heterologous nucleic acid in the form of an expression construct (e.g., for the production of a "knock-in" transgenic animal) or a heterologous nucleic acid that upon insertion within or adjacent a target gene results in a decrease in target gene expression (e.g., for production of a "knock-out" transgenic animal).

[0050] A "knock-out" of a gene means an alteration in the sequence of the gene that results in a decrease of function of the target gene, preferably such that target gene expression is undetectable or insignificant. Transgenic knock-out animals can comprise a

heterozygous knock-out of a target gene, or a homozygous knock-out of a target gene. "Knock-outs" as used herein also include conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g., Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.

[0051] A "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of a target gene, e.g., by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene. "Knock-in" transgenics can comprise a

heterozygous knock-in of the target gene or a homozygous knock-in of a target gene. "Knock-ins" also encompass conditional knock-ins.

[0052] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0053] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0055] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a GABAA receptor variant" includes a plurality of such variants and reference to "the ligand" includes reference to one or more ligands and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0056] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0057] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0058] The present disclosure provides a variant GABAA receptor having a substitution of a wild-type amino acid with a cysteine. The present disclosure provides a photoregulator comprising: i) a linker; ii) a photoisomerizable moiety; and iii) a GABAA receptor ligand. The present disclosure provides a light-regulated GABAA receptor comprising: a) the variant GABAA receptor; and b) the photoregulator covalently linked thereto. The present disclosure provides methods of modulating the activity of the light-regulated GABAA receptor. The present disclosure provides a treatment method comprising administering the variant GABAA receptor, or a nucleic acid encoding same; and administering the photoregulator. The present disclosure provides a transgenic non- human animal comprising in its genome a transgene encoding the variant GABAA receptor.

PHOTOREGULATOR

[0059] The present disclosure provides a photoregulator comprising: i) a linker (e.g., a cysteine- reactive linker moiety); ii) a photoisomerizable moiety; and iii) a GABAA receptor ligand. The linker, the photoisomerizable moiety and the GABAA receptor ligand are covalently linked to one another directly or via a linker.

Linker moiety

[0060] The linker group provides for covalent linkage to a cysteine residue present in the

GABAA-R. Suitable linkers will comprise a moiety such as, e.g., a vinylsulfone group, maleimide; a substituted maleimide, such as maleic anhydride; orthopyridyl-disulfide; a methanethio sulfonate; a disulfide; and the like. Cysteinyl residues can be reacted with a- haloacetates (and corresponding amides), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues can also be reacted with bromotrifluoroacetone, a-bromo-P-(4-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7- nitrobenzo-2-oxa-l,3-diazole. In some cases, the linker moiety comprises a maleimide group.

Photoisomerizable group

[0061] The photoisomerizable group is one that changes from a first isomeric form to a second isomeric form upon exposure to light of different wavelengths, or upon a change in exposure from dark to light, or from light to dark. For example, in some embodiments, the photoisomerizable group is in a first isomeric form when exposed to light of a first wavelength, and is in a second isomeric form when exposed to light of a second wavelength. Suitable photoisomerizable groups include stereoisomers and constitutional isomers.

[0062] The first wavelength and the second wavelength can differ from one another by from about 1 nm to about 2000 nm or more, e.g., from about 1 nm to about 10 nm, from about 10 nm to about 20 nm, from about 20 nm to about 50 nm, from about 50 nm to about 75 nm, from about 75 nm to about 100 nm, from about 100 nm to about 125 nm, from about 125 nm to about 150 nm, or from about 150 nm to about 200 nm, from about 200 nm to about 500 nm, from about 500 nm to about 800 nm, from about 800 nm to about 1000 nm, from about 1000 nm to about 1500 nm, from about 1500 nm to about 2000 nm, or more than 2000 nm.

[0063] In other embodiments, the photoisomerizable group is in a first isomeric form when exposed to light of a wavelength λι, and is in a second isomeric form in the absence of light (e.g., in the absence of light, the photoisomerizable group undergoes spontaneous relaxation into the second isomeric form). In these embodiments, the first isomeric form is induced by exposure to light of wavelength λι, and the second isomeric form is induced by not exposing the photoisomerizable group to light, e.g., keeping the photoisomerizable group in darkness. In other embodiments, the photoisomerizable group is in a first isomeric form in the absence of light, e.g., when the photoisomerizable group is in the dark; and the photoisomerizable group is in a second isomeric form when exposed to light of a wavelength λ_\. In other embodiments, the photoisomerizable group is in a first isomeric form when exposed to light of a first wavelength λι, and the photoisomerizable group is in a second isomeric form when exposed to light of second wavelength λ₂.

[0064] For example, in some embodiments, the photoisomerizable group is in a trans

configuration in the absence of light, or when exposed to light of a first wavelength; and the photoisomerizable group is in a cis configuration when exposed to light, or when exposed to light of a second wavelength that is different from the first wavelength. As another example, in some embodiments, the photoisomerizable group is in a cis configuration in the absence of light, or when exposed to light of a first wavelength; and the photoisomerizable group is in a trans configuration when exposed to light, or when exposed to light of a second wavelength that is different from the first wavelength.

[0065] The wavelength of light that effects a change from a first isomeric form to a second

isomeric form ranges from 10 -^"8 m to about 1 m, e.g., from about 10 -^"8 m to about 10 -^"7 m, from about 10^"7 m to about 10^"6 m, from about 10^"6 m to about 10^"4 m, from about 10^"4 m to about 10 -^"2 m, or from about 10 -^"2 m to about 1 m. "Light," as used herein, refers to electromagnetic radiation, including, but not limited to, ultraviolet light, visible light, infrared, and microwave.

[0066] The wavelength of light that effects a change from a first isomeric form to a second

isomeric form ranges in some embodiments from about 200 nm to about 800 nm, e.g., from about 200 nm to about 250 nm, from about 250 nm to about 300 nm, from about 300 nm to about 350 nm, from about 350 nm to about 400 nm, from about 400 nm to about 450 nm, from about 450 nm to about 500 nm, from about 500 nm to about 550 nm, from about 550 nm to about 600 nm, from about 600 nm to about 650 nm, from about 650 nm to about 700 nm, from about 700 nm to about 750 nm, or from about 750 nm to about 800 nm, or greater than 800 nm.

[0067] In other embodiments, the wavelength of light that effects a change from a first isomeric form to a second isomeric form ranges from about 800 nm to about 2500 nm, e.g., from about 800 nm to about 900 nm, from about 900 nm to about 1000 nm, from about 1000 nm to about 1200 nm, from about 1200 nm to about 1400 nm, from about 1400 nm to about 1600 nm, from about 1600 nm to about 1800 nm, from about 1800 nm to about 2000 nm, from about 2000 nm to about 2250 nm, or from about 2250 nm to about 2500 nm. In other embodiments, the wavelength of light that effects a change from a first isomeric form to a second isomeric form ranges from about 2 nm to about 200 nm, e.g., from about 2 nm to about 5 nm, from about 5 nm to about 10 nm, from about 10 nm to about 25 nm, from about 25 nm to about 50 nm, from about 50 nm to about 75 nm, from about 100 nm, from about 100 nm to about 150 nm, or from about 150 nm to about 200 nm.

[0068] The difference between the first wavelength and the second wavelength can range from about 1 nm to about 2000 nm or more, as described above. Of course, where the synthetic light regulator is switched from darkness to light, the difference in wavelength is from essentially zero to a second wavelength.

[0069] The intensity of the light can vary from about 1 W/m 2 to about 50 W/m 2 , e.g., from

about 1 W/m 2 to about 5 W/m 2 , from about 5 W/m 2 to about 10 W/m 2 , from about 10 W/m², from about 10 W/m² to about 15 W/m², from about 15 W/m² to about 20 W/m², from about 20 W/m² to about 30 W/m², from about 30 W/m² to about 40 W/m², or from about 40 W/m 2 to about 50 W/m 2. The intensity of the light can vary from about 1 μW/cm 2 to about 100 μW/cm 2 , e.g., from about 1 μW/cm 2 to about 5 μW/cm 2 , from about 5 μW/cm 2 to about 10 μW/cm 2 , from about 10 μW/cm 2 to about 20 μW/cm 2 , from about 20 μW/cm 2 to about 25 μW/cm 2 , from about 25 μW/cm 2 to about 50 μW/cm 2 , from about 50 μW/cm 2 to about 75 μW/cm 2 , or from about 75 μW/cm 2 to about 100 μW/cm 2. In some embodiments, the intensity of light varies from about 1 μψ/^ 2 to about 1 W/mm 2 , e.g., from about 1 μ /ηιηι 2 to about 50 μψ/^ 2 , from about 50 μW/mm 2 to about 100 μW/mm 2 , from about 100 μ /ηιηι 2 to about 500 μψ/^ 2 , from about 500 μW/mm 2 to about 1 mW/mm 2 , from about 1 mW/mm 2 to about 250 mW/mm 2 , from about 250 mW/mm 2 to about 500 mW/mm 2 , or from about 500 mW/mm 2 to about 1 W/mm².

[0070] The photoisomerizable moiety can be any of a variety of photoisomerizable moieties.

Suitable photoisomerizable groups include, but are not limited to, azobenzene and derivatives thereof; spiropyran and derivatives thereof; triphenyl methane and

derivatives thereof; 4,5-epoxy-2-cyclopentene and derivatives thereof; fulgide and derivatives thereof; thioindigo and derivatives thereof; diarylethene and derivatives thereof; diallylethene and derivatives thereof; overcrowded alkenes and derivatives thereof; and anthracene and derivatives thereof. Suitable spiropyran derivatives include, but are not limited to, 1,3,3-trimethylindolinobenzopyrylospiran; 1,3,3- trimethylindolino-6'-nitrobenzopyrylospiran; l,3,3-trimethylindolino-6'- bromobenzopyrylospiran; l-n-decyl-3,3-dimethylindolino-6'-nitrobenzopyrylospiran; 1- n-octadecy-l-3,3-dimethylindolino-6'-nitrobenzopyrylospiran; 3',3'-dimethyl-6-nitro- - [2-(phenylcarbamoyl)ethyl]spiro; [2H-l-benzopyran-2,2'-indoline]; 1,3,3- trimetnylindolino-8'-methoxybenzopyrylospiran; and l,3,3-trimetnylindolino-P- naphthopyrylospiran. Also suitable for use is a merocyanine form corresponding to spiropyran or a spiropyran derivative.

[0071] Suitable triphenylmethane derivatives include, but are not limited to, malachite green derivatives, specifically, there can be mentioned, for example,

bis[dimethylamino)phenyl] phenylmethanol, bis [4-

(diethylamino)phenyl]phenylmethanol, bis[4-(dibuthylamino)phenyl]phenylmethanol and bis[4-(diethylamino)phenyl]phenylmethane.

[0072] Suitable 4,5-epoxy-2-cyclopentene derivatives include, for example, 2,3-diphenyl-l- indenone oxide and 2',3'-dimethyl-2,3-diphenyl-l-indenone oxide.

[0073] Suitable azobenzene compounds include, e.g., compounds having azobenzene residues crosslinked to a side chain, e.g., compounds in which 4-carboxyazobenzene is ester bonded to the hydroxyl group of polyvinyl alcohol or 4-carboxyazobenzene is amide bonded to the amino group of polyallylamine. Also suitable are azobenzene compounds having azobenzene residues in the main chain, for example, those formed by ester bonding bis(4-hydroxyphenyl)dimethylmethane (also referred to as bisphenol A) and 4,4'-dicarboxyazobenzene or by ester bonding ethylene glycol and 4,4'- dicarboxyazobenzene.

[0074] Suitable fulgide derivatives include, but are not limited to, isopropylidene fulgide and adamantylidene fulgide.

[0075] Suitable diallylethene derivatives include, for example, l,2-dicyano-l,2-bis(2,3,5- trimethyl-4-thienyl)ethane; 2,3-bis(2,3,5-trimethyl-4-thiethyl) maleic anhydride; 1,2- dicyano-l,2-bis(2,3,5-trimethyl-4-selenyl)ethane; 2,3-bis(2,3,5-trimethyl-4-selenyl) maleic anhydride; and l,2-dicyano-l,2-bis(2-methyl-3-N-methylindole)ethane.

[0076] Suitable diarylethene derivatives include but are not limited to, substituted

perfluorocylopentene-bis-3-thienyls and bis-3-thienylmaleimides. [0077] Suitable overcrowded alkenes include, but are not limited to, czs-2-nitro-7-

(dimethylamino)-9-(2' ,3 ' -dihydro- 1 'H-naphtho[2, l-b]thiopyran- 1 '-ylidene)-9H- thioxanthene and iran5-dimethyl-[l-(2-nitro-thioxanthen-9-ylidene)-2,3-dihydro-lH- benzo[f]thiochromen-8-yl]amine. Overcrowded alkenes are described in the literature. See, e.g., terWiel et al. (2005) Org. Biomol. Chem. 3:28-30; and Geertsema et al. (1999) Agnew CHem. Int. Ed. Engl. 38:2738.

[0078] In some cases, the photoisomerizable moiety is azobenzene.

Ligands

[0079] The GAB A A receptor (GABA_A-R) ligand portion of the photoregulator

comprises a moiety comprising GAB A, guanidylated GAB A, guanidinium, or guanidine acetic acid. The GABAA-R ligand can be linked to the photoisomerizable moiety via a linker moiety that can be any of a variety of lengths, and can have e.g., 1, 2, 3, 4, 5, 6, 7, 8 9, or 10 carbons. In some cases, the ligand is selected from:

Exemplary photoregulators

[0080] Non-limiting examples of photoregulators of the present disclosure are as

follows. In some cases, a photoregulator of the present disclosure is of the formula: linker-azobenzene-R, where R is selected from:

[0081] In some cases, a photoregulator of the present disclosure is of the formula: maleimide-azobenzene-R where R is selected from:

[0082] In some cases, photoregulator of the present disclosure is of the formula: maleimide- azobenzene-R, where R is:

(PAG-1C). [0083] In some cases, photoregulator of the present disclosure is of the formula: maleimide-

(PAG-2A).

[0084] In some cases, photoregulator of the present disclosure is of the formula: maleimide- azobenzene-R where R is:

[0085] In some cases, photoregulator of the present disclosure is of the formula: maleimide- azobenzene-R where R is:

Compositions

[0086] The present invention further provides compositions comprising a photoregulator of the present disclosure. Compositions comprising a photoregulator of the present disclosure will in many embodiments include one or more of: a salt, e.g., NaCl, MgCl₂, KC1, MgS0₄, etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N- Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, Nonidet- P40, etc.; a protease inhibitor; and the like. In some cases, a subject composition comprising a photoregulator of the present disclosure is a pharmaceutical composition, as described in more detail below.

VARIANT GABA_A- ALPHA CHAINS AND VARIANT GABA_A-R

[0087] The present disclosure provides variant GABAA receptor (GABAA-R) alpha chain

polypeptides, where the variant GABAA-R alpha chain polypeptides comprise a substitution of an amino acid present in a wild-type GABAA-R alpha chain, such that the amino acid is substituted with a cysteine. The present disclosure provides a variant GABAA-R, where a variant GABAA-R of the present disclosure comprises a substitution of an amino acid present in the alpha chain of a wild-type GABAA-R, such that the amino acid is substituted with a cysteine. The cysteine substitution provides a linkage site for covalently linking the photoregulator. In many cases, the cysteine substitution is located in loop E of the alpha chain.

[0088] In some cases, the amino acid substitution is a T→C substitution of the amino acid

sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R alpha chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRITEDGCLLYT (SEQ ID NO:25). In some cases, the amino acid substitution is a T→C substitution of the amino acid sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R al chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R al chain comprises the amino acid sequence

LLRITEDGCLLYT (SEQ ID NO:25).

[0089] In some cases, the amino acid substitution is a Q→C substitution of the amino acid

sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

LLRICDDGTLLYT (SEQ ID NO:26). In some cases, the amino acid substitution is a Q→C substitution of the amino acid sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R a2 chain, such that the variant GABAA-R a2 chain comprises the amino acid sequence LLRICDDGTLLYT (SEQ ID NO:26).

[0090] In some cases, the amino acid substitution is a V→C substitution of the amino acid

sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

LLRLCDNGTLLYT (SEQ ID NO:27). In some cases, the amino acid substitution is a V→C substitution of the amino acid sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R a3 chain, such that the variant GABAA-R R a3chain comprises the amino acid sequence LLRLCDNGTLLYT (SEQ ID NO:27).

[0091] In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRIMRNGTILYT (SEQ ID NO:22) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

LFRICRNGTILYT (SEQ ID NO:28). In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRIMRNGTILYT (SEQ ID NO:22) present in a GABAA-R 4 chain, such that the variant GABAA-R 4 chain comprises the amino acid sequence LFRICRNGTILYT (SEQ ID NO:28).

[0092] In some cases, the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

LLRLCDDGTLLYT (SEQ ID NO:29). In some cases, the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R 4 chain, such that the variant GABAA-R 5 chain comprises the amino acid sequence LLRLCDDGTLLYT (SEQ ID NO:29).

[0093] In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

LFRLCHNGTILYT (SEQ ID NO:30). In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R 6 chain, such that the variant GABAA-R 6 chain comprises the amino acid sequence LFRLCHNGTILYT (SEQ ID NO:30).

[0094] In some cases, a variant GABAA-R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B, where the GABAA-R alpha chain comprises the amino acid substitution noted in the corresponding one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B .

[0095] In some cases, a variant GABAA-R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 16B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 153 of the amino acid sequence set forth in FIG. 16B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 16B .

[0096] In some cases, a variant GABAA-R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 17B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 149 of the amino acid sequence set forth in FIG. 17B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 17B .

[0097] In some cases, a variant GABAA-R R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 18B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 174 of the amino acid sequence set forth in FIG. 18B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 18B .

[0098] In some cases, a variant GABAA-R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 19B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 155 of the amino acid sequence set forth in FIG. 19B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 19B .

[0099] In some cases, a variant GABAA-R alpha chain of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 20B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 156 of the amino acid sequence set forth in FIG. 20B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 20B .

[00100] In some cases, a variant GABAA-R alpha chain of the present disclosure

comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 21B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 139 of the amino acid sequence set forth in FIG. 21B . In some cases, a variant GABAA-R alpha chain of the present disclosure comprises the amino acid sequence set forth in FIG. 21B . Nucleic acids and host cells

[00101] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure. In some cases, the nucleic acid is present in a recombinant expression vector. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to a transcriptional control element, e.g., a promoter, such as a promoter that is functional in a eukaryotic cell. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to a constitutive promoter. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to an inducible promoter. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to a repressible promoter. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to a neuron- specific promoter. In some cases, the nucleotide sequence encoding a variant GABAA-R alpha chain of the present disclosure is operably linked to a synapsin promoter.

[00102] Suitable promoters include, but are not limited to; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known promoters. Suitable regulatable promoters, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. [00103] Suitable neuron- specific control sequences include, but are not limited to, a neuron- specific enolase (NSE) promoter (see, e.g., EMBL HSEN02, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank

HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10): 1161-1166); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); an enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); a myelin basic protein (MBP) promoter; a Ca²⁺-calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93: 13250; and Casanova et al. (2001) Genesis 31:37); and a CMV enhancer/platelet-derived growth factor-β promoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60).

[00104] In some cases, a recombinant expression vector of the present disclosure is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S.

Patent No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, etc.

[00105] Suitable expression vectors that can be used to generate a recombinant

expression vector of the present disclosure include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94: 10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[00106] The present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid or a recombinant expression vector of the present disclosure. In some cases, the genetically modified host cell is a mammalian cell. In some cases, the genetically modified host cell is human cell. In some cases, the genetically modified host cell is a neuronal cell, e.g., a human neuronal cell, a non- human primate neuronal cell, a murine neuronal cell, and the like. In some cases, the genetically modified host cell is in vitro. In some cases, the genetically modified host cell is ex vivo. In some cases, the genetically modified host cell is in vivo.

[00107] Suitable mammalian cells include primary cells and immortalized cell lines.

Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC 12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No.

CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[00108] In some instances, the cell is not an immortalized cell line, but is instead a cell

(e.g., a primary cell) obtained from an individual. In some cases, the cell is a neuronal cell. In some cases, the neuronal cell is a neuronal cell that comprises endogenous GAB A A receptors. LIGHT-REGULATED GABA_A-R

[00109] The present disclosure provides a light-regulated GABAA receptor comprising: a) a variant GABAA-R alpha chain of the present disclosure; and b) a photoregulator of the present disclosure covalently linked to a cysteine in the variant GABAA-R alpha chain.

[00110] A light-regulated GABAA receptor comprises: i) two copies of a variant GABAA-

R alpha chain of the present disclosure; ii) two β chains; and iii) either a GABAA-R γ chain or a GABAA-R δ chain. The structure is depicted in FIG. 1A. A photoregulator of the present disclosure is covalently linked to a cysteine in both copies of the variant GABAA-R alpha chain.

[00111] A light-regulated GABAA receptor comprises: i) two copies of a variant GABAA-

R alpha chain of the present disclosure, where the variant GABAA-R alpha chain comprises a cysteine amino acid substitution, such that a wild-type amino acid is substituted with the cysteine.

[00112] In some cases, the amino acid substitution is a T→C substitution of the amino acid sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R alpha chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRITEDGCLLYT (SEQ ID NO:25). In some cases, the amino acid substitution is a T→C substitution of the amino acid sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R al chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R al chain comprises the amino acid sequence

LLRITEDGCLLYT (SEQ ID NO:25).

[00113] In some cases, the amino acid substitution is a Q→C substitution of the amino acid sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRICDDGTLLYT (SEQ ID NO:26). In some cases, the amino acid substitution is a Q→C substitution of the amino acid sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R a2 chain, such that the variant GABAA-R 2 chain comprises the amino acid sequence LLRICDDGTLLYT (SEQ ID NO:26).

[00114] In some cases, the amino acid substitution is a V→C substitution of the amino acid sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRLCDNGTLLYT (SEQ ID NO:27). In some cases, the amino acid substitution is a V→C substitution of the amino acid sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R 3 chain, such that the variant GABAA-R R a3chain comprises the amino acid sequence LLRLCDNGTLLYT (SEQ ID NO:27).

[00115] In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRIMRNGTILYT (SEQ ID NO:22) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence

[00116] In some cases, the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRLCDDGTLLYT (SEQ ID NO:29). In some cases, the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R 4 chain, such that the variant GABAA-R 5 chain comprises the amino acid sequence LLRLCDDGTLLYT (SEQ ID NO:29).

[00117] In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LFRLCHNGTILYT (SEQ ID NO:30). In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R 6 chain, such that the variant GABAA-R 6 chain comprises the amino acid sequence LFRLCHNGTILYT (SEQ ID NO:30).

[00118] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B, where the GABAA-R alpha chain comprises the amino acid substitution noted in the corresponding one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B. [00119] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 16B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 153 of the amino acid sequence set forth in FIG. 16B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 16B .

[00120] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 17B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 149 of the amino acid sequence set forth in FIG. 17B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 17B .

[00121] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 18B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 174 of the amino acid sequence set forth in FIG. 18B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 18B .

[00122] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 19B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 155 of the amino acid sequence set forth in FIG. 19B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 19B . [00123] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 20B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 156 of the amino acid sequence set forth in FIG. 20B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 20B .

[00124] In some cases, a variant GABAA-R alpha chain present in a light-regulated

GABAA-R of the present disclosure comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 21B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 139 of the amino acid sequence set forth in FIG. 21B . In some cases, a variant GABAA- R alpha chain present in a light-regulated GABAA-R of the present disclosure comprises the amino acid sequence set forth in FIG. 21B .

[00125] The ligand moiety of the photoregulator in a light-regulated GABAA-R of the present disclosure can function as an antagonist in the cis configuration or in the trans configuration, depending on where the photoregulator is attached. The photoregulator in a light-regulated GABAA-R of the present disclosure is in the trans configuration when exposed to light of a wavelength of from 480 nm to 520 nm, e.g., about 500 nm. The photoregulator in a light-regulated GABAA-R of the present disclosure is in the cis configuration when exposed to light of a wavelength of from 360 nm to 400 nm, e.g., about 380 nm.

[00126] In some cases, the ligand is PAG- 1C and, when the light-regulated GABAA-R comprising an alpha- 1 isomer variant (an alpha- 1 isomer variant of the present disclosure) is exposed to light of a wavelength of from 480 nm to 520 nm, the ligand is in the extended trans isomer configuration and binds to the ligand-binding site of the alpha chain, and inhibits GABA-elicited current mediated by the light-regulated

GABAA-R; and, when the light-regulated GABAA-R is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the cis isomer configuration and GABA- elicited current mediated by the receptor is restored. [00127] In some cases, the ligand is PAG-2A, PAG-2B, or PAG-3C, and, when the light- regulated GABAA-R is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the extended trans isomer configuration and binds to the ligand-binding site of the alpha chain (e.g., where the alpha chain is an alpha-2 isomer variant of the present disclosure, an alpha-3 isomer variant of the present disclosure, an alpha-4 isomer variant of the present disclosure, an alpha-5 isomer variant of the present disclosure, or an alpha-6 isomer variant of the present disclosure), and inhibits GABA-elicited current mediated by the receptor; and, when the light-regulated GABAA-R is exposed to light of a wavelength of from480 nm to 520 nm, the ligand is in the cis isomer configuration and GABA-elicited current mediated by the receptor is restored.

Compositions

[00128] The present invention further provides compositions comprising a light-regulated

GABAA-R of the present disclosure. Compositions comprising a light-regulated

GABAA-R of the present disclosure will in many embodiments include one or more of: a salt, e.g., NaCl, MgCl₂, KC1, MgSC>4, etc.; a buffering agent, e.g., a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N- Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, Nonidet-P40, etc.; a protease inhibitor; and the like. In some cases, a subject composition comprising a light- regulated GABAA-R of the present disclosure is a pharmaceutical composition, as described in more detail below.

METHODS OF MODULATING THE ACTIVITY OF A GABA_A RECEPTOR

[00129] The present disclosure provides methods of modulating the activity of a GABAA receptor. The present disclosure provides methods of modulating the activity of a neuron. The present disclosure provides treatment methods.

Methods of modulating the activity of a GABA_A receptor

[00130] The present disclosure provides methods of modulating the activity of a GABAA receptor, the method comprising exposing the GABAA receptor to light, wherein the GABAA receptor comprises: a) an alpha chain variant of the present disclosure, the alpha chain variant comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue at a surface accessible site in the alpha chain; b) a photo switchable group of the formula: linker-azobenzene-R, where R is a ligand for a GABAA receptor, and where the photoswitchable group is covalently linked to the cysteine via the linker; and where the light is of a wavelength in the range of from 360 nm to 400 nm, or where the light is of a wavelength in the range of from 480 nm to 520 nm.

[00131] In some cases R is selected from:

Treatment methods

[00132] The present disclosure provides a treatment method, the method comprising: a) administering to an individual in need thereof a variant GABAA receptor alpha chain of the present disclosure, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain; b) administering to the individual a

photoswitchable group of the formula: linker-azobenzene-R, wherein R is a ligand for a GABAA receptor, and wherein the photoswitchable group binds to the variant alpha chain and is covalently linked to a cysteine in the alpha chain via the linker, forming a photoregulated alpha chain, wherein the photoregulated alpha chain associates with two GABAA receptor β chains and either a GABAA receptor γ chain or a GABAA receptor δ chain, thereby generating a light-responsive GABAA receptor; and c) exposing the light- responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 480 nm to 520 nm.

[00133] In some cases R is selected from:

Methods of modulating the activity of a neuron

[00134] The present disclosure provides a method of modulating the activity of a neuron, the method comprising: a) introducing into the neuron a variant GABAA receptor alpha chain of the present disclosure, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain; b) administering to the individual a photo switchable group of the formula: linker-azobenzene-R, wherein R is a ligand for a GABAA receptor, and wherein the photoswitchable group binds to the variant alpha chain and is covalently linked to a cysteine in the alpha chain via the linker, forming a photoregulated alpha chain, wherein the photoregulated alpha chain associates with two GABAA receptor β chains and either a GABAA receptor γ chain or a GABAA receptor δ chain, thereby generating a light-responsive GABAA receptor; and c) exposing the light- responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 480 nm to 520 nm, wherein R is a GABAA receptor antagonist.

[00135] In some cases, R is selected from:

Formulations, dosages, and routes of administration

[00136] As discussed above, a method of the present disclosure involves administration to an individual in need thereof of an effective amount of one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure.

[00137] An "effective amount" of an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) is in some cases an amount that, when administered in one or more doses to an individual in need thereof, modulates the activity of a neuron (e.g., a GABAergic neuron) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the activity of the neuron in the absence of the agent. In some cases, "effective amount" of an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) inhibits the activity of a neuron by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 80%, compared to the activity of the neuron in the absence of the agent.

Formulations

[00138] In the subject methods, an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, an agent can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into

preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[00139] In pharmaceutical dosage forms, an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

[00140] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of an agent adequate to achieve the desired state in the subject being treated.

[00141] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[00142] For oral preparations, an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[00143] An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[00144] Pharmaceutical compositions comprising an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) are prepared by mixing the agent having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[00145] The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[00146] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a given agent may depend on the particular agent employed and the effect to be achieved, and the pharmacodynamics associated with each agent in the host. [00147] In some embodiments, an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) is formulated in a controlled release formulation. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent in which the matrices are in the form of shaped articles, e.g. films or microcapsules. Examples of sustained-release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers and poly-D-(-)- 3-hydroxybutyric acid. Possible loss of biological activity may be prevented by using appropriate additives, by controlling moisture content and by developing specific polymer matrix compositions.

Dosages

[00148] A suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular agent to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently. An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[00149] Those of skill will readily appreciate that dose levels can vary as a function of the specific agent, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

Routes of administration

[00150] An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) is administered to an individual using any available method and route suitable for drug delivery or nucleic acid delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.

[00151] Conventional and pharmaceutically acceptable routes of administration include intracranial, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be administered in a single dose or in multiple doses. In some embodiments, an agent is administered intracranially. In some embodiments, an agent is administered intravenously. In some embodiments, an agent is administered locally.

[00152] An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can be

administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

[00153] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, intracranial, and intravenous routes, i.e. , any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of an agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[00154] By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as cancer. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.

[00155] In some embodiments, an agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) is administered by injection and/or delivery, e.g., to a site in a brain artery or directly into brain tissue. An agent (where an "agent" can be one or more of: i) a variant GABAA receptor alpha chain of the present disclosure; ii) a nucleic acid (e.g., an expression vector) comprising a nucleotide sequence encoding variant GABAA receptor alpha chain of the present disclosure; and iii) a photoregulator of the present disclosure) can also be administered directly to a target site e.g., by direct injection, by implantation of a drug delivery device such as an osmotic pump or slow release particle, by biolistic delivery to the target site, etc.

TRANSGENIC NON-HUMAN ANIMALS

[00156] The present disclosure provides a non-human transgenic animal comprising a transgene in the genome of the animal, wherein the transgene comprises a nucleotide sequence encoding a variant GABAA receptor alpha chain of the present disclosure (e.g., comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain). In some cases, the non-human transgenic animal is homozygous for the transgene. In some cases, the non-human transgenic animal is heterozygous for the transgene. In some cases, the non-human transgenic animal is a rodent (e.g., a mouse; a rat). In some cases, the non-human transgenic animal is a rabbit.

[00157] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is a T→C substitution of the amino acid sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R alpha chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRITEDGCLLYT (SEQ ID NO:25). In some cases, the amino acid substitution is a T→C substitution of the amino acid sequence LLRITEDGTLLYT (SEQ ID NO: 19) present in a GABAA-R al chain, where the threonine that is substituted with a cysteine is indicated in bold, such that the variant GABAA-R al chain comprises the amino acid sequence LLRITEDGCLLYT (SEQ ID NO:25).

[00158] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is a Q→C substitution of the amino acid sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRICDDGTLLYT (SEQ ID NO:26). In some cases, the amino acid substitution is a Q→C substitution of the amino acid sequence LLRIQDDGTLLYT (SEQ ID NO:20) present in a GABAA-R a2 chain, such that the variant GABAA-R a2 chain comprises the amino acid sequence

LLRICDDGTLLYT (SEQ ID NO:26).

[00159] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is a V→C substitution of the amino acid sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRLCDNGTLLYT (SEQ ID NO:27). In some cases, the amino acid substitution is a V→C substitution of the amino acid sequence LLRLVDNGTLLYT (SEQ ID NO:21) present in a GABAA-R a3 chain, such that the variant GABAA-R R a3chain comprises the amino acid sequence

LLRLCDNGTLLYT (SEQ ID NO:27).

[00160] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is an M→C substitution of the amino acid sequence LFRIMRNGTILYT (SEQ ID NO:22) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LFRICRNGTILYT (SEQ ID NO:28). In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRIMRNGTILYT (SEQ ID NO:22) present in a GABAA-R a4 chain, such that the variant GABAA-R 4 chain comprises the amino acid sequence

LFRICRNGTILYT (SEQ ID NO:28).

[00161] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LLRLCDDGTLLYT (SEQ ID NO:29). In some cases, the amino acid substitution is an E→C substitution of the amino acid sequence LLRLEDDGTLLYT (SEQ ID NO:23) present in a GABAA-R a4 chain, such that the variant GABAA-R 5 chain comprises the amino acid sequence

LLRLCDDGTLLYT (SEQ ID NO:29).

[00162] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R alpha chain, such that the variant GABAA-R alpha chain comprises the amino acid sequence LFRLCHNGTILYT (SEQ ID NO:30). In some cases, the amino acid substitution is an M→C substitution of the amino acid sequence LFRLMHNGTILYT (SEQ ID NO:24) present in a GABAA-R a6 chain, such that the variant GABAA-R 6 chain comprises the amino acid sequence LFRLCHNGTILYT (SEQ ID NO:30).

[00163] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B, where the GABAA-R alpha chain comprises the amino acid substitution noted in the corresponding one of FIG. 16B, 17B, 18B, 19B, 20B, and 21B .

[00164] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 16B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 153 of the amino acid sequence set forth in FIG. 16B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 16B .

[00165] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 17B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 149 of the amino acid sequence set forth in FIG. 17B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 17B .

[00166] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 18B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 174 of the amino acid sequence set forth in FIG. 18B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 18B .

[00167] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 19B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 155 of the amino acid sequence set forth in FIG. 19B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 19B .

[00168] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 20B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 156 of the amino acid sequence set forth in FIG. 20B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 20B .

[00169] In some cases, a non-human transgenic animal of the present disclosure

comprises a transgene comprising a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution, where the variant GABAA-R alpha chain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the amino acid sequence depicted in FIG. 21B, where the alpha chain comprises a cysteine at a position corresponding to amino acid 139 of the amino acid sequence set forth in FIG. 21B . In some cases, the transgene encodes a variant GABAA-R alpha chain that comprises the amino acid sequence set forth in FIG. 21B . [00170] Methods of making transgenic non-human animals are known in the art; any known method can be used. For example, a CRISPR/Cas9 system can be used to introduce a mutation, which results in one of the aforementioned cysteine substitutions, into the endogenous GABAA receptor alpha chain-encoding gene of the non-human animal. A CRISPR/Cas9 system can be used to replace the endogenous GABAA receptor alpha chain-encoding gene of a non-human animal with a nucleic acid encoding a variant GABAA receptor alpha chain of the present disclosure.

EXAMPLES

[00171] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous (ly); and the like.

Example 1:

[00172] Exogenously-expressed opsins are valuable tools for optogenetic control of

neurons in circuits. A deeper understanding of neural function can be gained by bringing control to endogenous neurotransmitter receptors that mediate synaptic transmission. Here, a comprehensive optogenetic toolkit for controlling GABAA receptor-mediated inhibition in the brain is developed. A series of photoswitch ligands and the

complementary genetically-modified GABAA receptor subunits was synthesized. By conjugating the two components, light-sensitive versions of the entire GABAA receptor family were generated. These light-sensitive receptors were validated for applications across a broad range of spatial scales, from subcellular receptor mapping to in vivo photo-control of visual responses in the cerebral cortex. Finally, a knock-in mouse was generated, in which the "photo switch-ready" version of a GABAA receptor subunit genomically replaces its wild-type counterpart, ensuring normal receptor expression. This optogenetic pharmacology toolkit allows scalable interrogation of endogenous GABAA receptor function with high spatial, temporal, and biochemical precision.

MATERIALS AND METHODS

[00173] The photoswitch compounds were synthesized as trifluoroacetate salts. The

compounds were prepared as concentrated stocks (10-100 mM in anhydrous DMSO) and diluted in buffers for receptor conjugation (final DMSO concentration <1% v/v). AAV9 (10¹²-10¹³ vg/mL) encoding a mutant a-subunit (alT125C or a5E125C), an eGFP marker, and a human synapsin-1 promoter was prepared by UC Berkeley Gene Delivery Module (Lin et al., 2014). The al-GABA_A PhoRM mice were generated by UC Davis Mouse Biology program. All experiments were performed in accordance with guidelines and regulations of the ACUC at the University of California, Berkeley. Group data are reported as mean + SEM. Detailed experimental procedures and data analysis methods are provided in the Supplemental Information, below.

Mutant Expression and PTL Treatment

[00174] Ex vivo procedures (HEK cells, cultured neurons, and brain slices). HEK cells and dissociated hippocampal neurons were cultured on poly-L-lysine coated coverslips, maintained at 37 °C and 5% C0₂, and transfected via calcium phosphate precipitation. The mutant subunits were expressed in organotypic hippocampal slices by injecting AAV9 encoding eGFP-2A-alT125C or eGFP-2A-a5E125C in the CA1 pyramidal cell body layer. Viral transduction of mouse cerebral cortex was performed by neonatal injection (Figures 3 and 4) or stereotactic injection in adult mice (Figures 5 and 6).

[00175] Prior to electrophysiological experiments, the cells or slices were treated with tris(2-carboxyethyl)phosphine (TCEP; 2.5-5 mM, 5-10 min), washed, and then treated with PTL (25-50 μΜ, 25-45 min) at room temperature to convert the mutant receptors into LiGABARs.

[00176] In vivo PTL treatment. For experiments in Figures 5 and 6, a craniotomy of 2-3 mm in diameter was made, with subsequent duratomy on anesthetized mice. 100 μΐ of HEPES-aCSF, which contained PAG-1C (250 μΜ) and TECP (250-500 μΜ), was applied onto the exposed cortex for 1 hr. For multi-electrode recordings in awake mice (Figure 8), the skull was thinned and a small craniotomy (0.5-1.5 mm in diameter) was opened without duratomy over the visual cortex. The PTL solution was infused into the brain at a rate of 100 nl/min for 10 min with a glass micropipette attached to a microinfusion pump (UMP3 with SYS-Micro4 controller; World Precision Instrument). In control experiments, vehicle solution containing 500 μΜ TCEP without PAG-IC was infused.

Subcellular LiGABAR Mapping via Two-photon GABA uncaging

[00177] Imaging and uncaging were performed using a two-photon laser scanning

microscope (MOM; Sutter). The light source for fluorescence excitation (800 nm for Alexa Fluor 594 and 940 nm for gephyrin intrabody) and RuBi-GABA uncaging (800 nm) was a Ti:Sapphire laser (Chameleon XR; Coherent). LiGABAR-expressing hippocampal neurons were voltage clamped at 0 mV, with 25 μΜ DNQX, 50 μΜ D- AP5, and 0.5 μΜ TTX in the bath. Internal solution included 200 μΜ Alexa Fluor 594 (Life Technologies) for visualizing dendritic morphology. RuBi-GABA (200-400 μΜ; Abeam) was added to aCSF and re-circulated using a peristaltic pump (Idex). Uncaging was carried out at designated locations for 5-10 ms with a light intensity of -150 mW.

Full-field 390 nm (1.2 mW/mm 2 ) or 540 nm (3.2 mW/mm 2 ) conditioning flashes (5 sec) from an LED light source (Lumencor) were delivered through the objective.

Photo switching was calculated as 1 - (I540/I390), where I refers to the peak amplitude of GABA-elicited current.

Photo-control of LiGABAR in vivo

[00178] Visual stimulus generated with PsychToolbox was either a circular patch of

drifting square-wave gratings in full contrast (Figure 6) or a square full contrast checkerboard (Figure 8) against a mean luminance grey background. Targeted loose- patch recordings for Figure 6 were made from PV-tdTOM and LiGABAR-eGFP double positive cells in layer 2/3 (150-350 μιη below pia) of the visual cortex, using a two- photon laser-scanning microscope (Sutter) with a Ti:Sapphire laser (1050 nm; Coherent). For multi-electrode extracellular recordings (Figure 8), a 16-channel probe was used (NeuroNexus, Alxl6-3mm-25-177-A16). Recordings were amplified, digitized at 30 kHz (Spikegadget), and MClust was used for off-line sorting of the spike waveforms. Additional Experimental Procedures

[00179] Unless otherwise indicated, chemicals and buffers were obtained from Sigma,

Tocris, or Thermo-Fisher Scientific. All experiments were performed in accordance to the guidelines and regulations of the ACUC at the University of California, Berkeley. Cloning and Virus Preparation

[00180] The cDNAs of wild-type rat GABAAR al (in pGH19), a2 (in pRK7), a3 (in pRK7), β2 (in pGH19), and j2S (in pUNIV) were obtained from Professor Cynthia Czajkowski (University of Wisconsin, USA). The cDNAs of wild-type rat GABAAR a4 and a6 (both in pCMVNeo) were obtained from Professor Robert L. Macdonald (Vanderbilt University, USA). The pGH19 clones of wild-type al and β2 subunits were sub-cloned into vector pCDNA3.1 for expression in HEK293 cells. The wild-type rat GABAAR a5 in vector pRK5 was sub-cloned into vector pCDNA3.1 for further processing. Cysteine mutants of a-subunits were prepared by site-directed mutagenesis in the wild-type clones, and the mutations were confirmed by sequencing. The mutants used in the LiGABAR toolkit are (numbered based on the mature sequence): alT125, a2Q121, a3V146, a4M120, a5E125, and a6M120 (if counted from the start codon: alT152, a2Q149, a3V174, a4M155, a5E156, and a6M139).

[00181] The cDNA of GFP-fused intrabody against gephyrin (GPHN.FingR-GFP in vector pCAG; Gross, et al., 2013) was used (Addgene plasmid # 46296).

[00182] For neuronal expression, bi-cistronic pAAV constructs encoding a mutant a- subunit (alT125C or a5E125C) and an eGFP marker were prepared following the previously published procedures (Lin et al., 2014). Each mutant a-subunit has an N- terminal myc epitope tag which does not affect receptor function and synaptic targeting (Connolly, et al., 1996; Tretter et al., 2008). Gene expression is conferred by a human synapsin-1 promoter (Kugler et al., 2003). The resulting DNA clones were subsequently packaged into AAV9 at a titer of 10¹²-10¹³ vg/mL.

Animals

[00183] Pregnant female mice for neonatal viral injection (see below) and Sprague-

Dawley rats were obtained from Charles River Laboratories. PV-tdTOM mice were derived from crossing the two following mouse lines: PV-CRE (Jackson lab stock #008069) and Rosa-LSL-tdTOMATO (Allen Institute line Ai9, Jackson Labs #007905). [00184] alT125C knock- in mice were generated via UC Davis mouse biology program.

The genomic region of Gabral (NM_010250.4) was obtained from BAC clone RPCI-24 and was used to develop a targeting vector that contains the genomic region surrounding exons 5 and 6 of Gabral. A cysteine mutation was introduced for T152 (counting from the start codon) on exon 5 as well as a C to T silent mutation to create a Hind III site upstream of T152C for genotyping. The final targeting construct is shown in Figure 14. The construct was linearized and electroporated into ES cells from mouse strain 129. Cells were selected for transmitted neomycin resistance and homologous recombination was confirmed on flanking regions of the targeting vector. A loss of allele assay was performed to confirm a single recombination event. After karyotyping, ES cells were injected into C57/B6 mouse blastocysts and implanted into surrogates resulting in chimeras. After confirming germline transmission the F2 offspring were bred with a Cre recombinase expressing mouse to excise the neomycin cassette. The resulting progeny were bred to homozygosity of the Gabral knock-in and the Cre cassette was bred out. Culture and Transfection of HEK cells

[00185] Cells were maintained in Dulbecco's Minimum Essential Medium (Gibco)

supplemented with 10% fetal bovine serum (Gibco) at 37 °C and 5% C0₂. Cells were seeded in a 24- well plate (20-25 x 10" cells/well) on 12 -mm poly-L-lysine coated coverslips and were transfected by calcium phosphate precipitation at 40-50% confluence. A total of -1.1 μg DNA per coverslip was used. For al, a2, a3, and a5, the DNA mixture comprised ^g): 0.15 a, 0.15 β, 0.75 γ, and 0.05 eGFP. For a4 and a6, the DNA mixture comprised ^g): 0.25 a, 0.25 β, 0.50 γ, and 0.05 eGFP. Cells were used 1- 2 days after transfection.

Culture and Transfection of Dissociated Hippocampal Neurons

[00186] Cultures of dissociated hippocampal neurons were prepared from P0-P2 neonatal rats. In brief, pups were decapitated and the brains were removed to warmed HBSS++ solution (Ca²⁺- and Mg²⁺-free Hank's Balanced Salt Solution (Gibco) supplemented with 1 mM HEPES and 20 mM glucose). Hippocampi were dissected from brain and digested for 12 min in 0.25% trypsin (Gibco), washed five times in HBSS++ and transferred to warmed neural growth medium (NGM). NGM was made of Eagle's Minimum Essential Medium (Gibco) supplemented with 20 mM glucose, 5% FBS (Gibco), IX B27 supplement (Gibco), 2 mM glutamine (Gibco), and serum extender (BD Biosciences). Hippocampi were then triturated with fire-polished Pasteur pipettes and passed through a 40- μιη cell strainer to isolate individual cells. Cells were seeded in a 24-well plate (75-100 x 10 cells/well) on 12-mm poly-L-lysine coated coverslips, and were then maintained in NGM at 37 °C and 5% C0₂. Half of the culture medium was replaced with fresh NGM every 2-3 days. Cytosine arabinoside (araC; Sigma) was added on 4 DIV (to a final concentration of 2 μΜ in culture) to inhibit the proliferation of non-neuronal cells. On 7-9 DIV, neurons were transfected with al(T125C) (in pCDNA3.1, 0.8 μ^εΠ) and GPHN.FingR-GFP (in pCAG, 0.4 μ^ννεΐΐ) via calcium phosphate precipitation. Two-photon uncaging experiments were carried out 5-10 days thereafter.

Preparation and Viral Transduction of Organotypic Slice Cultures (Hippocampus)

[00187] Postnatal day 8 Sprague-Dawley rat pups were anaesthetized and decapitated.

Hippocampi were dissected and sliced into 350 μπι-thick sections using a tissue chopper (Stoelting). Slices were maintained at 34 °C on cell culture inserts (Milipore) in

Neurobasal-A medium (Life Technologies) supplemented with 20% horse serum

(Thermo Scientific), 0.03 units/mL insulin (Sigma), 0.5 mM ascorbic acid, IX Gluta- Max (Life Technolgies), 80 units/mL penicillin (Life Technolgies), 80 μg/ml streptomycin (Life Technolgies), and 25 mM HEPES. One day after preparation, slices were injected with AAV9 encoding eGFP-2A-alT125C or eGFP-2A-a5E125C. The CAl pyramidal cell body layer was injected at 1-2 sites/slice with 150 nL of virus with a fine glass pipette. Slices were used 5-14 days post-injection.

Viral Expression of Mutant a-Subunits in the Mouse Visual Cortex

[00188] Stereotactic Injection. Three to four weeks old wide-type or PV-tdTOM mice were injected stereotactic ally with a bi-cistronic AAV (see "Virus Preparation" above). Mice were anesthetized with isoflurane and a small craniotomy was made for the insertion of a beveled injection needle (Drummond) at 2.5 mm lateral and 0.5 mm anterior to lambda. The pipette was slowly lowered to 150-250 μιη below the brain surface. 120-150 nL of virus was injected over 4 min. The needle was left in place for an additional 2 min to allow viral diffusion. After removing the injection needle, the scalp was sutured. The animals were then given a dose of analgesics (buprenorphine, 0.1 mg/kg) and were allowed to recover for 2-3 weeks before the experiments. Infection was confirmed with an epifluorescent stereomicro scope and fluorescence was used to target all subsequent craniotomies and recordings in vivo.

[00189] Neonatal Injection. Neonates (P0-P3) of wild-type mice were anesthetized on ice, placed in a custom mold, and injected with 10-30 nL of virus at 1-2 sites in the visual cortex (1-1.5 mm lateral to lambda, 0 AP, 300-500 μιη DV). Experiments were carried out ~3 weeks thereafter.

Preparation of Acute Brain Slices

[00190] Mice (3 weeks to 2 months old) of both sexes were used for slice preparation.

For animals older than 1 month old, animals underwent intracardiac perfusion of ice cold cutting solution (see below) after katamine and xylazine induced anesthesia. Acute brain slices (350 μιη) from either visual cortex or cerebellar vermis were prepared in ice-cold cutting solution containing (in mM): 85 NaCl, 2.5 KCl, 0.5 CaCl₂, 4 MgCl₂, 1.25 NaH₂P0₄, 25 NaHC0₃, 75 sucrose, 0.4 ascorbic acid and 25 glucose (saturated with 95% 0₂ and 5% C0₂; pH 7.4). To record cerebellar Golgi cells, slices were prepared in (mM): 130 K-gluconate, 25 glucose, 15 KCl, 20 HEPES, 0.05 EGTA, and 1 kynurenic acid (Abeam), saturated with 95% 0₂ and 5% C0₂; pH 7.4 (ice-cold). After sectioning, slices were transferred to a holding chamber containing artificial cerebero spinal fluid (aCSF) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl₂, 1.3 MgCl₂, 1 NaH₂P0₄, 26.2 NaHC0₃, and 11 glucose (saturated with 95% 0₂ and 5% C0₂; pH 7.4) at 34 °C for 20 min and were then cooled down to room temperature. PTL treatment was carried out at room temperature thereafter (see below).

Ex vivo PTL Treatment

[00191] HEK cells and cultured neurons. Cells (in extracellular solution; see formula in the Electrophysiology section) were treated with tris(2-carboxyethyl)phosphine (TCEP; 2.5-5 mM, 5-10 min), washed, and then treated with PTL (25 μΜ, 25 min, pH 8.0 for HEK cells and 7.4 for cultured neurons) at room temperature.

[00192] Organotypic slices. Slices (in aCSF) were treated with TCEP (5 mM, 5 min), washed, and then incubated with PAG-1C (25-50 μΜ) and guanidinium hydrochloride (500 μΜ) for 60 min at room temperature.

[00193] Acute slices. Slices (in aCSF) were treated with TCEP (5 mM, 8-10 min),

washed, and incubated with either PAG-1C (25-50 μΜ, with 500 μΜ guanidinium hydrochloride) or PAG-2A (25-50 μΜ) for >35 min at room temperature. In vivo PTL Treatment

[00194] Custom-made chambers were implanted over the visual cortex of virus-injected or al(T125C) knock-in mice. For experiments in Figures 5 and 6, a craniotomy of 2-3 mm in diameter was made, with subsequent duratomy on anesthetized mice. 100

of HEPES-aCSF (in mM: 125 NaCl, 3 KCl, 10 HEPES, 10 glucose, 2 CaCl₂, 2 MgCl₂; pH 7.4, 290 mOsm) containing PAG-1C (250 μΜ) and TECP (250-500 μΜ) was applied onto the exposed cortex for 1 hr. The solution was then removed from the brain, and HEPES-aCSF was applied to rinse off residual drugs. The mice were either used in slice preparation (Figure 5) or covered with 1.5-2% low-melting temperature agarose (in HEPES-aCSF) on the brain for two-photon guided patching (Figure 6). For multi- electrode recording in awake knock-in mice (Figure 8), the skull was thinned and a small craniotomy (0.5-1.5 mm in diameter) was opened without duratomy over the visual cortex. The PTL qsolution was infused into the brain at a rate of 100 nL/min for 10 min with a glass micropipette attached to a microinfusion pump (UMP3 with SYS-Micro4 controller; World Precision Instrument). The surgery lasted less than 40 min and recordings started at least 45 min after the mice recovered from anesthesia. In control experiments, vehicle solution containing 500 μΜ TCEP without PAG-1C was infused. In vitro Electrophysiology

[00195] HEK cells. Recordings were carried out at room temperature using pipettes with

2.5-5 ΜΩ resistance. Cells were held at -70 mV. Pipettes were pulled from filamented borosilicate pipettes (G150TF-3, Sutter Instruments). The extracellular solution contained (in mM): 138 NaCl, 1.5 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 HEPES, 10 Glucose; pH 7.4. The intracellular solution contained (in mM): 140 CsCl, 4 NaCl, 10 HEPES, 2 MgC12, 2 Mg-ATP, and 10 EGTA; pH 7.2. Signals were amplified using a Patch Clamp PC-501A amplifier (Warner Instruments), low-pass filtered at 2 kHz, digitized at 10 kHz by a Digidata 1322A converter (Molecular Devices), and acquired with software Clampex 10 (Molecular Devices). Illumination for photo-control was provided by a Lambda-LS xenon lamp (Sutter Instruments) with 379 + 17 nm and 500 + 8 nm band pass filters.

[00196] Two-photon uncasing at cultured hippocampal neurons. For the uncaging

experiments in Figures 2H and 21, whole-cell voltage-clamp recordings were carried out at room temperature in re-circulated aCSF containing (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH₂P0₄, 10 Glucose, 1.3 MgCl₂, 26 NaHC0₃, 2.5 CaCl₂, 0.025 DNQX, 0.05 D-AP5, 0.0005 TTX, and 0.2-0.4 RuBi-GABA (Abeam) equilibrated with 95% 0₂ + 5% C0₂. Neurons were held at 0 mV. The internal solution contained (in mM): 108 Cs-gluconate, 2.8 NaCl, 20 HEPES, 5 TEA-C1, 0.4 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 10

phosphocreatine, and 0.2 Alexa-594 (for visualizing dendritic morphology; Life

Technologies); pH -7.2. See Subcellular LiGABAR Mapping via Two-photon GABA Uncaging below for details about two-photon imaging and RuBi-GABA photolysis.

[00197] IPSC recordings in hippocampal neurons. Slices were placed in a recording chamber mounted on an upright fixed-stage microscope (MOM, Sutter) with gradient contrast IR optics (Siskiyou) and GFP epifluorescence. Slices were perfused with aCSF at room temperature at 1-2 mL/min. ACSF contained (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH₂P0₄, 10 Glucose, 1.3 MgCl₂, 26 NaHC0₃ and 2.5 CaCl₂, equilibrated with 95% 0₂ + 5% C0₂. Whole-cell recordings were made from GFP-positive CA1 pyramidal cells with glass microelectrodes (R = 4-7 ΜΩ) filled with internal solution containing (in mM): 108 Cs-gluconate, 2.8 NaCl, 20 HEPES, 5 TEA-C1, 0.4 EGTA, 4 Mg-ATP, 0.3 Na-GTP and 10 phosphocreatine, adjusted to ~ 7.2 pH and -290 mOsm. To record isolated IPSCs, 25 μΜ DNQX and 50 μΜ D-AP5 were added to the aCSF and cells were held at the reversal potential of excitatory inputs (0 mV). A glass stimulating electrode (filled with aCSF) was placed in proximal stratum radiatum -100 μιη away from the recorded cell. Synaptic responses were evoked by a 0.2-ms, 10-100 μΑ current pulse delivered via a stimulus isolation unit (AMPI). Conditioning light (390 nm or 540 nm) was generated by a Spectra-X light engine under software control (Lumencor) and delivered through the microscope objective. Membrane currents were amplified

(Axopatch; Molecular Devices), digitized (Digidata; Molecular Devices) and recorded (pClamp; Molecular Devices) to a desktop computer.

[00198] Current-clamp recordings in hippocampal neurons. Whole-cell recordings from hippocampal neurons (in aCSF, without AP5 and DNQX) were performed using the instrumental setup described above. The internal solution contained (mM): 116 K- Gluconate, 6 KCl, 2 NaCl, 20 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP and 10 phosphocreatine, adjusted to - 7.2 pH and -290 mOsm. Conditioning light (390 nm or 540 nm) was applied 100 ms prior to Schaffer-collateral stimulation, with the recorded neuron at rest (around -70 mV). The stimulating electrode was placed in stratum radiatum -400 μηι from the recorded cell in CA1, and the stimulation was carried out at 0.5 Hz.

[00199] IPSC recordings in cortical and cerebellar neurons. Voltage-clamp recordings

( _hoi_d = 0 mV) were made on cortical or cerebellar neurons at room temperature.

Pipettes were pulled from filamented borosilicate glass with an open-tip resistance of 1.5-2.5 ΜΩ for cerebellar Purkine cells and of 3-5 ΜΩ for other neuron types. The internal solution contained (in mM): 108 Cs-gluconate, 2.8 NaCl, 20 HEPES, 1 EGTA, 5 TEA-C1, 4 Mg-ATP, 0.4 Na-GTP; pH. 7.3. Synaptic currents were amplified by Axoclamp 200A or Multiclamp 700B (Molecular Devices), recorded via pClamp 9.2/10 (Molecular Devices) or custom routines written with Matlab (Mathwork), and then filtered at 2 kHz and digitized at 10-20 kHz using a Digidata 1330/1440 (Molecular Devices) or BNC2090 (National Instrument) analog-to-digital converter. Monosynaptic IPSCs were evoked using patch pipette filled with aCSF placed 1-200 μιη away from the soma of the recorded neuron, with glutamatergic activities blocked by 3-4 mM kynurenic acid (Abeam). A constant current stimulus isolation unit was used (AMPI, Israel). Stimulus intensity was set between 250-750 μΑ to limit evoked IPSC amplitude below 750 pA. No correlation between the IPSC size and photoswitching magnitude were found (n = 6, Pearson correlation R= 0.77). LiGABAR photo-control was carried out using one of the following light sources: Polychrome (TILL Photonics), 380 nm (1 mW/cm 2") and 500 nm (1.6 mW/cm 2 ) delivered through a 4x objective; Spectra

(Lumencor), 390 nm (3.5 mW/cm 2 ) and 540 nm (15 mW/cm 2 ) delivered through a 20x objective; Prizmatix (Prizmatix, Israel), 385 nm (20 mW/cm 2 ) and 500 nm (15 mW/cm 2 ) delivered through optic fibers with 1-mm core diameter.

[00200] Effects ofPTL treatment on endogenous ion channels. Control experiments in

Figures 11A, 11C, 11D, HE, and 11F were performed on hippocampal CA1 pyramidal cells in acute slices, which were prepared from wild-type Sprague-Dawley rats (PI 4-21) and treated with PAG-1C. IPSCs were evoked with electrical stimulation in stratum pyramidale and recorded in whole-cell voltage-clamp mode at 0 mV, with GABA_B activities and glutamatergic inputs blocked with 5 μΜ CGP 54626, 10 μΜ DNQX, and 50 μΜ AP5. AMPAR-EPSCs were evoked with stimulation in stratum radiatum and recorded at -60 mV in the presence of 5 μΜ CGP 54626 and 100 μΜ picrotoxin to block all GABAergic activities. NMDAR-EPSCs were evoked with stimulation in stratum radiatum and recorded at +40 mV in the presence of 5 μΜ CGP 54626, 100 μΜ picrotoxin, and 10 μΜ DNQX. Voltage-gated sodium currents were evoked with a 200- ms step from -60 mV to -10 mV. Voltage-gated potassium currents were evoked with a 200-ms step from -60 mV to +40 mV in the presence of 3 μΜ TTX. Slices were perfused with aCSF at room temperature. Pipette solution for recording IPSCs, EPSCs, and sodium currents comprises (in mM): 108 Cs-gluconate, 2.8 NaCl, 20 HEPES, 5 TEA-C1, 0.4 EGTA, 4 Mg-ATP, 0.3 Na-GTP and 10 phosphocreatine, adjusted to ~ 7.2 pH and -290 mOsm. Pipette solution for recording potassium currents comprises (in mM): 116 K-Gluconate, 6 KCl, 2 NaCl, 20 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP and 10 phosphocreatine, adjusted to ~ 7.2 pH and -290 mOsm. GIRK-channel currents (for the measurement of GABA_B-receptor activation; Figure 1 IB) were evoked by applying 10 μΜ (K)-baclofen onto PAG-1C treated cultured hippocampal neurons. Whole-cell currents were recorded at room temperature with neurons held at -70 mV. The extracellular solution contained (in mM): 119.5 NaCl, 20 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 5 HEPES, 10 Glucose, 0.025 DNQX, 0.05 AP5, and 0.001 TTX; pH 7.4. The intracellular solution contained (in mM): 10 NaCl, 135 K-gluconate, 2 MgCl₂, 10 HEPES, 2 Mg-ATP, 0.35 NaGTP, and 1 EGTA; pH 7.2.

In vivo Electrophysiology

Targeted-cell loose-patch recordings for Figure 6 were made from PV-tdTOM and LiGABAR-eGFP double positive cells in layer 2/3 (150-350 μιη below pia) of the visual cortex using a two-photon laser scanning microscope (Sutter) with a Ti:Sapphire laser (Coherent) at 1050 nm. Two-photon images were acquired using Scanimage (Pologruto et al., 2003). Patch electrodes (4-5 ΜΩ) were filled with HEPES-aCSF and 50 μΜ Alexa Fluor 594 hydrazide (Invitrogen) for visualization. Neuron spiking activities were recorded as action potential currents, with neurons being clamped at the voltage that generated zero holding current (using Multiclamp 700B amplifier with custom routines written with Matlab). Data were filtered at 2 kHz and digitized at 20 kHz using BNC2090 analog-to-digital converter (National Instrument). For multi- electrode extracellular recordings in awake knock-in mice (Figure 8), 16-channel probe (NeuroNexus, Alxl6-3mm-25-177-A16) was used to record neuronal activities.

Recordings were amplified, digitized at 30 kHz (Spikegadget), and stored on a computer hard drive. MClust (http(colon)//redishlab(dot)neuroscience(dot)umn(dot)edu/MClust/MClust(dot)html) was used for off-line sorting of the spike waveforms.

[00202] Visual stimulus was either a circular patch of drifting square-wave gratings in full contrast (Figure 6) or a square full contrast checkerboard (Figure 8) against a mean luminance grey background. Gratings were presented at a temporal frequency of 2 Hz and a spatial frequency of 0.04 cycles/degrees (uncorrected for angle of monitor).

Checkerboards (12.5 degrees per square) were presented 10 times for 100 ms at 2 Hz per episode. Stimuli were generated with PsychToolbox and presented on a gamma- corrected 20" or 23" LCD screen with a 60 Hz refresh rate, -15 cm from the mouse, oriented -60 degree with respect to the body axis of the mouse.

Subcellular LiGABAR Mapping via Two-photon GABA Uncaging

[00203] Imaging and uncaging were performed using a two-photon laser scanning

microscope (MOM; Sutter) with a 20x objective (0.95 NA; Olympus). The light source for fluorescence excitation (1.6 W, 140 fs pulses, 90 MHz, 800 nm for Alexa Fluor 594 and 940 nm for GFP-fused gephyrin intrabody) was a Ti:Sapphire laser (Chameleon XR; Coherent). The same light source was used for RuBi-GABA uncaging. Intensity was controlled by a Pockels cell (Conoptics). Imaging and uncaging were controlled by MScan software (Sutter). LiGABAR-expressing hippocampal neurons were voltage clamped at 0 mV, with 25 μΜ DNQX, 50 μΜ D-AP5, and 0.5 μΜ TTX. Internal solution included 200 μΜ Alexa Fluor 594 (Life Technologies) for visualizing dendritic morphology. RuBi-GABA (200-400 μΜ; Abeam) was added to aCSF and re-circulated using a peristaltic pump (Idex). Baseline laser power was adjusted to excite Alexa Fluor 594 without uncaging RuBi-GABA (-2 mW). Scan mirror location and laser intensity were controlled with MScan to uncage at designated locations for 5-10 ms at -150 mW.

Full-field 390 nm (1.2 mW/mm 2 ) or 540 nm (3.2 mW/mm 2 ) conditioning flashes (5 s) were generated by a Spectra-X light engine under software control (Lumencor) and delivered through the objective. After each flash, a 2-min rest period allowed the uncaged GABA to diffuse away (Figure 12A-12B). Because a irans-antagonist was used for both al- and a5-LiGABARs, the receptors were antagonized by 540 nm and restored by 390 nm flashes, respectively. Photoswitching was thus calculated as 1 - (I540/I390), where I refers to the peak amplitude of GABA-elicited current. Immunocytochemistry

[00204] After being deeply anesthetized with ketamine and xylazine, wide-type and

homozygote littermates of alT125C knock-in mice (P30-P45) were perfusion-fixed with 2% paraformaldehyde in 0.1 M sodium acetate buffer (pH. 6) and post- fixed in the same solution for 2-4 h. The brains were transferred to 30% sucrose in 0.9% saline overnight for cryoprotection. Sagittal sections (40 μιη) were sliced using a microtome. Free-floating slices were incubated with TBS (0.05 M Tris and 0.15 M NaCl; pH 7.4) containing 10% normal goat serum (NGS, Jackson ImmunoResearch) for 1 h at room temperature. After blocking, slices were incubated with mouse anti-GABA_AR l (diluted 1:500; NeuroMAB, UC Davis) in TBS with 2% NGS and 0.1% Triton X-100 at 4 °C overnight. After three washes with TBS, slices were incubated with Alexa 594- conjugated secondary goat anti-mouse antibody (1:500; Invitrogen) in TBS for 1 h at room temperature. After washing off residual secondary antibody, slices were mounted with anti-fade reagent (Vectorshield; Vector Labs). Digital images were acquired using a 0.64x objective (NA, 0.15) on an Olympus MVX10 stereomiscroscope using a LED light source (Lumencor) with filter sets of 535-555 nm for excitation and 570-625 nm for emission to collect Alexa-594 fluorescence.

[00205] The fluorescence intensity data in Figure 7 were measured using a 10-pixel width line (region of interests, ROI) drawing across to the anatomical landmarks on both the images from wide-type and knock-in brain sections. 2-4 measurements were made in each section for 2-3 sections from 2 animals of each genotype. Data were processed and analyzed using ImageJ.

Data Analysis

[00206] The electrophysiology data were analyzed in Clampex 10.2 (Molecular Device),

Axograph X (AxoGraph) or customized routines in Matlab (Mathworks).

[00207] The dose-response curves in Figures ID and 10A-10D were measured using the following procedure. For each cell, the current elicited by each tested concentration was first normalized to a reference current elicited at -ECso (to correct for any possible drift in responses during the course of experiment). The normalized responses were then fitted by Hill Equation: I7I_max = 1/(1 + (ECso/[GABA])ⁿ), where I represents the current amplitude elicited by the given [GAB A], I_max represents the saturating response, EC₅₀ is the [GAB A] producing the half-maximal response, and n is the Hill coefficient. The GABA EC₅₀ and Hill coefficient for each tested receptor were calculated as mean + SEM from several analyzed cells. For group data presentation and fitting in Figures ID and 10A-10D, the initially normalized responses were further scaled (to fall between 0 and 1) using the I_max of each cell as reference. Note that in Figure ID, the Hill coefficient is reduced when al-LiGABAR is illuminated with 500 nm light (0.64 + 0.05, compared to 1.3 + 0.1 with 380 nm and 1.3 + 0.1 of the wild-type). This is likely due to incomplete PTL conjugation, which would result in a mixed population of fully and partially antagonized receptors.

[00208] Representative IPSC traces shown in Figures 3 and 7 are the average from 5-10 individual traces. For curve fitting in Figures 4C, 5C and 5F, single exponential fit y = a*exp(b*x) was performed to derive the time and depth constants in Matlab. 95% confidence bounds value were reported as the statistics of the goodness of fit. In Figures 5 C and 5F, the maximal photo switching was estimated from the y-intercept of the fit through back-extrapolation. All the data points were then normalized to the maximum value to construct the plots.

[00209] In Figure 6, a threshold method was used for spike detection. The minimum

threshold used was 4 times of the standard deviation of the baseline noise. PSTH plot was constructed with bin size of 25 ms.

[00210] Multi-electrode recording data (Figures 8 and 15) were analyzed in Matlab.

Spike waveforms were sorted into clusters off-line using MClust using features of peak, trough, energy and time of the waveforms. Clusters were accepted as single units if the cluster has less than 1.4% of the waveforms inter-spike intervals < 2 ms and was well separated from noises with its cluster separation distance > 5 (Schmitzer-Torbert et al., 2005). Waveforms were interpolated with cubic splines by a factor of 100 and spike half width and asymmetry index were extracted. The spike width is defined as the time from trough to the 2^nd peak. The asymmetry index is defined as: (Ai - A₂)/( Ai + A₂), where Ai and A₂ are the amplitude of the first and second peak, respectively (bottom left inset, Reyes-Puerta et al., 2015). Fast spike and regular spiking units are classified by £-means clustering for spike half width and asymmetry index (Figure 15). Average firing rates and average power of the local field potential (20-60 Hz) in each of the total 40 episodes were tested with Friedman tests to determine significance of modulation by conditioning light. In the PTL treated knock-in mice, only mice with significant change in both single unit firing rates and gamma power were included in the results (3 out of 5 mice, one mouse showed no firing rate change and one mouse showed no gamma change). In vehicle-treated knock- in mice, each of the 2 mice received 2 recording sessions with the probe placed at 2 different locations around the injections site.

[00211] Imaging data were processed and analyzed using ImageJ. Statistics were

performed using Microsoft Excel, GraphPad, and Matlab.

RESULTS

The LiGABAR Toolkit

[00212] The GABAA receptor has two GABA-binding sites, each at the interface of an a and β subunit (Figure 1A). LiGABAR is generated by conjugating a photoswitchable tethered ligand (PTL) onto a cysteine genetically engineered into the a-subunit near the GABA-binding site. The PTL molecule has three chemical modules (Figure IB): a cysteine-reactive maleimide (for receptor conjugation), an azobenzene (for

photoswitching), and a GABA-binding site ligand (for competitive antagonism). The azobenzene adopts an extended trans configuration in darkness and a twisted cis configuration in 360-400 nm light. The cis isomer slowly reverts to trans in darkness, but this can be accelerated with 460-560 nm light. Hence control is bi-directional.

Depending on where the PTL is attached, either the cis or the trans isomer antagonizes the receptor. Photoswitching to the alternative configuration alleviates antagonism (Figure 1A).

[00213] PTLs were previously developed with N-acyl muscimol as the parent ligand (Lin et al., 2014). While these compounds do impart light sensitivity on GABAA receptors, low efficacy limited the magnitude of photoswitching in vitro, and poor solubility (<50 μΜ) excluded their use in vivo. To improve efficacy, new PTLs were made with either GABA or its guanidinium analogs as the ligand. The diffuse positive charge of the guanidinium should enhance ionic, hydrogen-bond, and/or cation-π interactions with the receptor (Bergmann et al., 2013) and protonation at neutral pH should enhance water solubility (Figure 9A-9B). It is expected that these PTLs would be antagonists like other ester or amide derivatives of GABA (Matsuzaki et al., 2010).

[00214] The new PTLs were conjugated onto a series of cysteine mutants of al (Figure

9A-9B), co-expressed with wild-type β2 and γ2 in HEK-293 cells. The optimal combination of PTL and cysteine mutant was PAG-1C (Figure IB) tethered to residue 125 (alT125C; Figures 1C-1F and Figure 9A-9B). As expected, the GABA-elicited current was reduced in 500 nm {trans PTL) and restored completely in 380 nm light {cis PTL) (Figure 1C). Cis-to-trans photoisomerization reduced the response to half- saturating GABA by 78 + 2% (10 uM, n = 6; Figure IE), and to saturating GABA by 57 + 2% (300 μΜ, n = 6). Dose-response curves showed that the EC₅₀ increased from 15.3 + 6.0 μΜ (n = 6) to 583 + 139 μΜ (n = 4) when the PTL was converted from cis to trans (Figure ID), consistent with inducing competitive antagonism. Receptor activation was indistinguishable from wild-type with the PTL in the cis configuration (wild-type EC₅₀ = 9.5 + 2.3 μΜ, n = 7; p = 0.36, two-tailed t test). Taken together, the discovery of PAG- 1C for al-LiGABAR validates the PTL design and establishes effective photo-control of this receptor isoform.

[00215] The PTL strategy was applied to all other a-isoforms (a2-a6) to obtain the

complete LiGABAR toolkit. Cysteine mutants of a-subunits (focusing on loop E, where alT125C is located) were pairedwith a library of PTLs, and the resulting LiGABARs were evaluated in HEK-293 cells. These PTLs varied in their ligands (GABA, guanidinylated GABA, and guanidine acetic acid; Figure 9A-9B) and spacer lengths between the ligand and the azobenzene. For each isoform, the best PTL/mutant pair (Figure IE and IF and Figure 10A-19D) was selected based on two criteria: (1) GABA- elicited currents are robustly photo-controlled (preferably >50% photo-antagonism at EC₅₀), and (2) receptor function is unaffected by cysteine mutation and PTL conjugation.

[00216] Notably, a homologous mutation site was found that enables the reversed polarity of photo-control (i.e. antagonizing the receptor by cis PTL). When a longer PTL (e.g. PAG-2A, PAG-2B, or PAG-3C in Figure IB) is conjugated onto this site, GABA- elicited current is reduced in 380 nm light by 45-70% and is fully restored in 500 nm light (Figures IE and IF for α2-α6; 48 + 5% reduction by PAG-3C on alT121C, n = 3). Interestingly, some of the mutants enable either cis or trans mode of photo-antagonism when conjugated with a longer or a shorter PTL, respectively (e.g. a2 and a5

LiGABARs in Figure IE). This dual-option adds flexibility in whether or not the receptor will be turned off in the ground state (i.e. in darkness), an important

consideration for applications in neural circuits.

[00217] Even though all of the receptors have a cysteine point mutation, this change

appears to have minimal effects on receptor function, unless the PTL is conjugated and switched to the antagonizing configuration. None of the cysteine mutations, by themselves, alter receptor activation (Figure 10A-10D). Moreover, neither cysteine substitution nor PTL conjugation affects the characteristic properties of the parent receptor, such as allosteric modulation at the benzodiazepine site or anion permeability of the channel (Figure 10A-10D). Hence LiGABARs function as their normal receptor counterparts, until the moment they are photo-antagonized by a conjugated PTL.

[00218] Wild-type GABAA receptors, which lack a properly positioned cysteine near the

GABA-binding pocket, remain insensitive to light after PTL treatment (Figures 9A-9B and 1 lA-1 IF). Moreover, PTL treatment does not confer light sensitivity onto GABA_B receptors, glutamate receptors or voltage-gated Na⁺ and K⁺ channels (Figure 1 lA-1 IF), indicating that there are few, if any, acute off-target effects on proteins that govern the electrophysiology of a neuron.

Subcellular Mapping of GABAA Receptor Isoforms with Optogenetic

Pharmacology

[00219] Isoforms of GABAA receptors can be immunolocalized to distinct compartments of a dissociated neuron (Brunig et al., 2002), but subcellular localization can be problematic in intact neural tissue with intertwined cells. Moreover, antibody labeling cannot differentiate functional receptors from those that might be silent. Functional GABA receptors can be mapped with pinpoint accuracy via two-photon photolysis of "caged GABA" (Matsuzaki et al., 2010), but this cannot differentiate receptor isoforms. Optogenetic pharmacology with LiGABARs can overcome these limitations by allowing discrimination between functional receptor isoforms.

[00220] To validate this idea, the functional distributions of al- and a5-LiGABARs in hippocampal CA1 pyramidal neurons were mapped. The cysteine mutant of the al- or a5-subunit (alT125C or a5E125C) was virally co-expressed with GFP in a rat hippocampal slice. The transduced slice was then treated with PTL (PAG-1C), and fluorescent neurons were selected for whole-cell voltage-clamp recording. Responses to uncaged GABA were monitored when LiGABARs were either antagonized (by 540 nm) or relieved from antagonism (by 390 nm). By measuring the ratio of responses in these two conditions, the contribution of a particular a-isoform to the uncaging response, and control for potential sources of variability, were revealed. Other control experiments demonstrate that the two-photon uncaging response was unaltered by the conditioning light for receptor photo-control (Figure 12A, validated in the absence of the PTL), and that the two-photon light used for uncaging did not affect the state of the LiGABAR (Figure 12B).

[00221] A low-resolution view of where al- and a5-LiGABARs are present was obtained

(Figures 2A and 2B). Two locations were examined: one at or close to the soma (proximal site), and one on the primary apical dendrite (70-80 μιη from soma; distal site). Each uncaging site spanned 7-10 μιη. In cells expressing al-LiGABAR, photo switching (defined as the fraction of current antagonized by light) was more profound proximally than distally, with the effect decreasing from 0.47 + 0.02 at the proximal site to 0.11 + 0.07 at the distal site (p = 0.014, n = 5, paired t test; Figure 2A). In contrast, when a5-LiGABAR was expressed, photoswitching was not significantly different between the two sites (0.33 + 0.09 at the proximal site and 0.50 + 0.06 at the distal site; p = 0.271, n = 5, paired t test; Figure 2B). These results suggest that functionally active al- and a5-GABA_A receptors are differentially distributed, with al concentrated near the soma and a5 extending to more distal locations along the apical dendrite.

[00222] A higher resolution map was obtained of dendritic al- and a5-LiGABARs with smaller, more closely spaced uncaging spots (2.5 μιη, ~5 μιη apart; Figures 2D and 2E). It was found that the amplitude of the GABA-elicited current varied between these spots in neurons expressing either al or a5. Independent of this, however, there was a striking difference in the spatial pattern of photoswitching between these two isoforms (Figures 2D-2G). Photosensitivity appeared to be localized to "hot spots" for al (Figures 2D and 2F), but distributed evenly along the dendrite for a5 (Figures 2E and 2F). Group data show higher spatial variability of photoswitching for neurons expressing al-LiGABAR than for those expressing a5-LiGABAR, consistent with clustering of al-containing receptors (coefficient of variation: 0.59 for al vs. 0.18 for a5; p = 0.016, Levene's test, n = 22 and 18 uncaging sites from 5 and 6 cells, respectively).

[00223] Immunolabeling studies showed that the al isoform is concentrated at inhibitory synapses (Brunig et al. 2002; Kasugai et al., 2010). To verify that the photoswitching hot-spots of al-LiGABAR represent clusters of functional receptor at synapses, inhibitory synapses were targeted using a genetically-encoded fluorescent intrabody for gephyrin (a scaffolding protein that tethers GABAA receptors at synapses; Gross et al., 2013). Neurons expressing the gephyrin intrabody exhibit fluorescent puncta at postsynaptic sites. Significant photo switching of responses was found only when GAB A was uncaged at gephryin puncta (0.32 + 0.07 at puncta vs. -0.01 + 0.03 at ~4 μηι outside of puncta, n = 7 and 5 sites from 5 cells, respectively; p = 0.0008, paired t test; Figures 2H and 21). Hence by combining LiGABAR photo-control with two-photon uncaging, one can generate a functional map of a specific GABAA isoform on a neuron, resolved at the level of individual synaptic contacts.

Photo-control of Synaptic Inhibition with LiGABARs

[00224] It was tested whether LiGABARs can enable photo-control of inhibitory

postsynaptic currents (IPSCs). Mutant a-subunits were exogenously expressed by viral transduction in mouse cerebral cortex. Brain slices were treated with PTLs to generate LiGABARs. Monosynaptic IPSCs were evoked by electrical stimulation of local inhibitory inputs while blocking excitatory glutamate receptors.

[00225] When a LiGABAR that exhibits irans-antagonism (PAG-1C on al) was

employed, it was found that IPSC amplitude was 63 + 3% smaller in 500 nm light than in 380 nm light (p < 0.05, paired t test; n = 6; Figure 3A). When a LiGABAR that exhibits czs-antagonism (PAG-2A on a5) was used, the opposite effect was observed; IPSC amplitude was 52 ± 2% smaller in 380 nm light than in 500 nm light (p < 0.05, paired t test; n = 6; Figure 3B). Hence, synaptic inhibition can be photo-control with either polarity LiGABAR.

[00226] In principle, the amplitude of IPSCs can be changed by altering presynaptic

GAB A release or postsynaptic GABAA receptors. To verify that the observed effects are entirely postsynaptic, the paired-pulse ratio (PPR) at the two photo switching

wavelengths was compared. Changes in PPR would reflect changes in presynaptic release probability (Zucker and Regehr, 2002). It was found that PPR was the same under 380 and 500 nm illumination (0.9 ± 0.1 vs. 0.9 ± 0.1, p > 0.05, n = 11, paired t test), indicating that photo switching was entirely a postsynaptic phenomenon.

[00227] Extrasynaptic GABAA receptors mediate tonic inhibition, important for setting the tone of excitability in the brain (Farrant and Nusser, 2005). To test whether

LiGABARs enable photo-control of tonic inhibition, recordings from hippocampal pyramidal neurons expressing a5-LiGABAR (conjugated with PAG-1C) were made. To magnify GABA-mediated currents, neurons were clamped at 0 mV, far from EQ (-70 mV), and a small volume of artificial corticospinal fluid (aCSF; -30 mL) was recirculated to avoid wash-out of extracellular GABA. Under these conditions, a brief flash of 390 nm light caused an outward current increase of 52 ± 13 pA (n = 5) that was reversed by 540 nm light (Figure 3C). The effect of light was abolished after applying picrotoxin (100 μΜ), confirming that it was mediated by GABAA receptors.

[00228] The results suggest that viral expression of the LiGABAR mutant alone, in the absence of the photoswitch, did not significantly alter synaptic properties. The ratio of excitatory and inhibitory synaptic currents (E/I ratio) were compared in alT125C- expressing vs. non-expressing neurons in cortical slices. The E/I ratio was the same in mutant-expressing neurons and in control neurons, and there was no difference in the kinetics of IPSCs between the two groups (Figure 13A-13D). Taken together,

LiGABARs can be exogenously introduced into brain tissue without changing the balance between synaptic excitation and inhibition.

Kinetics of LiGABAR Photo-control

[00229] Optogenetic tools allow rapid manipulations of neuronal activities with temporal precision. To test the speed of LiGABAR photo-control, the minimal illumination time required for full IPSC photo switching was measured in CAl pyramidal neurons with al- LiGABAR. A flash of 540 nm (28 mW/mm²) or 390 nm (4.5 mW/mm²) light was applied 100 ms prior to presynaptic stimulation to antagonize or restore the receptor, respectively. LiGABAR was fully antagonized with a fixed duration of 540 nm light (500 ms) and restored receptor activity with various durations of 390 nm light (ranging from 10 ms to 500 ms; Figure 4A). Photo switching (relief of antagonism) increased with increasing duration of 390 nm light, and approached maximal (>95 %) with a 100-ms flash. The experiment was repeated with different durations of 540 nm light (and fixed 390 nm flashes; Figure 4A). In this case, photo switching (induction of antagonism) approached maximal with a 200-ms flash of 540 nm light.

[00230] It was tested whether rapid control of synaptic inhibition could change the spike output of a neuron in response to synaptic stimulation. Current-clamp recordings were carried out in CAl pyramidal neurons expressing al-LiGABAR. Shaffer collaterals were electrically stimulated, recruiting overlapping excitatory and inhibitory synaptic inputs that have opposite effects on spiking. Each stimulus elicited a single spike when LiGABAR was photoantagonized. The spike was eliminated when LiGABAR was relieved from antagonism. The spiking response could be gated with a flash of light as brief as 100 ms, delivered immediately before the presynaptic stimulus (Figure 4B). Collectively, these results (Figure 4 A and 4B) suggest that inhibition can be photo- controlled at a time scale of 100-200 ms. Because the speed of photo-control is largely determined by light intensity, LiGABAR manipulation may be accelerated further with a brighter light source.

[00231] LiGABARs can also be used as a bi-stable switch. To illustrate this feature, the

IPSC amplitude was monitored after transient conditioning with 380 nm or 500 nm light (Figure 4C). The IPSC amplitude was elevated by 380 nm light, and slowly decreased upon returning to darkness with a time constant of 30 + 6 min (95% confidence bounds: 26 + 4 min and 38 + 8 min; n = 4 cells). Exposure to 500 nm light quickly reduced the IPSC back to the initial amplitude, where it remained steady over 10 min. Hence, LiGABAR can be stably toggled between antagonized and antagonism-relieved states with brief flashes of conditioning light. This feature minimizes photo-toxicity and enables the use of other optical manipulations in the same experiment (e.g. GABA uncaging; Figure 2).

Spatial Reach of LiGABAR Photo-control in the brain

[00232] Before implementing LiGABAR in vivo, experiments were conducted to define how far the PTL and the light can penetrate through brain tissue to enable photo-control. It was determined how deep into the cerebral cortex the PTL can penetrate to form LiGABAR (Figures 5A-5C). To evaluate this parameter, the mutant a-subunit was expressed by stereotactically injecting a virus (encoding alT125C and eGFP) into mouse visual cortex. After 10-14 days, the mouse was anesthetized, and a craniotomy was performed to expose the cortex where neurons expressed the mutant receptor.

Following the subsequent duratomy, a droplet of aCSF containing the PTL (250 μΜ PAG-1C) was applied onto the exposed brain surface (Figure 5A).

[00233] After one hour of treatment, cortical slices were prepared and recordings from

GFP-positive neurons were made at various depths beneath the craniotomy region. The degree of IPSC photo switching was assessed as an index of LiGABAR formation. It was found that the degree of IPSC photo switching declined with the depth from the pia, decreasing from -40% near the surface to -0% at 400 μιη away from the surface (Figure 5B). This decline in IPSC photosensitivity could be fit with a single exponential function with a depth constant of 371 μηι (95% confidence bounds: 239 μηι and 824 μηι; n = 15 cells from 3 mice; Figure 5C).

[00234] A brain slice was used as a surrogate for intact brain tissue to evaluate how far the light can penetrate to photo-control LiGABAR (Figures 5D-5F). Acute cortical slices were prepared from virally transduced mice, and incubated the slices in PTL-containing aCSF to allow uniform receptor conjugation. In each neuron, the ratio of IPSC photo switching was measured under two different illumination conditions: first, with light projected directly into the slice axially from the pia surface and second, with light projected directly onto the slice in cross-section (Figure 5D). Axial illumination should photo-control LiGABAR maximally near the pia surface where light intensity is the highest. Cross-sectional illumination should photo-control LiGABAR uniformly, with variability attributable to other factors, such as differences in the expression of the mutant subunit. Hence the ratio of IPSC photoswitching by axial versus cross-sectional illumination reflects the efficiency of LiGABAR photo-control, calibrating for other factors that could cause cell-to-cell variation. It was found that IPSC photoswitching by axial illumination decreased from -41 % near the pia surface to ~11% at -400 μιη from the surface (Figure 5E). The depth-dependent decrease of photoswitching ratio (axial vs. cross-sectional) could be fit with a single exponential function with a depth constant of 352 μηι (95% confidence bounds: 255 μιη and 568 μιη; n = 12 cells from 3 mice; Figure 5F). These experiments utilized an unfocused light source for axial illumination, which emitted at -15 mW/cm for both wavelengths of light. A brighter or more focused light source, or an implanted optrode system, should allow an even deeper photo -control.

[00235] Taken together, these experiments suggest that both the PTL and the light can effectively reach as deep as -350 μιη from the brain surface, extending through layer 2/3 of the mouse cerebral cortex.

Photo-control of Cortical Visual Responses in vivo

[00236] Once it was established that both the PTL and the light can penetrate into brain tissue to control inhibition at a sufficient depth, it was tested whether photo-control is effective in vivo. Specifically, it was asked whether photo-control of LiGABAR could alter information processing in the primary visual cortex (VI) of a mouse as it is responding to a visual stimulus (Figure 6). The LiGABAR mutant was virally introduced into mice two weeks before the experiments. After the mouse underwent anesthesia, craniotomy, and PTL treatment, extracellular loose-patch recordings were made from LiGABAR-expressing, parvalbumin-positive (PV+) interneurons in layer 2/3 (Figures 6 A and 6B). It was confirmed that the visual stimulus, a 100% contrast drifting square grating, evoked spikes in the recorded neurons. To toggle LiGABAR between the antagonized and non- antagonized states, a full-field spot of conditioning light (390 nm or 470 nm) was delivered into the cortex through a microscope objective. Because LiGABAR is bi-stable (Figure 4C), a brief illumination of conditioning light (10 s) was sufficient to switch the receptor state for several minutes. This provided a time window for any spurious response to the conditioning light to decay before the onset of the visual stimulus.

[00237] It was found that the pattern of spiking in PV+ neurons, during the visual

response, changed from burst-firing after conditioning with 470 nm light (antagonism induced) to sustained-firing after conditioning with 390 nm light (antagonism relieved; Figure 6C). Moreover, the average increase in spike rate during the visual stimulus was larger when LiGABAR was antagonized. Changes in spike rate evoked by the visual stimulus could be modulated up and down repeatedly by switching the conditioning light back and forth (n = 7 cells from 4 mice, p < 0.05, one-way ANOVA; Figure 6D).

Control experiments showed that neither the LiGABAR mutant alone nor the PTL alone enabled photo-control of visual responses (n = 6 cells from 2 mice, p > 0.05, one-way ANOVA; PTL alone, n = 9 cells from 2 mice, p > 0.05, one-way ANOVA; Figure 6D). Taken together, these results show that LiGABAR can be introduced into a mouse brain for in vivo photo-control. Furthermore, these findings support the notion that

GABAergic inhibition in PV+ neurons plays a role in information processing in the visual cortex, such as setting the gain and determining the temporal dynamics of the visual response (Katzner et al. 2011).

Genomic Substitution of a Wild-type GABAA a-isoform with the Photoswitch- Ready Mutant in a Knock-in Mouse

[00238] The results described herein suggest that in cortical pyramidal neurons, over- expression of a mutant a-subunit causes no significant changes in IPSC kinetics or E/I ratio (Figure 13A-13D). However, unadulterated expression in all neurons can only be assured by replacing the gene encoding the wild-type a-subunit with its mutant counterpart. [00239] To bring about exact genomic substitution, a knock-in mouse was generated in which a single point mutation (T125C) was introduced into the gene of the al-subunit through homologous recombination. This mouse was named the al-GABAswitch mouse. Immunohistochemical analysis confirmed that the expression pattern of the mutant al was identical to that of the wild-type. Immunolabeling profiles through tissue slices from cerebral cortex, hippocampus, and cerebellum (Figures 7A-7C) were the same for the al-GABA_A PhoRM mouse as for the wild-type.

[00240] Functionally, the expression of alT125C was examined by measuring IPSC

photo switching in PAG-1C treated brain slices. Photoswitching was compared in neuronal cell types that differ in the relative abundance of al with respect to other a isoforms (Figures 7D and 7E). Cell types thought to express only the al-isoform

(cerebellar molecular layer interneurons (MLIs) and Purkinje cells (PCs) were used; Eyre et al., 2012; Fritschy et al., 2006), a cell type that expresses al along with other isoforms (pyramidal neurons in layer 5 of cerebral cortex (L5 PYNs); Ruano et al., 1997), and a cell type devoid of al (cerebellar Golgi cells (GoCs); Fritschy and Hohler, 1995). Photoswitching was the strongest in MLIs and PCs (51 ± 2% and 50 ± 2%, n = 7 and 6 cells from 2 and 3 mice, respectively), intermediate in L5 PYNs (30 ± 2%, n = 6 cells from 2 mice), and non-existent in GoCs (-2 ± 3%, n = 5 cells from 3 mice). Hence the degree of photoswitching is correlated with the relative abundance of al in a neuron. Photo-control of Sensory Responses and Gamma Oscillations in the al-GABA_A PhoRM Mouse

[00241] Understanding the role of inhibition in the cortex has often relied on non-specific blockers or antagonists of GAB A A receptors. The al-GABA_A PhoRM mouse provides the unprecedented opportunity to selectively and reversibly remove a particular endogenous receptor from a functional neural circuit both in vitro and in vivo. A multi- electrode probe was used to record extracellular spiking activity in neurons in the visual cortex of the awake al-GABA_A PhoRM mouse. The PTL was applied by intracranial infusion through a micropipette inserted -275 μιη into the cortex (Figure 8A), an alternative approach to topical application on the brain surface.

[00242] The response of neurons to a visual stimulus train, which consisted of 10 full contrast checkerboard images, was examined. Brief conditioning flashes were applied to switch al-LiGABAR 5 sec before each episode of the stimulus train. In many neurons (15/43 cells in 3 PTL-treated mice, p < 0.05, Friedman test over episodes; Supplemental Information), conditioning flashes that either induced or relieved antagonism reliably changed visually-evoked spiking activity. Owing to its non-homogenous distribution pattern in the brain (Figure 7; Fritschy and Mohler, 1995), it was surmised that photo- controlling al-LiGABAR might result in heterogeneous effects on cortical neurons. Indeed, some neurons showed a significant increase in firing rate after photo-antagonism (Figure 8B, top), while other neurons showed a significant decrease (Figure 8B, bottom). Photo switching occurred in a larger fraction of fast-spiking neurons (FS cells; 12/28) than regular spiking neurons (RS; 3/15) (Figure 8C; see classification of FS and RS cells in Figure 15). In control mice infused with vehicle alone, only 1/28 FS cells and 1/16 RS cells exhibited photosensitivity (2/44 cells in 2 mice, Friedman test over episodes, p < 0.05), confirming that spike modulation was specifically a consequence of LiGABAR photo-control.

[00243] FS cells have been identified as mostly PV+ interneurons (Averamnn et al.,

2012), which express a high level of al-containing receptors (Hu et al., 2014), whereas RS cell are largely pyramidal neurons, which express multiple a isoforms (Bosnian et al, 2002). The bimodal effect of light is consistent with the inhibitory microcircuit of the cortex, which includes an extensive network of interneuron-interneuron synaptic connections. Hence spike rate in an interneuron will tend to decrease when its own

GABAA receptors are more active, and increase when GABAA receptors on presynaptic interneurons are more active. Understanding when and where direct inhibition or disinhibition dominates in the circuit is an important question that LiGABAR will help to answer.

[00244] Gamma (γ) oscillations are thought to be mediated primarily by reciprocal

interactions between excitatory and inhibitory (E-I) neurons or by reciprocal interactions within networks of inhibitory neurons (I-I) (Bartos et al., 2007; Buzskai and Wang, 2012). Consistent with a crucial role for GAB A, Non-selective blockade of all GABAA receptor isoforms dampens the γ-oscillation (Welle, 2010). Surprisingly, the opposite effect was observed when GABAA receptors that contain the al subunit were specifically photo-antagonized: enhancement of γ-power (increase of 28 ± 10%, n = 3, Friedman test over episodes, p < 0.05; Figures 8D and 8E). Experiments on control mice infused with vehicle alone showed no significant change in γ-power (increase of 2 ± 1%, n = 4, Friedman test over episodes, p < 0.05; Figures 8D and 8E). Inhibitory synapses between PV cells (I-I connections) are highly enriched with al-containing receptors (Klausberger et al., 2002). Hence these results support a crucial role of I-I in γ-rhythmogenesis.

[00245] Figure 1A-1F. Optogenetic Toolkit for the GABA_A Receptor Family (A) The operating principle of LiGABAR. A PTL is conjugated onto the a-subunit near the GABA-binding site. Photoisomerization with different wavelengths antagonizes or allows GABA binding, thereby controlling whether the receptor can be activated. (B) PTLs consist of a cysteine-reactive maleimide group, a photosensitive azobenzene core, and a GABA-site ligand (blue; linked to azobenzene directly or via a short spacer). (C) Photo-control of a representative LiGABAR (PAG-IC conjugated alT125C, co- expressed with the wild-type β2 and y2S). Currents were elicited by 30 μΜ GABA in 380 nm (purple) or 500 nm (green) light. (D) al-LiGABAR functions like the wild-type receptor in 380 nm and is strongly inhibited in 500 nm. Data points are mean + SEM. Dose-response curves are fits to the Hill Equation. Black: wild-type, 7 cells; purple: LiGABAR/380 nm, 6 cells; green: LiGABAR/500 nm, 4 cells. (E) Quantification of LiGABAR photosensitivity for each a-isoform. Currents were elicited by GABA at -EC50 of the wild-type receptors (see [GABA]_test values listed in Figure 10A-10D). Photosensitivity is percent decrease of peak current by photo-antagonism. Data are plotted as mean + SEM (n = 4-6). (F) The PTL attachment site for each a-isoform. The sequences of loop E in rat al-a6 subunits are aligned. Cysteine substitution sites are shown in bold orange. C-E were recordings from HEK-293 cells voltage-clamped to -70 mV.

[00246] FIG. 2A-2I. Mapping Subcellular Distributions of Specific GAB A A Isoforms

(A-C) Low-resolution mapping of al- and a5-LiGABARs (both antagonized by trans PAG-IC). (A) Left: Neuron (filled with Alexa Fluor 594) expressing al-LiGABAR. Red boxes indicate the proximal (prox) and distal (dist) locations for two-photon RuBi- GABA uncaging (800 nm, 5-10 ms). Scale bar = 10 μιη. Right: Currents elicited by uncaging at 2 min after a 5-sec flash of 390 nm (purple) or 540 nm (green) light. Scale bars = 20 pA, 200 ms. Photo switching is less at the distal site. (B) Measurements from a neuron expressing a5-LiGABAR. Note that photo switching is large at the distal site. (C) Group data of photoswitching at proximal and distal sites (5 cells for each isoform). (D- G) Higher-resolution mapping of al- and a5-LiGABARs along the apical dendrites. (D) Top: Soma and proximal dendrite from a neuron expressing al-LiGABAR. RuBi- GABA was uncaged at 7 sites (each spanning 2-3 μιη) along the dendrite. Bottom:

Currents elicited at each site after 390 nm (purple) or 540 nm (green) conditioning flashes. Scale bars = 50 pA, 500 ms. (E) Measurements from a neuron expressing a5- LiGABAR. Note the more uniform photoswitching across the entire region. (F)

Photoswitching (mean + SEM) quantified for each uncaging site shown in panels D and E. (G) Photoswitching values pooled from 22 and 18 uncaging sites in neurons expressing al- and a5-LiGABAR, respectively (5 cells each). (H-I) Probing al- LiGABAR localization to inhibitory synapses in cultured hippocampal neurons co- expressing al-LiGABAR and GFP-fused gephyrin intrabody. Two-photon uncaging of RuBi-GABA at single pixel resolution, either at GFP-positive puncta (p) or at adjacent GFP-negative locations (np). (H) Representative images and recording traces. Cells were filled with Alexa Fluor 594 (red). GFP-positive puncta (yellow) are inhibitory synapses. Scale bars: 2 μιη (images) and 2 pA, 100 ms (traces). (I) Group data (5 cells) showing that photoswitching of al-LiGABAR is detectable only at GFP-positive puncta. Neurons were held at 0 mV. Traces are averages from 3-5 trials. Photoswitching is the fraction of current photo-antagonized. For panels C and I, individual measurements (average of each site) are open symbols, mean values for each group are filled symbols (error bars = SEM).

[00247] Figure 3A-3C. LiGABARs Enable Photo-control of Synaptic and Tonic

inhibition. (A) Photo-control by a irans-antagonist (PAG-1C, conjugated to al-T125C mutant). (B) Photo-control by a czs-antagonist (PAG-2A, conjugated to a5E125C). Left: representative traces. Right: Changes in peak IPSC amplitudes in darkness (white), 380 nm (purple), and 500 nm light (green). (C) Photo-control of tonic currents (PAG- 1C, conjugated to a5E125C) in a CA1 pyramidal neuron. Light intensity was 4.5 mW/mm 2" for 390 nm and 28 mW/mm 2" for 540 nm. Current levels were sustained after light flashes due to the bi-stability of LiGABAR (see Figure 4C). Photo-control was abolished after all of the GABAA receptors (including a5-LiGABAR) are blocked by picrotoxin (100 μΜ). Error bars = SEM.

[00248] Figure 4A-4C. Kinetics of LiGABAR Photo-control. (A) Purple: Illumination time required for restoring LiGABAR from antagonism. Pairs of IPSCs were recorded, one measured with a fixed duration of 540 nm (500 ms) and the other with a variable duration of 390 nm. Green: Illumination time required for imposing LiGABAR antagonism. The same measurements were made with a fixed duration of 390 nm (500 ms) and variable durations of 540 nm. Conditioning flashes were delivered 100 ms prior to synaptic stimulation. Inset: Representative photosensitive IPSC component (IPSC₃9₀ - IPSC₅₀₀) from the same neuron receiving different durations of conditioning light.

Fractional photo switching (i.e. normalized amplitude of photo-controlled IPSC) over different duration flashes were plotted (symbols; mean + SEM, n = 2-4) and fit with a single exponentials (curves). (B) Photo-control of synaptically-stimulated action potential firing with a brief flash of light. With the neuron at rest (around -70 mV;

current-clamp), a brief flash of conditioning light (colored squares) was applied 100 ms prior to Schaffer-collateral stimulation (triangles). A 100-ms flash of each conditioning light was sufficient for photo-controlling spike generation. Scale bar = 20 mV, 1 s. (C) Bi-stability of LiGABAR. Prior to illumination, LiGABAR was antagonized by the trans-PTL in darkness (a). The amplitude of IPSC increased upon the illumination of 380 nm (b), which then decreased slowly in darkness after the conditioning light was turned off (c). The amplitude of IPSC reduced to the initial level upon 500 nm

illumination (d), which remained steady in darkness thereafter. The time course of IPSC decrease in darkness (post-380 nm) is fitted with a single exponential decay (red curve; τ = 30 + 6 min) to depict the thermal relaxation of czs-PTL.

Figure 5A-5F. Accessible Depth of LiGABAR Photo-control from the Surface of the Brain. (A) Strategy for measuring the penetration depth of a PTL into an intact brain. (B) Map of the depth-dependence of IPSC photoswitching. Each point indicates the location of a recorded cell in cortical layers (L1-L5), with the magnitude of IPSC photoswitching color-coded. (C) Depth-dependent decrease in IPSC photoswitching (n = 15 cells). The data were normalized and fit with an exponential decay (Supplemental Information) to calculate the depth constant (λ) of PTL penetration from the brain surface. (D) Strategy for estimating the penetration depth of light into the brain. The axial light mimicked the in vivo illumination (with light penetrating into the brain from the pia surface). The cross-sectional light photo-controlled LiGABARs regardless of the cell position, providing a scale factor for estimating the effectiveness of the axial light. (E) Depth-dependence of IPSC photoswitching, with either axial or cross-sectional illumination. (F) Depth-dependent decrease in IPSC photoswitching. The data (ratio of axial vs. cross-sectional photo switching from 12 cells) were normalized and fit with an exponential decay to calculate the depth constant (λ) of photo switching from the brain surface. The virus used in these experiments encodes mutant alT125C and eGFP. PTL = PAG-1C.

[00250] Figure 6A-6D. In vivo Photo-control of Visual Responses in the Mouse Cortex.

(A) Schematic illustration of the experimental procedures. (B) Two-photon image of a recorded PV+ neuron. The cell was identified by the co-expression of tdTomato (red, marker of PV+ cell) and eGFP (green, marker of LiGABAR expression). (C)

Experimental sequence. The raster plots and peristimulus time histograms show the spike activity of a PV+ neuron before any conditioning illumination (black), and after 10 sec exposure to either 390 nm (purple) or 470 nm (green) light. Note that the

conditioning lights were delivered between visual stimulation trials. (D) Summary of visually evoked spike activities in PV+ neurons (circles), showing higher firing rate when LiGABAR is antagonized (dark and 470 nm) than is relieved from antagonism (390 nm). n = 7 cells from 4 mice. Control experiments with PTL treatment alone (squares; n = 6 cells from 2 mice) or viral injection alone (triangles; n = 9 cells from 2 mice) show no significant difference in spike activities after exposure to 390 nm vs. 470 nm light. Data are plotted as mean + SEM.

[00251] Figure 7A-7H. Characterizations of the OII-GABAA Photo switch-ready Mutant

(PhoRM) Knock-in Mouse (A-C) Fluorescent images of antibody labeling showing the expression pattern of the al-subunit in the wild-type and the homozygous OII-GABAA PhoRM mice in visual cortex (A), hippocampus (B), and cerebellum (C). (D-F)

Quantification of al expression in different brain regions. Fluorescence intensity (F. I., in arbitrary unit) profile was measured along the yellow dash arrows in (A-C), showing similar expressing patterns between the wild-type and the OII-GABAA PhoRM mice in all of the three brain regions analyzed. In each genotype, the profiles were obtained from 2-3 sections in each mouse (2 wild type and 3 knock- in mice). (G) Representative recording traces from a cerebellar molecular layer interneuron and a Golgi cell of the al- GABAA PhoRM mouse, showing differential photo-control of IPSC in these cell types. (H) Scatter plots summarizing the magnitude of IPSC photoswitching in different types of neurons. CB: cerebellum. MLI: molecular layer interneuron. PC: Purkinje cell. GoC: Golgi cell. PYN: pyramidal neuron. [00252] Figure 8A-8E. In vivo Photo-control of Visually-evoked Responses and Gamma

Oscillations in the Awake OII-GABAA PhoRM Mouse (A) Schematic illustration of the experimental procedures. (B) Top: A neuron with an increased firing rate when al- LiGABAR was antagonized (green points), compared to its firing rate when LiGABAR was relieved from antagonism (purple points). Bottom: A neuron with a decreased firing rate when al-LiGABAR was antagonized. (C) Average firing rates from A) and B) in each illumination condition (20 episodes per condition). Data are mean + SEM. Error bars are SEM of each episode in the left panels and are SEM of each condition in the right panels. (C) Summary plot all of the cells recorded in PTL-treated (PTL) and vehicle-treated (Ctrl) (XI-GABAA PhoRM mice. The number of cells that have significant photo switching in firing rate is shown in blue for the PTL group and in red for the Ctrl group. See Figure 15 for the classification of FS (fast spiking) and RS (regular spiking) cells. (D) Example power spectrum of local field potential in one of the PTL-treated (XI-GABAA PhoRM mice. Photo-antagonizing al-LiGABAR in vivo (green) increased the power of visually-evoked γ oscillation, compared to the γ-power when antagonism is relieved (purple). Data are plotted as mean + SEM for each illumination condition. (E) Example recording of γ-power (averaging between 20-60 Hz) in episodes when al-LiGABAR was antagonized (green points) and in those when the receptor was relieved from antagonism (purple points). Data are plotted as mean + SEM for each episode.

[00253] FIG. 9A-9B. The design and screening of new LiGABARs. (A) The chemical modules comprising a photo switchable tethered ligand (PTL) and the proposed model of PTL-mediated receptor antagonism. The top panel illustrates a LiGABAR occupied by its conjugated PTL. PTL conjugation is achieved via a Michael-addition reaction between the maleimide (of the PTL) and the sulfhydryl group (of the cysteine residue). Putative PTL conjugation sites (yellow sticks; i.e. residues subject to cysteine substitution) on the al-subunit (blue ribbon) are shown in a homology model (O'Mara et al., 2005). The docking of GABA (spheres) in this receptor model suggests that the carboxyl group of GABA faces toward the complementary face of the al-subunit. Thus an optimized scaffold (maleimide + cis or trans azobenzene + spacer; thick brown arrow in the model) can efficiently deliver the ligand moiety (GABA or its analogue) into the binding pocket once conjugated at an appropriate location. The new PTLs are soluble in water due to their positive charges formed at physiological pH. Note that the negatively charged carboxylate groups (at physiological pH) of the free ligands will be converted into neutral amide groups in the PTLs. Because this negative charge is critical for receptor activation, the tethered ligands are expected to antagonize rather than activate the receptor. Finally, the guanidinium groups of GABA analogues are expected to form more ionic and/or hydrogen-bond interactions inside the binding pocket, compared to the ammonium group of GABA, and enhance receptor antagonism. The optimal

LiGABARs (PTL+mutant) for individual a-isoforms are listed in Figures IE and IF. (B) Screening of PAG-1C conjugation sites for the al-isoform. The al subunit (wild-type or one of the six cysteine-substituted mutants indicated in panel A) was co-assembled with β2 and y2S subunits in HEK cells, and the cells were treated with PAG-1C prior to electrophysiological evaluations. Currents elicited by 10 μΜ GABA in either 380 nm or 500 nm light were recorded. Photosensitivity of PAG-1C treated receptors was indexed as the ratio of peak current amplitude in 380 nm vs. in 500 nm (i.e. Lsonm/Isoonm)- A ratio of 1 indicates that the treated receptor was not functionally affected by light switching. The results show that receptor photosensitization requires not only the presence but also the proper location of a cysteine in the al subunit. Whole-cell voltage-clamp recordings were carried out with cells held at -70 mV. Data are plotted as mean + SEM (n = 3-6).

FIG. 10A-10D. Receptor functions are not affected by cysteine mutation or PTL conjugation. (A) Defining testing conditions based on the EC50 of GABA for wild-type receptors. The dose-response curve of each wild-type a-isoform (co-assembled with wild-type β2 and y2S) is plotted by fitting the respective grouped data (mean + SEM; n = 4-7) with Hill Equation. The GABA EC50 for each isoform is calculated as mean + SEM from 4-7 individually analyzed cells. Details of data analysis are described in "Data Analysis" of the Supplemental Experimental Procedures. [GABA]_test, the concentration of GABA used for indexing LiGABAR photosensitivity (Figure IE) and receptor activation (panel B, see below), was set to be -ECso of the wild-type receptor. (B) Receptor activation is not affected by cysteine mutation or PTL conjugation. For each isoform, the impacts of receptor modifications were assayed by comparing the fraction of receptor activation at [GABA]_test among the wild-type (blue), the untreated mutant (orange), and the PTL-conjugated mutant receptors (dark red; measured when the receptor was not photo-antagonized). The fraction of receptor activation is defined as the ratio of peak current amplitude at [GABA]_test vs. at a saturating concentration (i.e. [GABA]r_ef in panel A). Data are plotted as mean + SEM (n = 3-7). For a2 and a5, the PTL used for LiGABAR was PAG-1C. The p value for each a-isoform is >0.05 (two- tailed t test) when comparing the untreated mutant or uninhibited LiGABAR with the wild-type. (C) Channel permeability to bicarbonate (relative to chloride) is not affected by cysteine mutation or PTL conjugation. The relative permeability (P_HCO3-/P_CI-) through wild-type alp2y2S, al(T125C)p2y2S, and al-LiGABAR (uninhibited) was measured in HEK cells using a previously described method (Wotring et al., 1999). In brief, reversal potential was measured in a modified saline with 75 mM of NaCl (of the control saline, i.e. regular extracellular solution) replaced by NaHC0₃. P_HCO3-/P_CI- was calculated using a rearranged Goldman-Hodgkin-Katz equation: P_HCO3-/P_CI- = { [CI ]iexp(-V_revF/RT) - [Cr]₀}/[HC031_o, where [CL]i is the intracellular chloride concentration, V_rev is the reversal potential, [CL]₀ is the extracellular chloride concentration, and [HC03 ]₀ is the extracellular bicarbonate concentration. F, R, T are Faraday's constant, gas constant, and temperature, respectively. The results show that the reversal potentials in control and modified saline, as well as P_Hco₃- Pci-, are not altered by cysteine mutation or PTL conjugation (p >0.05, two-tailed t test). Data are shown as mean + SEM (n = 4-6). (D) Receptor modulation through the benzodiazepine site is not altered by cysteine mutation or PTL conjugation, (left) Representative recording traces showing receptor inhibition mediated by DMCM, an inverse agonist targeting the benzodiazepine site. A 20-s pre-application of DMCM was carried out prior to the co-application of GABA and DMCM. When testing on al-LiGABAR, a 5-s flash of 380 nm light was illuminated before the application of GABA or DMCM (to relieve LiGABAR from photo-antagonism), (right) Group data (mean + SEM; n = 4-5) showing that there is no difference in DMCM-mediated inverse agonism between wild- type alp2y2S, al(T125C)p2y2S, and al-LiGABAR (p >0.05, two-tailed t test).

Recordings for panels A, B, and D were carried out in HEK-293 cells held at -70 mV.

FIG. 1 lA-1 IF. PTL treatment does not confer light sensitivity onto endogenous ion channels in hippocampal neurons. (A) PAG-1C treatment does not photosensitize endogenous GABAA receptors in hippocampal slices. Representative evoked IPSC traces are shown. I500 I380 = 1.00 + 0.03 (in peak amplitude; V_hoi_d = 0 mV; n = 5, p = 0.89). (B) PAG-1C treatment does not photosensitize endogenous GABA_B receptors in cultured hippocampal neurons. Representative GIRK currents elicited by 10 μΜ (R)- baclofen are shown. I500/I380 = 1.00 + 0.04 (V_hoi_d = -70 mV; n = 4, p = 0.90). (C) PAG- 1C treatment does not photosensitize endogenous AMPA receptors in hippocampal slices. Representative evoked EPSC traces are shown. I500/I380 = 1.01 ± 0.01 (in peak amplitude; Vhoid = -60 mV; n = 4, p = 0.16). (D) PAG-1C treatment does not photosensitize endogenous NMDA receptors in hippocampal slices. Representative evoked EPSC traces are shown. I500/I380 = 0.97 + 0.05 (in peak amplitude; Vhoid = +40 mV; n = 3, p = 0.59). (E) PAG-1C treatment does not photosensitize endogenous voltage-gated Na⁺ channels in hippocampal slices. Representative inward currents (elicited by a 200-ms depolarization step from -60 to -10 mV) are shown. I500/I380 = 1.00 + 0.01 (in peak amplitude; n = 11, p = 0.72). (F) PAG-1C treatment does not photosensitize endogenous voltage-gated K⁺ channels in hippocampal slices.

Representative outward currents (elicited by a 200-ms depolarization step from -60 to +40 mV) are shown. I500/I380 = 0.997 + 0.0041 (steady-state amplitude; n = 5, p = 0.41). Recordings for panels A, C, D, E, and F were carried out in CA1 pyramidal neurons. Recording traces acquired under 380 nm and 500 nm illumination are plotted in purple and green, respectively. Group data of I500/I380 are reported as mean + SEM, and p values were calculated from two-tailed t tests. Additional details of PTL treatment and electrophysiology are described in the Supplemental Experimental Procedures.

FIG. 12A- 12B . LiGABAR photo switching can be paired with two-photon GABA uncaging. (A) Conditioning light for LiGABAR photoswitching can be orthogonal to GABA uncaging. Whole-cell voltage-clamp recordings were carried out from hippocampal pyramidal neurons held at 0 mV, in the presence of 25 μΜ DNQX, 50 μΜ AP5, and 0.5 μΜ TTX. Responses to two-photon photolysis of RuBi-GABA (by 800 nm) were identical before (back arrows) and after (blue arrows) a conditioning flash (390 nm; purple bar). Lite/ ore = 1.02 + 0.03, n = 4 cells, p = 0.65. The conditioning flash caused some photolysis of RuBi-GAGA, but the released GABA can diffuse away in 0.5-2 min after the flash. Scale bars = 500 pA, 1 s. (inset) Magnification of uncaging responses before (black) and after (blue) conditioning flash. Scale bars = 25 pA, 500 ms. (B) Two-photon illumination for GABA uncaging does not affect the state of the LiGABAR. Current amplitudes from an example cell over multiple trials in response to RuBi-GABA photolysis. Response amplitude was stable after photo-antagonism was relieved (by a 390-nm conditioning flash (purple bar); Iiast I&st = 0.94 + 0.06, n = 6 cells, p = 0.35) or triggered (by a 540-nm conditioning flash (green bar); Iiast I&st = 1.01 ± 0.03, n = 6 cells, p = 0.89). Currents are normalized to the mean amplitude after 390 nm illumination. Whole-cell voltage-clamp recordings were carried out from hippocampal pyramidal neurons expressing a5-LiGABAR (conjugated with PAG-1C; trans- antagonism). Cells were held at 0 mV and treated with 25 μΜ DNQX, 50 μΜ AP5, and 0.5 μΜ TTX. Iafte/Ibefore (panel A) and Iiast I&st (panel B) are reported as mean + SEM; p values are calculated from two-tailed t tests.

[00257] FIG. 13A-13D. Exogenously expressing a cysteine mutant of GABAA receptors does not significantly change neuronal excitability or the kinetics of inhibitory postsynaptic currents. (A) Fluorescence image of a cortical slice prepared from a mouse injected with a bi-cistronic AAV encoding alT125C and eGFP. Virally infected neurons were identified by the green fluorescence. (B) Recording traces from layer 5 pyramidal neurons expressing (GFP+) or not expressing (GFP-) alT125C. Postsynaptic excitatory and inhibitory currents were evoked by electrical stimulation, with each neuron voltage-clamped at -65 mV and 0 mV, respectively. (C) The ratio of excitatory vs. inhibitory currents (E/I ratio; mean + SEM) in alT125C-expressing neurons (green; 0.32 ± 0.06, n = 6) is not significantly different from that in control neurons (black; 0.34 ± 0.08, n = 6; p > 0.05, two-tailed t test). (C) IPSCs in alT125C-expressing neurons (green) and control neurons (black) showed no significant differences in either the rise time or decay time constant. GFP+ vs. GFP- neurons: 20-80% rise time, 1.32 + 0.26 ms vs. 1.52 + 0.37; decay time constant, 11.9 + 0.9 ms vs. 13.5 + 0.8 ms, n = 6 and 6, respectively; p > 0.05, two-tailed t test.

[00258] FIG. 14. Strategy of gene targeting for generating the alT125C knock-in mouse.

The genomic region of Gabral (NMJ310250.4) was obtained from BAC clone RPCI-24 and was used to develop a targeting vector that contains the genomic region surrounding exons 5 and 6 of Gabral . A cysteine mutation was introduced to T 152 (counting from the start codon, i.e. T125 in the mature peptide sequence) on exon 5 as well as a C to T silent mutation to create a Hind III site upstream of T152C for genotyping. A loxP site flanked neomycin gene and a diphtheria toxin A-chain (DTpA) gene were introduced in the targeting vector upstream of exon 5 for positive-negative ES cell selection. [00259] FIG. 15. Separation of FS and RS neurons. £-means clustering was used to categorize all of the recorded units. Top and bottom panels show the scatter plot of the spike width and the asymmetry ratio from all units recorded in PTL- treated and vehicle- treated knock-in mice, respectively. Inset in the bottom left illustrates the definition of spike width (S) and amplitude (A) of peaks to construct the plot (detailed in Data analysis section). The average waveforms of all FS (red) or RS (blue) units in each group are shown in the top and bottom right insets. In the PTL group, FS and RS cells have S of 0.30 + 0.021 and 0.57 + 0.043 ms, respectively. In the vehicle group, FS and RS cells have S of 0.31 + 0.016 and 0.60 + 0.037 ms, respectively. Data are mean + SEM

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[00304] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A light-regulated gamma amino butyric acid (GAB A) receptor (GABAA receptor), the light-regulated GABAA receptor comprising:

a) an alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain;

b) a photoswitchable group of the formula: linker-azobenzene-R,

wherein R is a ligand for the GABAA receptor, and

wherein the photoswitchable group is covalently linked to the cysteine via the linker.

2. The li ht-regulated GABAA receptor of claim 1, wherein R is selected from:

3. The light-regulated GABAA receptor of claim 1, wherein the alpha chain is an isoform 1 alpha chain, and wherein the cysteine substitution is a T153C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO: l.

4. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain is an isoform 2 alpha chain, and wherein the cysteine substitution is a Q149C substitution, based on the amino acid numbering of the isoform 2 alpha chain amino acid sequence set forth in SEQ ID NO:3.

5. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain is an isoform 3 alpha chain, and wherein the cysteine substitution is a V174C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO:5.

6. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain is an isoform 4 alpha chain, and wherein the cysteine substitution is a M155C substitution, based on the amino acid numbering of the isoform 4 alpha chain amino acid sequence set forth in SEQ ID NO:7.

7. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain is an isoform 5 alpha chain, and wherein the cysteine substitution is an E156C substitution, based on the amino acid numbering of the isoform 5 alpha chain amino acid sequence set forth in SEQ ID NO:9.

8. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain is an isoform 6 alpha chain, and wherein the cysteine substitution is a M139C substitution, based on the amino acid numbering of the isoform 6 alpha chain amino acid sequence set forth in SEQ ID NO: l l.

9. The light-regulated GABAA receptor of claim 1 , wherein the alpha chain comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:2, 4, 6, 8, 10, and 12.

10. The light-regulated GABAA receptor of claim 1 , wherein, when the ligand is PAG- 1C and when the GABAA receptor comprising the alpha- 1 isomer is exposed to light of a wavelength of from 480 nm to 520 nm, the ligand is in the extended trans isomer configuration and binds to the ligand-binding site of the alpha chain, and inhibits GABA-elicited current mediated by the receptor, and wherein, when the GABAA receptor is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the cis isomer configuration and GABA- elicited current mediated by the receptor is restored.

11. The light-regulated GABAA receptor of claim 1, wherein, when the ligand is PAG-2A, PAG-2B, or PAG-3C, and when the GABAA receptor is exposed to light of a wavelength of from 360 nm to 400 nm, the ligand is in the extended trans isomer configuration and binds to the ligand-binding site of the alpha chain, and inhibits GABA-elicited current mediated by the receptor, and wherein, when the GABAA receptor is exposed to light of a wavelength of from 480 nm to 520 nm, the ligand is in the cis isomer configuration and GABA- elicited current mediated by the receptor is restored.

12. An isomerizable light-responsive ligand for a GABAA receptor, wherein the ligand is of the formula: maleimide-azobenzene-R,

wherein R is a ligand for the GABAA receptor.

13. The isomerizable li ht-responsive ligand of claim 12, wherein R is selected from:

14. A method of modulating the activity of a GAB A A receptor, the method comprising exposing the GABAA receptor to light, wherein the GAB AA receptor comprises: a) an alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue at a surface accessible site in the alpha chain; b) a photoswitchable group of the formula: linker-azobenzene-R,

wherein R is a ligand for the GABAA receptor, and

wherein the photoswitchable group is covalently linked to the cysteine via the linker, wherein the light is of a wavelength in the range of from 360 nm to 400 nm, or wherein the light is of a wavelength in the range of from 480 nm to 520 nm.

15. The method of claim 14, wherein R is selected from:

o o PAG-2C; and

16. A variant GABAA receptor alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain.

17. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 1 alpha chain, and wherein the cysteine substitution is a T153C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO: l.

18. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 2 alpha chain, and wherein the cysteine substitution is a Q149C substitution, based on the amino acid numbering of the isoform 2 alpha chain amino acid sequence set forth in SEQ ID NO:3.

19. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 3 alpha chain, and wherein the cysteine substitution is a V174C substitution, based on the amino acid numbering of the isoform 1 alpha chain amino acid sequence set forth in SEQ ID NO:5.

20. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 4 alpha chain, and wherein the cysteine substitution is an M155C substitution, based on the amino acid numbering of the isoform 4 alpha chain amino acid sequence set forth in SEQ ID NO:7.

21. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 5 alpha chain, and wherein the cysteine substitution is an E156C substitution, based on the amino acid numbering of the isoform 5 alpha chain amino acid sequence set forth in SEQ ID NO:9.

22. The variant GABAA receptor alpha chain of claim 16, wherein the alpha chain is an isoform 6 alpha chain, and wherein the cysteine substitution is an M139C substitution, based on the amino acid numbering of the isoform 6 alpha chain amino acid sequence set forth in SEQ ID NO: l l.

23. A GABAA receptor comprising the variant alpha chain of any one of claims 16-

22.

24. A non-human transgenic animal comprising a transgene in the genome of the animal, wherein the transgene comprises a nucleotide sequence encoding a variant GABAA receptor alpha chain comprising an amino acid substitution such that a cysteine is substituted for a wild-type amino acid residue in loop E of the alpha chain.

25. A treatment method comprising:

a) administering to an individual in need thereof a variant GABAA receptor alpha chain of any one of claims 16-22, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain;

b) administering to the individual a photoswitchable group of the formula: linker- azobenzene-R,

wherein R is a ligand for a GABAA receptor, and

wherein the photoswitchable group binds to the variant alpha chain and is covalently linked to a cysteine in the alpha chain via the linker, forming a photoregulated alpha chain, wherein the photoregulated alpha chain associates with two GABAA receptor β chains and either a GABAA receptor γ chain or a GABAA receptor δ chain, thereby generating a light- responsive GABAA receptor;

c) exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 480 nm to 520 nm.

26. The method of claim 25, wherein R is selected from:

27. A method of modulating the activity of a neuron, the method comprising:

a) introducing into the neuron a variant GABAA receptor alpha chain of any one of claims 16-22, or a nucleic acid comprising a nucleotide sequence encoding the variant GABAA receptor alpha chain;

wherein R is a ligand for a GABAA receptor, and

c) exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 360 nm to 400 nm, or exposing the light-responsive GABAA receptor to light of a wavelength in the range of from 480 nm to 520 nm, wherein R is a GABAA receptor antagonist.

28. The method of claim 27, wherein R is selected from: