INHIBITORY SLEEP-CIRCUIT NEURONS AND METHODS OF MODULATING A STABLE STATE BY

REGULATING THE SAME

CROSS-REFERENCE

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

62/311,119, filed March 21, 2016, which application is incorporated herein by reference in its entirety.

INTRODUCTION

[0002] Sleep is a naturally recurring state of the body and mind characterized by altered

consciousness, relatively inhibited sensory activity, inhibition of nearly all voluntary muscles, and reduced interactions with surroundings. It is distinguished from wakefulness by a decreased ability to react to stimuli, but is more easily reversed than the state of hibernation or of being comatose. Mammalian sleep occurs in repeating periods, in which the body alternates between two highly distinct modes known as non-REM and REM sleep. REM stands for "rapid eye movement" but involves many other aspects including virtual paralysis of the body.

Literature

[0003] Weber et al. (2015) Nature 526:435

SUMMARY

[0004] Provided herein are inhibitory neurons of a neural sleep circuit, and methods of

modulating a sleep circuit-regulated state of an individual by selectively regulating activity of the neural sleep circuit. The method may include illuminating with a light stimulus a region of a brain of an individual, the brain region containing a first subtype of sleep circuit neurons, wherein a light-activated polypeptide is selectively expressed in neurons of the first subtype, and wherein the light-activated polypeptide is configured to modulate activity of neurons of the first subtype when illuminated by the light stimulus, thereby promoting or suppressing a sleep circuit- regulated state of the individual, wherein the sleep circuit includes: a second subtype of neurons in a preoptic area (POA) of the brain, wherein neurons of the second subtype (a) are gamma aminobutyric acid-producing (GABAergic), and (b) project to a tuberomamrnillary nucleus (TMN) of the brain; and one or more third subtypes of neurons that synapse onto, and/or receive a synaptic connection from, neurons of the second subtype. Also provided are methods of identifying an agent that promotes or suppresses sleep.

The method may include i) administering to a non-human mammal a test agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in sleep-promoting and/or sleep-activated sleep circuit neurons in a POA of a brain compared to the average level of expression of the gene in neurons of the POA, wherein the sleep circuit neurons (a) are gamma aminobutyric acid-producing (GABAergic), and (b) project to a

tuberomammillary nucleus (TMN) of the brain; and ii) monitoring a sleep circuit-regulated state of the non-human mammal, wherein the sleep circuit-regulated state comprises REM sleep, non- REM sleep and wakefulness, wherein a test agent that changes the probability of REM sleep, non-REM sleep, and/or wakefulness in the non-human mammal is identified as an agent that promotes or suppresses sleep.

[0006] The present disclosure provides a method of modulating a neural sleep circuit-regulated state of an individual, the method comprising: illuminating with a light stimulus a first region of a brain of an individual, the first region comprising a first subtype of sleep circuit neurons, wherein a light-activated polypeptide is selectively expressed in neurons of the first subtype, and wherein the light-activated polypeptide modulates activity of neurons of the first subtype when illuminated by the light stimulus, thereby promoting or suppressing a sleep circuit-regulated state of the individual, wherein the sleep circuit comprises: a second subtype of neurons in a preoptic area (POA) of the brain, wherein neurons of the second subtype (a) are gamma aminobutyric acid-producing (GABAergic), and (b) project to a tuberomammillary nucleus (TMN) of the brain; and/or one or more third subtypes of neurons that synapse onto, and/or receive a synaptic connection from, neurons of the second subtype. In some cases, the illuminating step induces i) a first change in a probability of REM sleep and/or non-REM sleep, and ii) a second change in a probability of wakefulness, wherein the first and second change are of opposite sign. In some cases, neurons of the first subtype comprise neurons in the POA that express one or more neuropeptide genes selected from the group consisting of: corticotropin releasing hormone, galanin, neurotensin, tachykinin 1 , tachykinin 2, prepronociceptin, calcitonin-related polypeptide alpha, prodynorphin, and cholecystokinin. In some cases, the second subtype of neurons comprises the first subtype of neurons. In some cases, the illuminating step increases the probability of REM sleep and/or non-REM sleep, and reduces the probability of wakefulness. In some cases, the one or more third subtypes of neurons comprise the first subtype of neurons. In some cases, neurons of the third subtype comprise hypothalamic or amygdala neurons. In some cases, the amygdala neurons are GABAergic neurons of the central nucleus of the amygdala (CEA). In some cases, the illuminating step reduces the probability of REM sleep and/or non- REM sleep, and increases the probability of wakefulness. In some cases, the light-activated polypeptide is an ion channel or an ion pump. In some cases, the method further comprises monitoring a sleep circuit-regulated state of the individual before and/or after the illuminating step, wherein the sleep circuit-regulated state comprises REM sleep, non-REM sleep and/or wakefulness. In some cases, the monitoring comprises using electroencephalography (EEG) and/or electromyography (EMG) to determine the sleep circuit-regulated state. In some cases, the individual has a sleeping disorder. In some cases, the individual is sleep deprived. In some cases, the method further comprises, before the illuminating step, genetically modifying neurons of the first subtype with a nucleic acid comprising a nucleotide sequence encoding the light- activated polypeptide, wherein the nucleic acid is configured to express the light-activated polypeptide selectively in neurons of the first subtype. In some cases, the genetically modifying step comprises administering to a second region of the brain an expression vector comprising the nucleic acid, wherein the second region comprises one or more subtypes of neurons of the sleep circuit. In some cases, the expression vector is a recombinant viral expression vector, and wherein the administering comprises administering a virion comprising the viral expression vector. In some cases, the second region is the TMN or the POA. In some cases, the first region and second region are same regions of the brain. In some cases, the first region and second region are different regions of the brain, wherein the second region comprises neurons of a third subtype that receive a synaptic connection from neurons of the second subtype, and wherein the recombinant viral vector is derived from a lenti viral vector. In some cases, the virion is pseudotyped with a rabies glycoprotein. In some cases, the individual is a transgenic, non-human mammal expressing a site-specific recombinase in GABAergic neurons or glutamatergic neurons of the brain.

The present disclosure provides a method of identifying an agent that promotes or suppresses sleep, the comprising: i) administering to a non-human mammal a test agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in circuit neurons in a POA of a brain compared to the average expression level of the gene in neurons of the POA, wherein the sleep circuit neurons (a) are gamma aminobutyric acid-producing (GABAergic), and (b) project to a tuberomammillary nucleus (TMN) of the brain; and ii) monitoring a sleep circuit-regulated state of the non-human mammal, wherein the sleep circuit-regulated state comprises REM sleep, non-REM sleep and/or wakefulness, wherein a test agent that changes the probability of REM sleep, non-REM sleep, and/or wakefulness in the non-human mammal is identified as an agent that promotes or suppresses sleep. In some cases, the test agent that causes i) a first change in the probability of REM sleep and/or non- REM sleep, and ii) a second change in the probability of wakefulness, wherein the first and second change are of opposite sign, is identified as an agent that promotes or suppresses sleep. In some cases, the test agent specifically binds to the gene product. In some cases, the test agent is a small molecule, nucleic acid, polypeptide or a combination thereof. In some cases, the gene product is a post-transcriptional gene product. In some cases, the gene product is a post- translational gene product. In some cases, the gene encodes a cell-surface receptor. In some cases, the gene encodes a G-protein coupled receptor. In some cases, the gene encodes a polypeptide gene product selected from a polypeptide gene product listed in Table 3, in Figures 18A-18AI. In some cases, the monitoring step comprises using electroencephalography (EEG) and/or electromyography (EMG) to determine the sleep circuit-regulated state. In some cases, the non-human mammal has a sleeping disorder. In some cases, the non-human mammal is sleep deprived. In some cases, the non-human mammal is a rodent (e.g., a mouse; a rat). In some cases, the non-human mammal is a non-human primate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures 1A-1B are a collection of images, diagrams and graphs showing retrograde labeling and c-Fos staining of sleep-active preoptic area (POA) neurons, according to embodiments of the present disclosure.

[0009] Figures 2A-2C are a collection of images, diagrams and graphs showing that optogenetic activation of GAD ^{POA→T N} neurons induces sleep, according to embodiments of the present disclosure.

[0010] Figures 3A-3F are a collection of images, diagrams and graphs showing that optogenetic activation of GAD ^ROA or VGL ^T JT ^poa→ ™ ⁿ neurons promotes wakefulness.

[0011] Figures 4A-4F are a collection of images and graphs showing that optogenetically

identified Q^r ^POA→ ™ ^N neurons are active during sleep, according to embodiments of the present disclosure.

[0012] Figures 5A-5F are a collection of images, diagrams and graphs showing mapping of monosynaptic inputs to Q^r ^POA→ ™ ^N neurons, according to embodiments of the present disclosure.

[0013] Figures 6A-6F are a collection of images, diagrams and graphs showing identification of molecular markers for POA sleep-promoting neurons, according to embodiments of the present disclosure.

[0014] Figures 7A-7D are a collection of images, diagrams and graphs showing identification of molecular markers for POA sleep-promoting neurons, according to embodiments of the present disclosure. [0015] Figure 8 is a collection of images and graphs showing c-Fos staining of sleep-active

POA neurons in GADl-GFP or VGLUT2-GFP mice, according to embodiments of the present disclosure.

[0016] Figures 9A-9B are a collection of a diagram and a graph showing the effect of laser stimulation in GAD ^POA→TMN -nlsPelican control mice.

[0017] Figures 1 OA- IOC are a collection of graphs and diagrams showing the effect of laser stimulation on transition probability between each pair of brain states in GAD ^POA→TMN -ChR2,

GAD ^POA→TMN -Ctrl and GAD ^POA -ChR2 mice, according to embodiments of the present disclosure.

[0018] Figures 11 A and 1 IB are a collection of graphs showing optogenetic identification of

GAD ^POA→1MN neurons, according to embodiments of the present disclosure.

[0019] Figure 12 is a graph showing the firing rates of unidentified POA neurons.

[0020] Figures 13A and 13B are a collection of graphs showing the identification of

differentially expressed genes in TRAP, according to embodiments of the present disclosure.

[0021] Figures 14A and 14B are a collection of images showing the overlap of identified molecular markers in POA, according to embodiments of the present disclosure.

[0022] Figure 15 is a graphs showing that optogenetic stimulation of POA GAL neurons promotes wakefulness

[0023] Figures 16 shows Table 1, showing neuropeptide-encoding genes enriched in

GAD ^POA→1MN neurons, according to embodiments of the present disclosure.

[0024] Figure 17 shows Table 2, showing G-protein coupled receptors (GPCRs) whose

expression is upregulated in Q^r ^POA→ ™ ^N neurons, according to embodiments of the present disclosure.

[0025] Figures 18A-18AI shows Table 3, showing genes whose expression is upregulated in

GAD ^POA→1MN neurons, according to embodiments of the present disclosure.

[0026] Figures 19A-19B are a collection of amino acid sequences of light-activated

polypeptides that find use in embodiments of the present disclosure.

DEFINITIONS

[0027] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA -RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0028] 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.

[0029] By "hybridizable" or "complementary" or "substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non- covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, "anneal", or

"hybridize," to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule: guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA.

[0030] Hybridization and washing conditions are well known and exemplified in Sambrook, J.,

Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0031] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides) the position of mismatches can become important (see Sambrook et al., supra, 11.7-11.8). Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more). Temperature, wash solution salt concentration, and other conditions may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0032] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with

complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), and the like.

[0033] "Gene product" as used herein, may refer to any physiologically relevant biological molecule that is produced based on transcription and/or translation of a nucleic acid (e.g., DNA) containing a nucleotide sequence. The physiologically relevant biological molecule may be an RNA (e.g., mRNA, micro RNA, other regulatory RNAs, etc.), or a polypeptide encoded by the nucleic acid.

[0034] "Binding" as used herein (e.g. with reference to an active (e.g., active compound)

binding to a gene product, and the like) refers to a non-covalent interaction between molecules. Binding interactions are generally characterized by a dissociation constant (K _D ) of 10 ⁶ M or less, 10 ⁷ M or less, 10 ^s M or less, 10 ⁹ M or less, 10 ¹⁰ M or less, 10 ¹¹ M or less, 10 ¹² M or less, 10 ¹³ M or less, 10 ¹⁴ M or less, or 10 ¹⁵ M or less. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower K _D . "Specific binding" as used herein may refer to a non-covalent interaction between molecules mediated by one or more features (e.g., specific sequence of nucleotides, position/presence of specific amino acid side chains, specific structure of a molecule, etc.) from each of the molecules, where the absence of a feature (e.g., change in the nucleotide sequence, change in amino acid sequence, change in molecular structure, etc.) from one or more of the molecules reduces the binding affinity.

[0035] The term "genetic modification" refers to a permanent or transient genetic change

induced in a cell following introduction into the cell of a heterologous nucleic acid (e.g., a nucleic acid exogenous to the cell). Genetic change ("modification") can be accomplished by incorporation of the heterologous nucleic acid into the genome of the host cell, or by transient or stable maintenance of the heterologous nucleic acid as an extrachromosomal element. Where the cell is a eukaryotic cell, a permanent genetic change can be achieved by introduction of the nucleic acid into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.

[0036] An "individual" as used herein, may be any suitable animal amenable to the methods and techniques described herein, where in some cases, the individual may be a vertebrate animal, including a mammal, bird, reptile, amphibian, etc. The individual may be any suitable mammal, e.g., human, mouse, rat, cat, dog, pig, horse, cow, monkey, non -human primate, etc.

[0037] As used herein, the terms "treat," "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 health condition, disease or symptoms thereof and/or may be therapeutic in terms of a partial or complete cure for a health condition, disease and/or adverse effect attributable to the health condition or disease. "Treatment," as used herein, covers any treatment of a health condition or disease in a mammal, particularly in a human, and includes: (a) preventing the health condition or disease from occurring in a subject which may be predisposed to the health condition or disease but has not yet been diagnosed as having it; (b) inhibiting the health condition or disease, i.e., arresting its development; and (c) relieving the health condition or disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the health condition or disease.

[0038] A "therapeutically effective amount" or "effective amount" means the amount of an agent that, when administered to a cell, a tissue, a mammal or other individual for obtaining a desired change in a physiological parameter, e.g., for treating a disease, is sufficient to effect such desired change, e.g., treatment for the disease or condition. The "therapeutically effective amount" will vary depending on the agent, the disease or condition and its severity and the age, weight, etc., of the subject to be treated. [0039] By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to an individual without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

[0040] An "agent" as used herein, may refer to a chemical compound, or combinations of chemical compounds. The physical entity may be a chemical compound (e.g., small molecule, organic compound, nucleic acid, polypeptide, inorganic molecule, etc.), a complex of chemical compounds (e.g., antibody, nucleoprotein complex, etc.), and the like.

[0041] By "small molecule" is meant a non-peptidic, non-oligomeric organic compound that may be synthetic or natural. A small molecule may contain one or more carbon-carbon bonds, and may have a molecular weight of 1500 daltons or less than 1500 daltons.

[0042] "Synapse" as used herein, may refer to the presence of a directional, chemical

communication between a first neuron and a target cell, which may be a second neuron, that causes an electrical change in the target cell.

[0043] "Subtype of neuron," or "neuronal subtype" as used herein, may refer to a classification of neurons based on a static identity of a neuron, and/or a dynamic property of a neuron. A static identity of a neuron may include marker expression (e.g., mRNA transcript or protein expression), anatomical location and/or connectivity, etc. A dynamic property of a neuron may include the pattern (e.g., frequency, duration, timing, etc.) of depolarization/action potential firing. In some cases, a region of a brain may include two or more subtypes of neurons that are intermingled with each other such that the two or more subtypes of neurons are represented in a monolithic, three-dimensional volume (e.g., a substantially spherical volume) of the brain region containing a population of neurons (e.g., a population of 10 or more, e.g., 20 or more, 50 or more, 100 or more, 1,000 or more, 10,000 or more, including 100,000 or more, neurons).

"Represent" as used herein may be used to indicate that a member of an ensemble is present in the ensemble in sufficient amount so as to functionally contribute to a property of the ensemble as a whole. In some cases, the member may be present at 0.1% or more, e.g., 1% or more, 5% or more, 10% or more, 20% or more, including 50% or more, of the entire ensemble.

[0044] "Circuit" as used herein, may refer to a collection of interconnected neurons through which activity of individual neurons is propagated to produce a physiological and/or behavioral effect on an individual. In some cases, an activity pattern (e.g., frequency, amplitude, etc.) of neurons of the circuit may represent a state, e.g., a stable but reversible state, of the circuit (e.g., sleep, rapid eye movement (REM) sleep, non-REM sleep, wakefulness, etc.) that is distinguished from another state having a different activity pattern. The neurons may be organized into different subtypes, e.g., based on their functional role in the circuit, connectivity to other neurons, and/or gene expression profile.

[0045] "Selective" as used herein, may be used to describe an action that is applied to, or a property that is present in, a larger proportion of members within a first group compared to the proportion of members of a second group to which the action is applied, or in which the property is present, where the relative proportions are such that the difference in functional consequence of the action or property between the first and second groups can be attributed to the action or the property.

[0046] A "sleeping disorder" as used herein, may refer to a condition where a temporal

progression through different sleep circuit-regulated state (e.g., REM sleep, non-REM sleep and/or wakefulness) of an individual deviates from a physiologically normal progression as exhibited by average members of the species to which the individual belongs in given light-dark cycle. "Sleep deprivation" may refer to a condition where an individual is unable to or prevented from being in REM sleep and/or non-REM sleep for an amount of time due to an disruption (either physiological or external) that would have been achieved but for the disruption. A sleeping disorder or sleep deprivation may be characterized by deterioration in the ability of an individual to perform a task relative to when in the absence of the sleeping disorder of sleep deprivation.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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 neuron" includes a plurality of such neurons and reference to "the agent" includes reference to one or more agents 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.

[0051] 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 subcombination. 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.

[0052] 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

[0053] As summarized above, methods of modulating a neural sleep circuit-regulated state of an individual, and methods of identifying an agent that modulates a neural sleep circuit-regulated state in an individual are provided. In general terms, the present methods are based on the identification of a subpopulation of sleep circuit neurons in the preoptic area (POA) of the brain that can promote a sleep state (e.g., rapid-eye movement (REM) sleep or non-REM sleep) when selectively activated, e.g., activated using optogenetic tools, and/or that are selectively active when the individual is in REM or non-REM sleep state. The present subtype of sleep circuit neurons of the POA are characterized by having an inhibitory projection to the

tuberomammiUary nucleus (TMN). The present subtype of sleep circuit neurons of the POA are spatially intermingled among other neurons of the POA having distinct functions, and many of these other neurons promote arousal. As a result, non-specific activation of neurons in the POA suppresses sleep and increase wakefulness. Thus, a sleep-circuit regulated state (e.g. REM sleep, non-REM sleep or wakefulness) can be modulated, to specifically promote or suppress sleep, by the neuronal subtype-specific regulation of neural activity of the present subtype of sleep circuit neurons of the POA, among a plurality of subtypes of neurons that are intermingled with each other in the POA, or by the neuronal subtype-specific modulation of neural activity of other subtypes of neurons that are functionally connected to (e.g., synapse onto or receive synaptic input from) the present subtype of sleep circuit neurons of the POA.

GABAERGIC, TMN-PROJECTING NEURONS OF THE PREOPTIC AREA

[0054] Aspects of the present disclosure include a subtype of neurons in a preoptic area (POA) of a brain of an individual, which subtype is characterized by inhibitory projections to the tuberomammiUary nucleus (TMN) of the brain. Thus, inhibitory, TMN-projecting neurons of the present subtype use gamma aminobutyric acid (GAB A) as neurotransmitter and express genes that are used for GAB A biosynthesis and processing (i.e., are GABAergic neurons). The present subtype of sleep circuit neurons are GABAergic neurons and may express any suitable gene related to the production of GABA as a neurotransmitter, such as, but not limited to, glutamate decarboxylase (GAD 1/2), vesicular GABA transporter (VGAT), GABA transporter 1 (GAT1). In some cases, the present subtype of GABAergic, TMN-projecting neurons of the POA may be identified by expression of a gene operatively linked to a regulatory sequence for GAD2, and by their projection to the TMN, and may be referred to herein as Q^r ^POA→ ™ ^N neurons. In some cases, the neurons are isolated. In some cases, the neurons are genetically modified to express a light-activated polypeptide. In some cases, the neurons are in vitro, and are genetically modified to express a light-activated polypeptide.

[0055] The inhibitory projection of neurons in the POA may be determined based on, e.g., anterograde or retrograde tracing methods. A suitable retrograde tracing method is described in, e.g., Cetin et al., J. Neurophysiol. Ill, 2150-2159.

[0056] GABAergic, TMN-projecting POA neurons, e.g., QAD ^roA→TMN neurons, of the present disclosure may be characterized by a transcriptome that is distinct from the average

transcriptome of neurons of the POA. "Transcriptome" as used herein, may refer to the profile of the identity and corresponding expression levels of 100 or more, e.g., 200 or more, 300 or more, 400 or more, 500 or more, 800 or more, 1,000 or more, 5,000 or more, 10,000 or more, or substantially all genes in the genome of the individual from whom the neurons originate. In some embodiments, the GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→1mn neurons, have an expression level of a gene that is higher by 1.5 fold or more, e.g., 1.8 fold or more, 2.0 fold or more, 2.2 fold or more, 2.5 fold or more, 3.0 fold or more, 4.0 fold or more, 5.0 fold or more, including 10 fold or more, and in some cases higher by 1,000 fold or less, e.g., 500 fold or less, 100 fold or less, 50 fold or less, 20 fold or less, 10 fold or less, including 5.0 fold or less than the average expression level in neurons of the POA (i.e., the average expression level across neurons of the POA regardless of the neuronal subtype, which may include a relatively small fraction of the GABAergic, TMN-projecting POA neurons compared to the fraction of neurons that are not the GABAergic, TMN-projecting POA neurons). In some embodiments, the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons, have an expression level of a gene that is higher by a range of 1.5 to 1,000 fold, e.g., 1.8 fold to 500 fold, 2.0 fold to 100 fold, 2.0 fold to 50 fold, 2.0 fold to 20 fold, including 2.0 fold to 10 fold, compared to the average expression level in neurons of the POA. The fold expression of a gene may be determined, e.g., by high- throughput sequencing of gene transcripts expressed in neurons of interest, followed by comparison of the value for fragments per kilobase of exon per million fragments mapped (FPKM) for each gene.

[0057] In some cases, the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→1MN

neurons, have an upregulated expression of neuropeptide genes (i.e., genes encoding a neuropeptide gene product), such as those listed in Table 1 of Figure 16. In some cases, the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, have upregulated expression of genes encoding a polypeptide as shown in Table 3 (Figures 18A-18AI), e.g., when the individual to which the neurons belong is a murine individual, or a homologue thereof, e.g., when the individual is a non-murine individual. [0058] A further aspect of the present disclosure includes a neural sleep circuit that includes neurons that form a neural pathway such that the activity of the neurons are reflected in the output of the circuit, which output are functionally linked (e.g., via synaptic connections, endocrine signals, etc.) to other components of the body (e.g., other neurons of the brain and body, muscle, internal organs, etc.) to promote one stable state over one or more other stable states (e.g., promote rapid eye movement (REM) and/or non-REM sleep over arousal) of the individual. The sleep circuit may include neurons that are found in any suitable anatomical region of the brain that can control a stable state (e.g. REM sleep, non-REM sleep or wakefulness) of the individual. The sleep circuit may include, without limitation, neurons of the brainstem, thalamus, hypothalamus, the basal forebrain, and the limbic system, etc. The sleep circuit may include, without limitation, neurons of the pedunculopontine and laterodorsal tegmental nucleus (PPT/LDT), locus coeruleus, dorsal and median raphe nucleus,

tuberomammillary nucleus (TMN), preoptic area (POA), amygdala, ventral periaqueductal grey matter, lateral hypothalamus, etc. Neurons of the sleep circuit may be characterized by having a specific type of neurotransmitter, such as GAB A, glutamate, acetylcholine, serotonin, dopamine, histamine, noradrenaline, etc., and may be characterized by having a specific peptide hormone, such as orexin/hypocretin, melatonin-concentrating hormone, etc.

[0059] In some cases, the sleep circuit includes GABAergic, TMN-projecting POA neurons, e.g., GAE) ^POA→1MN neurons, as described above. The sleep circuit may also include pre- and/or post-synaptic partners of the GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons. In some cases, the sleep circuit includes neurons of the POA that are characterized by having expression of one or more genes that are upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, compared to the average expression level of the respective genes in neurons of the POA overall. The upregulated genes are described, e.g., in Table 1 of Figure 16, Table 2 of Figure 17, and Table 3 of Figures 18A-18AI. In some embodiments, the upregulated gene has an expression level in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, that is 1.5 fold or more, e.g., 1.8 fold or more, 2.0 fold or more, 2.5 fold or more, including 3.0 fold or more, and in some cases, by 100 fold or less, e.g., 50 fold or less, 30 fold or less, 20 fold or less, 10 fold or less, 5.0 fold or less, including 4.0 fold or less, higher compared to its respective average expression level in the POA. In some embodiments, the upregulated gene has an expression level in the GABAergic, TMN-projecting POA neurons, e.g., QAD ^roA→TMN neurons, that is in the range of 1.5 fold to 100 fold, e.g., 1.5 fold to 50 fold, 1.5 fold to 30 fold, 2.0 fold to 30 fold, 2.5 fold to 30 fold, including 3.0 fold to 30 fold, higher compared to its respective average expression level in the POA. [0060] In some embodiments, the sleep circuit includes neurons of the POA that have upregulated expression of one or more neuropeptide genes selected from: corticotropin releasing hormone, galanin, neurotensin, tachykinin 1, tachykinin 2, prepronociceptin, calcitonin-related polypeptide alpha, prodynorphin, and cholecystokinin, compared to their respective average expression level in neurons of the POA. In some embodiments, the sleep circuit includes neurons of the POA that have upregulated expression of one or more GPCR genes selected from: 5- hydroxytryptamine (serotonin) receptor IF; cholinergic receptor, muscarinic 4; arginine vasopressin receptor 1A, adrenergic receptor, beta 1; progestin and adipoQ receptor family member VII; G protein-coupled receptor 83; and G protein-coupled receptor 83, compared to their respective average expression level in neurons of the POA.

[0061] The sleep circuit may function as a switch between different stable behavioral,

physiological and neurological states of the individual. The stable states may include rapid eye movement (REM) sleep, non-REM sleep and wakefulness, and the different states of the individual may be determined using a suitable method for measuring neuronal and/or muscle activity. In some cases, the state of the individual is monitored using electroencephalography, electromyography, or a combination thereof. Thus, in some cases, REM sleep is defined by having a high power at theta frequencies as measured by EEG; non-REM sleep is defined by a synchronized EEG with high amplitude and low frequency; and wakefulness is defined by desynchronized EEG. In some embodiments, REM sleep is defined by a low EMG activity; non- REM sleep is defined by low EMG activity; and wakefulness is defined by high EMG activity. REM sleep may be defined by a high power at theta frequencies as measured by EEG, and low EMG activity; non-REM sleep may be defined by a synchronized EEG with high amplitude and low frequency, and low EMG activity; and wakefulness may be defined by desynchronized EEG, and high EMG activity. The threshold for EEG frequency may vary depending on, e.g., the species to which the individual belongs. For example, if the individual is a mouse, the low EEG frequency may include a range of 0.5-4 Hz, and a theta frequency may include a range of 6-9 Hz. If the individual is a human, a theta frequency may include a range of 4-7 Hz.

[0062] In some cases, the state of the individual may be monitored by visual observation, e.g., visually determining whether the individual is awake or not. In some cases, the state of the individual may be monitored subjectively, e.g., based on the individual's subjective experience of sleep and/or wakefulness. METHODS

Method of Modulating a Neural Sleep Circuit- Regulated State

[0063] As summarized above, methods modulating a neural sleep circuit-regulated state of an individual are provided. In general terms, the present method may include optogenetically stimulating a subtype of neurons of a neuronal sleep circuit of the individual to promote or suppress a sleep circuit-regulated state (e.g., REM sleep, non-REM sleep, wakefulness). The sleep circuit may include GABAergic, TMN-projecting POA neurons, e.g., Q^r ^POA→ ™ ^N neurons, and other neurons having a synaptic connection thereto, as described above. By "optogenetic stimulation" is meant any one of a number of methods of modulating the electrical activity of a neuron that expresses a light-activated polypeptide (e.g., a depolarizing light- activated polypeptide such as channelrhodopsin 2 (ChR2), CI VI, SFO, SSFO, etc., as described further below) by illuminating the neuron with a light stimulus suitable for activating the light- activated polypeptide.

[0064] A suitable optogenetic stimulation may target and selectively modulate the neural

activity of a functionally-defined subtype of neurons in the sleep circuit such that the optogenetic stimulation preferentially changes the activity of the targeted subtype of neurons without directly modulating the neural activity of another functionally distinct subtype of neuron of the sleep circuit. In some cases, the optogenetically targeted subtype of neurons is intermingled within a population of different subtypes of neurons having distinct and/or opposite function as the targeted subtype, and the optogenetic stimulation can modulate the activity of the targeted subtype of neurons without substantially modulating the activity of the non-targeted subtype of neurons. Such selectivity may be achieved using any suitable method, e.g., by selectively expressing a light-activated polypeptide in the subtype of neurons of interest, as described further below.

[0065] The level of activity of neurons of the sleep circuit may control the likelihood or

probability that an individual is in any given stable state. In some embodiments, the activity of a subtype of neurons in the sleep circuit promotes REM sleep, by increasing the transition probability from another state (e.g., non-REM sleep or wakefulness) to REM sleep, and/or by reducing the transition probability from REM sleep to another state, when neurons of the subtype are more active as compared to when they are less active.

[0066] In some embodiments, the activity of a subtype of neurons in the sleep circuit suppresses

REM sleep, by reducing the transition probability from another state (e.g., non-REM sleep or wakefulness) to REM sleep, and/or by increasing the transition probability from REM sleep to another state, when neurons of the subtype are more active as compared to when they are less active. [0067] In some embodiments, the activity of a subtype of neurons in the sleep circuit promotes non-REM sleep, by increasing the transition probability from another state (e.g., REM sleep or wakefulness) to non-REM sleep, and/or by reducing the transition probability from non-REM sleep to another state, when neurons of the subtype are more active as compared to when they are less active.

[0068] In some embodiments, the activity of a subtype of neurons in the sleep circuit suppresses non-REM sleep, by reducing the transition probability from another state (e.g., REM sleep or wakefulness) to non-REM sleep, and/or by increasing the transition probability from non-REM sleep to another state, when neurons of the subtype are more active as compared to when they are less active.

[0069] In some embodiments, the activity of a subtype of neurons in the sleep circuit promotes arousal, by increasing the transition probability from another state (e.g., REM sleep or non-REM sleep) to wakefulness, and/or by reducing the transition probability from wakefulness to another state, when neurons of the subtype are more active as compared to when they are less active.

[0070] In some embodiments, the activity of a subtype of neurons in the sleep circuit suppresses arousal, by reducing the transition probability from another state (e.g., REM sleep or non-REM sleep) to wakefulness, and/or by increasing the transition probability from wakefulness to another state, when neurons of the subtype are more active as compared to when they are less active.

[0071] The activity of a subtype of neurons in the sleep circuit may suppress a sleep circuit- regulated state (e.g. REM sleep, non-REM sleep, or wakefulness) at the expense of one or more of the other sleep circuit-regulated states. In some embodiments, a change in the probability that the individual is in REM sleep and/or non-REM sleep is accompanied by a change of opposite sign in the probability that the individual is in wakefulness. In some cases, an increase in the probability that the individual is in REM sleep and/or non-REM sleep is accompanied by a reduction in the probability that the individual is in wakefulness. In some cases, a reduction in the probability that the individual is in REM sleep and/or non-REM sleep is accompanied by an increase in the probability that the individual is in wakefulness. The magnitude of change in one sleep circuit-regulated state relative a change of opposite sign in another sleep circuit-regulated state may be different (e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 75% or more, including 100% or more different), similar (e.g., up to 10%, up to 5%, including up to 3% different) or may be substantially the same.

[0072] The optogenetic stimulation may include illuminating with a light stimulus, a region of the brain of the individual (e.g., a human; a non-human mammal such as a non-human primate, a rat, a mouse, etc.), which region includes sleep circuit neurons of a particular subtype that has been genetically modified to express a light-activated polypeptide, e.g., a light-activated ion channel such as channelrhodopsin 2 (ChR2), as described further below, where activation of the light-activated polypeptide by the light stimulus induces changes in the neuronal activity (e.g., depolarization or hyperpolarization) of the neurons expressing the light-activated polypeptide. The light stimulus may vary, and may depend on, e.g., the absorption spectrum, expression level, subcellular localization, etc., of the light-activated polypeptide. For example, the light stimulus may have a wavelength that is close to the optimal wavelength for activating the light-activated polypeptide (e.g., around 480 nm or ChR2). The light stimulus may include one or more pulses of light, each pulse having a duration of 0.1 milliseconds (ms) or more, e.g., 0.5 ms or more, 1.0 ms ormore, 2.0 ms or more, including 5.0 ms or more, and in some cases a duration of 100 ms or less, e.g., 50 ms or less, 25 ms or less 20 ms or less, including 15 ms or less. In some embodiments, the light stimulus may include one or more pulses of light, each pulse having a duration in the range of 0.1 to 100 ms, e.g., 0.5 to 100 ms, 1.0 to 50 ms, including 2.0 to 25 ms.

[0073] The light stimulus may include pulses of light applied at a regular frequency. In some cases, the light stimulus may include pulses of light applied at a frequency of 1 Hz or more, e.g., 2 Hz or more, 5 Hz or more, 10 Hz or more, 15 Hz or more, including 20 Hz or more, and in some cases, at a frequency of 100 Hz or less, e.g., 80 Hz or less, 60 Hz or less, 40 Hz or less, including 30 Hz or less. In some embodiments, the light stimulus may include pulses of light applied at a frequency in the range of 1 Hz to 100 Hz, e.g., 2 Hz to 80 Hz, 5 Hz to 60 Hz, 10 Hz to 40 HZ, including 10 Hz to 30 Hz.

[0074] The light stimulus may include one or more pulses of light having any suitable power. In some embodiments, the light stimulus may include one or more pulses of light having a power of 0.1 mW or more, e.g, 0.5 mW or more, 1.0 mW or more, 2.0 mW or more, including 4.0 mW or more, and in some cases, a power of 50 mW or less, e.g., 40 mW or less, 30 mW or less, 20 mW or less, including 10 mW or less, at the tip of an optical fiber used to deliver the light stimulus to the brain region. In some embodiments, the light stimulus may include one or more pulses of light having a power in the range of 0.1 to 50 mW, e.g., 0.5 to 40 mW, 1.0 to 30 mW, 2.0 to 20 mW including 4.0 to 10 mW.

[0075] The light stimulus may be delivered to the brain region using any suitable method. In some cases, the light stimulus is delivered to the brain region using an optical fiber having an end implanted into the brain region of interest, e.g., the brain region that includes a subtype of neurons belonging to the present sleep circuit, where the subtype of neurons express a light- activated polypeptide. The optical fiber may be any suitable optical fiber for delivering the light stimulus. The optical fiber may be a multimode or single mode optical fiber. [0076] The change in a sleep circuit-regulated state induced by optogenetic stimulation may occur within a time interval that varies depending on, e.g., the light-activated polypeptide expressed in neurons of the sleep circuit, the light stimulus properties and stimulation protocol, the nature of the neurons being activated by the optogenetic stimulation, etc. In some cases, the sleep circuit-regulated state is promoted or suppressed substantially immediately after illuminating the neurons with a light stimulus. In some cases, the sleep circuit-regulated state is promoted or suppressed within 1 second (s) or more, e.g., within 5 seconds (s) or more, within 10 s or more, within 20 s or more, within 30 s or more, within 1 minute (min) or more, within 5 minutes (min) or more, within 10 min or more, including within 30 min or more, and in some cases, within 1 day or less, e.g., within 18 hours (hr) or less, within 12 hr or less, within 6 hr or less, within 3 hr or less, within 1 hour (hr) or less, within 30 min or less, within 15 min or less, within 5 min or less, within 1 min or less, 45 s or less, including 30 s or less, upon illumination of the neurons. In some embodiments, the sleep circuit-regulated state is promoted or suppressed within a time ranging from 1 s to 30 s, 5 s to 30 s, 10 s to 30 s, 10 s to 45 s, 20 s to 45 s, 20 s to 1 min, lmin to 5 min, 5 min to 15 min, 10 min to 15 min, 10 min to 30 min, 30 min to 1 hr, 30 min to 3 hr, 30 min to 3 hr, 30 min to 6 hr, 30 min to 12 hr, 30 min to 18 hr, or 30 min to 1 day, upon illumination of the neurons.

[0077] The changes in the state of the individual, as described above, induced by activation of neurons of a subtype of neurons in the sleep circuit may be defined relative to a suitable reference condition. In some cases, the measured states (e.g., probability of states) are compared before and after performing a method step (e.g., before and after illuminating a neuron). The comparison may be made on an individual basis (i.e., comparison between before and after performing a method step for each individual), or may be made on a population basis (i.e., comparison between a population average before and a population average after performing a method step on a number of individuals). In some cases, the measured states (e.g., probability of states) are compared to a predetermined standard, e.g., a predetermined threshold value. The predetermined standard may be obtained by monitoring sleep-circuit regulated states in a control group of individuals (i.e., a group of individuals to whom a method step has not been performed).

[0078] The subtype of neurons of the sleep circuit to be targeted may vary depending on the desired outcome. In some cases, the optogenetic stimulation selectively modulates the activity of the GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→ ™ ⁿ neurons, as described above. An optogenetic stimulation that selectively activates GABAergic, TMN-projecting neurons of the POA, e.g., GAD ^roA→ ™ ^N neurons, may promote REM sleep and/or non-REM sleep and suppresses arousal in the individual. [0079] In some embodiments, the optogenetic stimulation selectively modulates the activity of

GABAergic POA neurons that also express a gene that is upregulated in the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons. Examples of such upregulated genes are shown in, e.g., Table 3, in Figures 18A-18AI. In some cases, the optogenetic stimulation selectively modulates the activity of GABAergic POA neurons that also express a gene that is upregulated by 1.5 fold or more, e.g., 1.8 fold or more, 2.0 fold or more, 2.5 fold or more, including 3.0 fold or more, and in some cases, by 100 fold or less, e.g., 50 fold or less, 30 fold or less, 20 fold or less, 10 fold or less, 5.0 fold or less, including 4.0 fold or less, compared to its respective average expression level in the POA. In some embodiments, the optogenetic stimulation selectively modulates the activity of GABAergic POA neurons that also express a gene that is upregulated by a range of 1.5 fold to 100 fold, e.g., 1.5 fold to 50 fold, 1.5 fold to 30 fold, 2.0 fold to 30 fold, 2.5 fold to 30 fold, including 3.0 fold to 30 fold, compared to its respective average expression level in the POA.

[0080] In some cases, the optogenetic stimulation selectively modulates the activity of

GABAergic POA neurons that also express one or more neuropeptide genes selected from corticotropin releasing hormone, galanin, neurotensin, tachykinin 1 , tachykinin 2,

prepronociceptin, calcitonin-related polypeptide alpha, prodynorphin, and cholecystokinin. In some cases, the gene upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^POA→1MN neurons, including any one or more of the neuropeptide genes listed above, may be expressed at higher level, compared to the average expression level of respective genes in the POA, in the GABAergic POA neurons selectively modulated by the optogenetic stimulation. In some embodiments, an optogenetic stimulation that selectively activates the GABAergic neurons of the POA promotes REM sleep and/or non-REM sleep and suppresses arousal in the individual.

[0081] In some embodiments, the optogenetic stimulation selectively modulates activity of neurons that synapse onto, or which receives synaptic input from, the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons. Neurons that synapse onto the

GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, may include any neurons with such connectivity, and may include, without limitation, a subtype of neurons in other areas of the hypothalamus, or the amygdala. The functional connectivity between the neurons may be activating (e.g., glutamatergic) or inhibitory (e.g., GABAergic). In some cases, the optogenetic stimulation selectively modulates activity of GABAergic neurons of the central nucleus of the amygdala (CEA). In some embodiments, the optogenetic stimulation selectively activates GABAergic neurons of the CEA to suppress REM sleep and/or non-REM sleep, and to promote arousal. In some embodiments, the optogenetic stimulation selectively activates a subtype of neurons of the CEA to promote REM sleep and/or non-REM sleep, and to suppress arousal.

[0082] Neurons that receive input from the GABAergic, TMN-projecting POA neurons, e.g.,

GAD ^POA→1MN neurons, may include any neurons with such connectivity, and may include, without limitation, a subtype of neurons in other areas of the hypothalamus (e.g., the dorsomedial hypothalamus), the TMN, or the ventrolateral periaqueductal grey (vIPAG).

Optogenetic Stimulation for Selective Modulation of the Activity of a Subtype of Sleep Circuit Neurons

[0083] An optogenetic stimulation that selectively modulate the activity of a subtype of neurons in the sleep circuit includes a light stimulus, and the subtype of neurons in the sleep circuit targeted by the light stimulus is genetically modified to express a light-activated polypeptide configured to depolarize or hyperpolarize the neuron in which the light-activated polypeptide is expressed when activated by the light stimulus. Selective modulation of the subtype of neurons in the sleep circuit may be achieved by expression of the light-activated polypeptide selectively in the subtype of neurons in the sleep circuit through, e.g., the use of appropriate regulatory elements in a nucleic acid encoding the light-activated polypeptide, and/or localized delivery of and genetic modification by the nucleic acid encoding the light-activated polypeptide, etc. Localized delivery of the light stimulus may also provide for selective modulation of the subtype of neurons in the sleep circuit.

[0084] Any suitable light-activated polypeptide may be used in the present method, as described further below. In some embodiments, the light-activated polypeptide is a light-activated ion channel (e.g., channelrhodopsin 2 (ChR2); C1V1; SFO; SSFO; etc.) where administering the light stimulus depolarizes the subtype of neurons in the sleep circuit and that express the light- activated ion channel. In some embodiments, the light-activated polypeptide is a light-activated ion pump, where administering the light stimulus hyperpolarizes the subtype of neurons in the sleep circuit and that express the light-activated ion pump.

Method of Identifying an Agent that Promotes or Suppresses Sleep

[0085] Also provided herein are methods, e.g., screening methods, for identifying an agent that promotes or suppresses sleep in an individual. The present method may include administering to an individual (e.g., a non-human mammal, such as a rat, a mouse, a non-human primate, etc.) a test agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons, as described above; and monitoring a sleep circuit-regulated state (e.g., REM sleep, non-REM sleep and/or wakefulness) of the individual. If the test agent changes one or more probabilities for the individual being in a sleep circuit-regulated state (e.g., REM sleep, non- REM sleep and/or wakefulness), the test agent may be determined to be an agent that modulates sleep, e.g., promotes or suppresses sleep. For example, if the test agent increases the probability of REM sleep and/or non-REM sleep, and reduces the probability of wakefulness in the individual, the test agent may be determined to be an agent that promotes sleep. Likewise, if the test agent reduces the probability of REM sleep and/or non-REM sleep, and increases the probability of wakefulness in the individual, the test agent may be determined to be an agent that suppresses sleep. Test agents identified according to the present method may find use in promoting or suppressing sleep in an individual in need, e.g., an individual with a sleeping disorder.

[0086] The gene product may be any suitable gene product of a gene whose expression is

upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→ ™ ⁿ neurons. Suitable genes are listed in, e.g., Table 3, in Figures 18A-18AI. In some embodiments, the test agent targets a gene product of a gene that is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, by 1.5 fold or more, e.g., 1.8 fold or more, 2.0 fold or more, 2.5 fold or more, including 3.0 fold or more, and in some cases, by 100 fold or less, e.g., 50 fold or less, 30 fold or less, 20 fold or less, 10 fold or less, 5.0 fold or less, including 4.0 fold or less, compared to its respective average expression level in the POA. In some embodiments, the test agent targets a gene product of a gene that is upregulated in GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons, by a range of 1.5 fold to 100 fold, e.g., 1.5 fold to 50 fold, 1.5 fold to 30 fold, 2.0 fold to 30 fold, 2.5 fold to 30 fold, including 3.0 fold to 30 fold, compared to its respective average expression level in the POA.

[0087] In some embodiments, the gene product encoded by a gene whose expression is

upregulated in the GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→1mn neurons, includes a cell-surface receptor, such as a G-protein coupled receptor (GPCR). The GPCR targeted by the test agent in the present method may include one or more GPCRs selected from 5-hydroxytryptamine (serotonin) receptor IF; cholinergic receptor, muscarinic 4; arginine vasopressin receptor 1A, adrenergic receptor, beta 1; progestin and adipoQ receptor family member VII; G protein-coupled receptor 83; and G protein-coupled receptor 83 (see also, Table 2 in Figure 17).

[0088] The test agent may be any suitable agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. The test agent may be, for example, a protein or polypeptide, nucleic acid, lipid, carbohydrate, antibody, small molecule, inorganic molecule, etc., that modulates a functional activity of the gene product. In some embodiments, the test agent selectively or specifically interacts with the gene product to modulate the function of the gene product. The specific interaction may include a specific binding interaction between the test agent and the gene product (e.g., RNA or polypeptide). The specific binding may be

characterized by a dissociation constant (K _D ) of 10 ⁶ M or less, 10 ⁷ M or less, 10 ⁸ M or less, 10 ⁹ M or less, 10 ¹⁰ M or less, 10 ¹¹ M or less, including 10 ¹² M or less.

[0089] The test agent of the present disclosure may specifically interact with a gene product encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, as described above, in a manner sufficient to modulate, e.g., inhibit or enhance, a functional activity of the gene product. The functional activity modulated by the test agent may vary depending on the whether the gene product with which the test agent specifically interacts is a post-transcriptional nucleic acid gene product, e.g., RNA, or post-translational gene product, e.g., polypeptide, and/or may depend on the functional role of the gene product, e.g., polypeptide, in the neuronal subtype and/or the sleep circuit. In some embodiments, where the gene product targeted by the test agent is a post-transcriptional nucleic acid gene product, e.g., RNA, the test agent may specifically interact with the gene product to reduce or prevent translation of the transcribed RNA, and/or reduce the level of the transcribed RNA in the neuron. In some embodiments, where the gene product is a post-translational gene product, e.g, polypeptide, the test agent may specifically interact with the gene product to reduce or suppress a function of the polypeptide, e.g., reduce or suppress an enzymatic function, binding function, structural function, signaling function, etc.

[0090] In some embodiments, the gene product is a nucleic acid gene product, such as an RNA transcribed from the gene, or a polypeptide gene product, such as a protein translated from an mRNA encoded by the gene. In some embodiments, the functional activity of a nucleic acid gene product, such as an RNA, may be modulated by a test agent that specifically interacts with the nucleic acid gene product. A test agent that specifically interacts with, e.g., specifically binds to, the nucleic acid gene product may be any suitable test agent, such as, but not limited to, a nucleic acid that hybridizes to the nucleic acid gene product, a nucleic acid-binding polypeptide that binds to the nucleic acid gene product in a sequence-specific manner, etc. In some cases, the test agent is an inhibitory RNA, e.g., a small-interfering RNA (siRNA) or short hairpin RNA (shRNA) that is designed to target the nucleic acid gene product, e.g., RNA, encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^POA→TMN neurons.

[0091] Thus, in some embodiments, the test agent can specifically bind to a post-transcriptional nucleic acid gene product, e.g., RNA, encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→ ™ ⁿ neurons. The specificity of the interaction may be determined by the structural relationship between a nucleotide sequence of the post-transcriptional nucleic acid gene product, e.g., RNA, and the test agent. In some cases, the test agent is a nucleic acid containing a nucleotide sequence that can hybridize with the post-transcriptional nucleic acid gene product, e.g., RNA, encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons. In some cases, the test agent is an inhibitory RNA, e.g., a small-interfering RNA (siRNA) or short hairpin RNA (shRNA) that is designed to target the post-transcriptional nucleic acid gene product, e.g., RNA, encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. Any suitable method may be used to design and administer a nucleic acid to target the post-transcriptional nucleic acid gene product, e.g., RNA, encoded by the gene whose expression is upregulated in the

GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. Suitable methods are described, e.g., in US App. Pub. Nos. 20100143450; 20110009468; 2011020779; 20110319475; 20120165397; 20120177723; PCT Pub. No. WO2013/138783; Baker-Herman et al., 2004 Nat Neurosci. 7:48; Pettersson et al., 2014 PLoS One 9:el00730; Jacobs et al., 1999 Neoplasia 1:402, which are incorporated herein by reference. Commercial sources of siRNA and/or shRNA include Santa Cruz Biotech and Thermo Fisher.

[0092] In some embodiments, the test agent specifically binds to a post-translational gene

product, e.g., polypeptide, encoded by the gene whose expression is upregulated in the

GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. The specificity of the interaction may be determined by the structural relationship between the post-translational gene product, e.g., polypeptide, and the test agent. In some cases, the test agent is an antibody that specifically binds to an epitope on the post-translational gene product, e.g., polypeptide, encoded by the gene whose expression is upregulated in the GABAergic, TMN-projecting POA neurons, e.g., GAE) ^POA→1MN neurons. In some cases, the test agent is a small molecule that specifically binds to one or more interaction pockets in or on the post-translational gene product, e.g., polypeptide, encoded by the gene whose expression is upregulated in the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons.

[0093] In some embodiments, the functional activity of a polypeptide gene product, such as an enzyme, scaffold protein, structural protein, signaling protein, etc., may be modulated by a test agent that specifically interacts with the polypeptide gene product. The specific interaction in some cases may be a specific binding interaction. A test agent that specifically binds to the polypeptide gene product may be any suitable test agent, such as, but not limited to, a polypeptide, an antibody or a small molecule. For example, if the polypeptide gene product binds a binding partner for functional activity, the test agent may be a polypeptide containing at least the binding domain on the binding partner that is sufficient for the specific binding interaction. In another example, an antibody test agent may specifically bind to an epitope on the polypeptide gene product to functionally modulate the activity of the polypeptide gene product. The test agent may be an agent that functionally modulates the activity of a polypeptide gene product in any other suitable manner.

[0094] The modulation of the functional activity of the gene product by the test agent through its specific interaction with the gene product, as described above, may in turn result in selective modulation of the activity of a subtype of neurons of a sleep circuit of a brain of the individual, to promote or suppress a sleep circuit-regulated state, as described herein. In some embodiments, where the gene product normally promotes the activity of the subtype of neurons of the sleep circuit to promote a sleep circuit-regulated state, a test agent that inhibits the functional activity of the gene product may suppress the sleep circuit-regulated state. In some embodiments, where the gene product normally suppresses the activity of the subtype of neurons of the sleep circuit to promote a sleep circuit-regulated state, a test agent that inhibits the functional activity of the gene product may suppress the sleep circuit-regulated state. In some embodiments, where the gene product normally promotes the activity of the subtype of neurons of the sleep circuit to promote a sleep circuit-regulated state, a test agent that enhances the functional activity of the gene product may further promote the sleep circuit-regulated state. In some embodiments, where the gene product normally suppresses the activity of the subtype of neurons of the sleep circuit to promote a sleep circuit-regulated state, a test agent that enhances the functional activity of the gene product may suppress the sleep circuit-regulated state.

[0095] The monitoring may be done in any suitable manner to determine a sleep circuit- regulated state (e.g., REM sleep, non-REM sleep and/or wakefulness) of the individual. In some cases, the state of the individual is monitored using electroencephalography, electromyography, or a combination thereof. Thus, in some cases, REM sleep is defined by having a high power at theta frequencies as measured by EEG; non-REM sleep is defined by a synchronized EEG with high amplitude and low frequency; and wakefulness is defined by desynchronized EEG. In some embodiments, REM sleep is defined by a low EMG activity; non-REM sleep is defined by low EMG activity; and wakefulness is defined by high EMG activity. In some embodiments, REM sleep is defined by a high power at theta frequencies as measured by EEG, and low EMG activity; non-REM sleep is defined by a synchronized EEG with high amplitude and low frequency, and low EMG activity; and wakefulness is defined by desynchronized EEG, and high EMG activity. The threshold for EEG frequency may vary depending on, e.g., the species to which the individual belongs. For example, if the individual is a mouse, the low EEG frequency may include a range of 0.5-4 Hz, and a theta frequency may include a range of 6-9 Hz. If the individual is a human, a theta frequency may include a range of 4-7 Hz.

[0096] The individual may be any suitable individual, and in some cases, the individual is an experimental animal, e.g., a mouse, rat, etc., that can be genetically manipulated to express, e.g., a heterologous gene product, such as a light-activated ion channel or ion pump, in one or more cells of interest, e.g., a subtype of neurons of a sleep circuit that includes GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons. In some embodiments, the individual is a non-human individual, e.g, a non-human mammal. In some cases, the individual is a human.

[0097] The test agent may be administered to an individual in a suitable pharmaceutical

composition, as desired. In some embodiments, the test agent is in a pharmaceutical composition which, when administered to the individual, specifically modulates the functional activity of a gene product encoded by a gene whose expression is upregulated in the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons, e.g., in the brain.

Pharmaceutical compositions and methods of administration thereof

[0098] The pharmaceutical composition administered to the individual may include any other suitable components for administering, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate salts, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), and the like.

[0099] In some cases, a subject pharmaceutical composition will be suitable for injection into a subject, e.g., will be sterile. For example, in some embodiments, a subject pharmaceutical composition will be suitable for injection into a subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins.

[00100] In some embodiments, a test agent modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, is formulated in a sustained release dosage form that is designed to release the test agent at a predetermined rate for a specific period of time. Such sustained release formulations may include, for example, formulations for use in drug delivery implants or devices, e.g., ingestible devices.

[00101] For oral preparations, a test agent modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^→ neurons, 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.

[00102] In certain embodiments, a test agent modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, 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.

[00103] Unit dosage forms for oral administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, or tablet, contains a predetermined amount of the test agents of the present disclosure. Similarly, unit dosage forms for injection or intravenous administration may include one or more test agents that selectively modulate activity of a subtype of neurons of a sleep circuit in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00104] 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 the test agent of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present disclosure depend on the particular test agent or test agents employed and the effect to be achieved, and the pharmacodynamics associated with each agent in the subject.

[00105] Any suitable pharmaceutically acceptable excipients, such as vehicles, adjuvants,

carriers or diluents may be employed in the subject methods. Moreover, any suitable pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, may be used.

[00106] Any of the test agents of the present disclosure may be formulated for use in any route of administration or dosage form disclosed herein. [00107] In practicing the methods of the present disclosure, routes of administration may be selected according to any of a variety of factors, such as properties of the test agent(s) to be delivered, the type of condition to be treated (e.g., type of sleeping disorder), and the like. For instance the pharmaceutical composition containing aa test agent of the present disclosure, e.g., a test agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons, may be delivered systemically or locally. In some instances, pharmaceutical composition containing a test agent of the present disclosure are administered orally, such as through the digestive tract (enteral administration), buccal, sublabial, or sublingual

administration. Such dosage forms may be pills, tablets, capsules, time-release formulations, osmotic controlled release formulations, solutions, softgels, hydrogels, suspensions, emulsions, syrups, orally disintegrating tablets, films, lozenges, chewing gums, mouthwashes, ointments, and the like.

[00108] Pharmaceutical compositions of the present disclosure, e.g., compositions that include a test agent that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons, can be administered by direct injection into a target tissue or into the blood stream, including intradermal, subcutaneous, intravenous, intramuscular, intraosseous, or intraperitoneal injection. Pharmaceutical compositions of the present disclosure can be administered by intracavernous or intravitreal delivery to organs or tissues, or administered by intracerebral, intrathecal, or epidural delivery to tissues of the central nervous system.

[00109] For some conditions, it may be necessary to formulate test agents to cross the blood- brain barrier (BBB). One strategy for drug delivery through the blood-brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or

biochemically by the use of vasoactive substances such as bradykinin. The potential for using BBB opening to target specific agents to brain regions is also an option. A BBB disrupting agent can be co-administered with test agents of the present disclosure when the test agents are administered by intravascular injection. Other strategies to go through the BBB may entail the use of endogenous transport systems, including Caveolin-1 mediated transcytosis, carrier- mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. Active transport moieties may also be conjugated to the test agents for use in the invention to facilitate transport across the endothelial wall of the blood vessel.

[00110] Local administration of the pharmaceutical compositions may include intrathecal

administration, which may be carried out through the use of an Ommaya reservoir, in accordance with known techniques (F. Balis et al., Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989), see also, e.g. US Patent Nos. 5,222,982 and 5385582, incorporated herein by reference).

[00111] Where the pharmaceutical compositions are locally administered in the brain, one

method for administration of the pharmaceutical compositions of the present disclosure is by deposition into or near the site by any suitable technique, such as by direct injection (aided by stereotaxic positioning of an injection syringe, if necessary) or by placing the tip of an Ommaya reservoir into a cavity, or cyst, for administration. Alternatively, a convection-enhanced delivery catheter may be implanted directly into the site, into a natural or surgically created cyst, or into the normal brain mass {see e.g. US Application No. 20070254842, incorporated here by reference). Such convection-enhanced pharmaceutical composition delivery devices greatly improve the diffusion of the composition throughout the brain mass. The implanted catheters of these delivery devices utilize high-flow microinfusion (with flow rates in the range of about 0.5 to 15.0 μΐ/minute), rather than diffusive flow, to deliver the test agent to the brain. Such devices are described in U.S. Patent No. 5,720,720, incorporated fully herein by reference.

[00112] In the methods of the present disclosure, the test agents may be administered to the patient using any convenient routes of administration that are capable of resulting in the desired modulation of a sleep-circuit regulated states, or treatment of a sleeping disorder. Thus, the test agents can be incorporated into a variety of formulations for therapeutic administration. More particularly, the test agents of the present disclosure 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 or liquid forms, such as tablets, capsules, powders, granules, ointments, solutions and injections.

[00113] In pharmaceutical dosage forms, the test agents of the present disclosure may 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 previously-described routes of administration and dosage forms are merely exemplary and are in no way limiting.

[00114] In certain embodiments, a test agent that modulates a functional activity of a gene

product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, may be administered at a dosage that is sufficient to modulate the activity of the sleep circuit. Thus, the dosage of the test agent will vary, depending upon the mode of action of the test agent, the frequency of administration, the manner of administration, the clearance of the test agent from the subject, and the like. [00115] In certain embodiments, a small molecule that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, is administered at a dose ranging from 0.1 g/kg to 200 mg/kg body weight, e.g., 0.5 g/kg to 175 mg/kg body weight, 1 μg/kg to 150 mg/kg body weight, 5 g/kg to 125 mg/kg body weight, 10 μg/kg to 100 mg/kg body weight, 20 μg/kg to 50 mg/kg body weight, 50 μg/kg to 20 mg/kg body weight, 75 μg/kg to 10 mg/kg body weight, 100 μg/kg to 5 mg/kg body weight, including 100 μg/kg to 1 mg/kg body weight, to modulate the activity of the sleep circuit in an individual. In certain embodiments, a small molecule that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^poa→ ™ ⁿ neurons, may be administered at a dose of 0.1 μg/kg or more, e.g., 0.5 μg/kg or more, 1 μg/kg or more, 5 μg/kg or more, 10 μg/kg or more, 20 μg/kg or more, 50 μg/kg or more, 100 μg/kg or more, 200 μg/kg or more, 500 μg/kg or more 1 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 50 mg/kg or more, 75 mg/kg or more, 100 mg/kg or more, by body weight, and in some cases the dose may be 200 mg/kg or less, e.g., 150 mg/kg or less, 100 mg/kg or less, 75 mg/kg or less, 50 mg/kg or less, 25 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 750 μg/kg or less, 500 μg/kg or less, 250 μg/kg or less, 100 μg/kg or less, 75 μg/kg or less, 50 μg/kg or less, 20 μg/kg or less, 10 μg/kg or less, 5 μg/kg or less or 1 μg/kg or less, by body weight, to modulate the activity of the sleep circuit in an individual. In certain embodiments, a small molecule that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons, is administered at a dose or in a dosage regimen that provides for a target tissue and/or blood concentration in the range of 0.1 to 1000 nM, e.g., 1 to 500 nM, 10 to 400 nM, 20 to 300 nM, 25 to 250 nM, 30 to 200 nM, including 50 to 150 nM.

[00116] In certain embodiments, a polypeptide or antibody that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons, may be administered at a dose ranging from 1 μg/kg to 150 mg/kg body weight, e.g., 5 μg/kg to 125 mg/kg body weight, 10 μg/kg to 100 mg/kg body weight, 20 μg/kg to 75 mg/kg body weight, 50 μg/kg to 50 mg/kg body weight, 75 μg/kg to 20 mg/kg body weight, 100 μg/kg to 10 mg/kg body weight, including 100 μg/kg to 1 mg/kg body weight, to modulate the activity of the sleep circuit in an individual. In certain embodiments, a polypeptide or antibody that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, may be administered at a dose of 1 μg/kg or more, e.g., 5 μg/kg or more, 10 μg/kg or more, 50 μg/kg or more, 100 μg/kg or more, 200 μg/kg or more, 500 μg/kg or more 1 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 50 mg/kg or more, 75 mg/kg or more, 100 mg/kg or more, by body weight, and in some cases the dose may be 200 mg/kg or less, e.g., 150 mg/kg or less, 100 mg/kg or less, 75 mg/kg or less, 50 mg/kg or less, 25 mg/kg or less, 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 750 μg/kg or less, 500 g/kg or less, 250 g/kg or less, 100 μg/kg or less, 75 μg/kg or less, 50 μg/kg or less, 20 μg/kg or less, or 10 μg/kg or less, by body weight, to modulate the activity of the sleep circuit in an individual. In certain embodiments, a polypeptide that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons, is administered at a dose or in a dosage regimen that provides for a target tissue and/or blood concentration in the range of 1 to 1 ,000 nM, e.g., 5 to 900 nM, 10 to 800 nM, 20 to 750 nM, 50 to 700 nM, 100 to 600 nM, including 200 to 600 nM. In certain embodiments, an antibody that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, is administered at a dose or in a dosage regimen that provides for a target tissue and/or blood concentration in the range of 0.01 to 1 ,000 μg/ml, e.g., 0.05 to 500 μ^ιηΐ, 0.1 to 250 μ^ητΐ, 0.5 to 100 μg/ml, 1 to 50 μg/ml, 1 to 40 μ^ητΐ, including 1 to 25 μg/ml.

[00117] A test agent modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in GABAergic, TMN-projecting POA neurons, e.g., QAD ^POA→1MN neurons, may be administered according to any suitable dosage regimen, including, but not limited to, daily administration, weekly administration, biweekly administration, monthly administration, semiannual administration, etc.

GENETICALLY MODIFIED ANIMALS

[00118] Also provided herein are non-human animals (e.g., non-human mammals; e.g., rats; mice; rabbits; non-human primates; etc.) that find use in performing methods of the present disclosure, and methods for generating the non-human animals. The present non-human animals have a subtype of neurons genetically modified to selectively express light-activated

polypeptide, e.g. a light-activated ion channel, such as channelrhodopsin 2, where the subtype of neurons belong to a neural sleep circuit that contains GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, as described above. In some embodiments, the light- activated polypeptide is expressed in GABAergic, TMN-projecting POA neurons, e.g.,

GAD ^POA→1MN neurons. In some embodiments, the light-activated polypeptide is expressed in a subtype of neurons that are synaptically connected to the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. In some embodiments, the light-activated polypeptide is expressed in a subtype of neurons that synapse onto the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons, which subtype of neurons may include neurons of the hypothalamus or the amygdala. In some cases, the amygdala neurons are GABAergic neurons of the CEA.

[00119] Also provided herein are methods of generating the present genetically modified non- human animals, e.g., genetically modified non-human mammals. The present method of generating a genetically modified non-human mammal of the present disclosure may include administering an expression vector, e.g., a viral expression vector, containing a nucleic acid that encodes a light-activated polypeptide, to a region of the brain that includes one or more subtypes of neurons of the present sleep circuit, e.g., a sleep circuit that includes the GABAergic, TMN- projecting POA neurons, e.g., QAD ^poa→1mn neurons. To deliver the expression vector, e.g., a viral expression vector, to the target neurons, the expression vector may be packaged I any suitable viral particle (i.e., virions). In some cases, the host animal may be a transgenic animal that expresses a site-specific recombinase (e.g., Cre or Flp recombinase) under a cell type- specific promoter. In some cases, a viral vector that includes a nucleic acid encoding a site- specific recombinase may be administered to a brain region of the host animal, to provide the recombinase activity in neurons of the brain region. The viral vector may include recombination sites specific for the site-specific recombinase (e.g., Lox site, for Cre; FRT site for Flp), where the recombination sites are configured relative to a nucleic acid sequence encoding the light- activated polypeptide in such a way as to provide for expression of the light-activated polypeptide specifically in cells that express the site-specific recombinase. The viral vector may include any suitable control elements (e.g., promoters, enhancers, etc.) to control expression of the light-activated polypeptide according to neuronal subtype, timing, presence of an inducer, etc.

[00120] In some cases, the expression vector encoding the light-activated polypeptide may

include a regulatory element, e.g., a promoter, that is specific for the target neurons, at least within the region of the brain in which the expression vector is administered.

[00121] Any suitable arrangement of recombination sites and other regulatory elements may be used in the viral vector, such as, but not limited to, a double inverted open reading frame (DIO) strategy. Suitable methods are described in, e.g, Fenno et al., Nat Methods. 2014 Jul;l l(7):763; Gompf et al., Front Behav Neurosci. 2015 Jul 2;9:152; Cetin et al., J. Neurophysiol. Ill, 2150- 2159.

[00122] Neuron-specific promoters and other control elements (e.g., enhancers) are known in the art. Suitable neuron-specific control sequences include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSEN02, X51956; see also, e.g., U.S. Pat. No. 6,649,811, U.S. Pat. No. 5,387,742); 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: 1161); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363- 2384 (1987) and Neuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al, Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMV enhancer/platelet-derived growth factor-β promoter (see, e.g., Liu et al. (2620) Gene Therapy 11 :52-60); a motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2620) Development 131 :3295-3306); and an alpha subunit of Ca( ²⁺ )-calmodulin-dependent protein kinase II (CaMKIIoc) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93: 13250). Other suitable promoters include elongation factor (EF) la and dopamine transporter (DAT) promoters.

[00123] Any suitable viral vector may be used to deliver a nucleic acid, e.g., viral vector,

encoding the light-activated polypeptide. Suitable viral vectors include, without limitation, adeno-associated viral vector and lentiviral vectors. The viral particle (i.e., virion) may be any suitable viral particle. In some cases, the envelope of the viral particle is derived from more than one type of virus. Thus, in some cases, the viral particle of one type of virus is pseudotyped with an envelope protein from another type of virus. In some embodiments, the viral particle is pseudotyped with a rabies glycoprotein. In some embodiments, the viral particle is an equine infectious anemia virus (EIAV) pseudotyped with a rabies glycoprotein.

[00124] The site-specific recombinase may be under the control of any suitable cell type-specific promoter. In some embodiments, the site-specific recombinase is expressed in, without limitation, GABAergic neurons, glutamatergic neurons, cholinergic neurons, histaminergic neurons, serotonergic neurons, or dopaminergic neurons, etc. Suitable cell type-specific promoters include, without limitation, promoter elements for GAD1, GAD1, VGAT, GAT1, VGLUT2, CCK, CRH, TAC1, GAL, etc.

[00125] The brain region to which the viral particles are administered may be any suitable brain region with functional connectivity to the sleep circuit, e.g., a sleep circuit that contains the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. In some cases, the brain region is the TMN. In some embodiments, the brain region is the POA. In some embodiments the brain region is the CEA. The brain region may be any other region of the brain with a subtype of neurons that have synaptic connections to the GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→TMN neurons.

[00126] The present method of generating a transgenic non-human mammal of the present

disclosure can involve use of a gene-editing composition, e.g., a CRISPR/Cas system comprising a CRISPR/Cas endonuclease (e.g., Cas9) and a guide RNA. Methods of using a CRISPR/Cas system to generate make genome modifications in non-human mammals are known in the art; see, e.g., Lie et al. (2013) Nat. Biotechnol. 31 :684; Yang et al. (2013) Cell 154: 1370; and Niu et al. (2014) Cell 156:836.

LIGHT-ACTIVATED POLYPEPTIDES

[00127] A light-activated polypeptide for use in methods of the present disclosure may be any suitable light-activated polypeptide for selectively modulating activity of a subtype of neurons by illuminating the neurons with a light stimulus. In some instances, the light-activated polypeptide is a light-activated ion channel polypeptide. The light-activated ion channel polypeptides are adapted to allow one or more ions to pass through the plasma membrane of a target cell when the polypeptide is illuminated with light of an activating wavelength. Light- activated proteins may be characterized as ion pump proteins, which facilitate the passage of a small number of ions through the plasma membrane per photon of light, or as ion channel proteins, which allow a stream of ions to freely flow through the plasma membrane when the channel is open. In some embodiments, the light-activated polypeptide depolarizes the cell when activated by light of an activating wavelength. In some embodiments, the light-activated polypeptide hyperpolarizes the cell when activated by light of an activating wavelength. Suitable hyperpolarizing and depolarizing polypeptides, such as opsins, are known in the art and include, e.g., a channelrhodopsin (e.g., ChR2), variants of ChR2 (e.g., C128S, D156A, C128S + D156A, E123A, E123T), iClC2, C1C2, GtACR2, NpHR, eNpHR3.0, C1V1, VChRl, VChR2, SwiChR, Arch, ArchT, KR2, ReaChR, ChiEF, Chronos, ChRGR, CsChrimson, and the like. In some cases, the light-activated polypeptide includes bReaCh-ES, as described herein and described further in, e.g., Rajasethupathy et al., Nature. 2015 Oct 29;526(7575):653, which is incorporated by reference. Hyperpolarizing and depolarizing opsins have been described in various publications; see, e.g., Berndt and Deisseroth (2015) Science 349:590; Berndt et al. (2014) Science 344:420; and Guru et al. (July 25, 2015) Intl. J. Neuropsychopharmacol. pp. 1-8 (PMID 26209858).

[00128] The present disclosure provides for the modification of light-activated polypeptides, e.g., opsins, expressed in a cell by the addition of one or more amino acid sequence motifs which enhance transport to the plasma membranes of mammalian cells. Light-activated polypeptides having components derived from evolutionarily simpler organisms may not be expressed or tolerated by mammalian cells or may exhibit impaired subcellular localization when expressed at high levels in mammalian cells. Consequently, in some embodiments, the light-activated polypeptides expressed in a cell can be fused to one or more amino acid sequence motifs selected from the group consisting of a signal peptide, an endoplasmic reticulum (ER) export signal, a membrane trafficking signal, and/or an N-terminal golgi export signal. The one or more amino acid sequence motifs which enhance light-activated polypeptide transport to the plasma membranes of mammalian cells can be fused to the N-terminus, the C-terminus, or to both the N- and C-terminal ends of the light-activated polypeptide. Optionally, the light-activated polypeptide and the one or more amino acid sequence motifs may be separated by a linker. In some embodiments, the light-activated polypeptide can be modified by the addition of a trafficking signal (ts) which enhances transport of the protein to the cell plasma membrane. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In other embodiments, the trafficking signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: l).

[00129] Trafficking sequences that are suitable for use can comprise an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to an amino acid sequence such as a trafficking sequence of human inward rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: l)).

[00130] A trafficking sequence can have a length of from about 10 amino acids to about 50

amino acids, e.g., from about 10 amino acids to about 20 amino acids, from about 20 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[00131] Signal sequences that are suitable for use can comprise an amino acid sequence having

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to an amino acid sequence such as one of the following:

[00132] 1) the signal peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ

ID NO:2))

[00133] 2) the β2 subunit signal peptide of the neuronal nicotinic acetylcholine receptor (e.g.,

MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO:3));

[00134] 3) a nicotinic acetylcholine receptor signal sequence (e.g.,

MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO:4)); and [00135] 4) a nicotinic acetylcholine receptor signal sequence (e.g., MRGTPLLLVVSLFSLLQD

(SEQ ID NO:5)).

[00136] A signal sequence can have a length of from about 10 amino acids to about 50 amino acids, e.g., from about 10 amino acids to about 20 amino acids, from about 20 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[00137] Endoplasmic reticulum (ER) export sequences that are suitable for use in a modified opsin of the present disclosure include, e.g., VXXSL (where X is any amino acid) (SEQ ID NO:6) (e.g., VKESL (SEQ ID NO:7); VLGSL (SEQ ID NO:8); etc.); NANSFCYENEVALTSK (SEQ ID NO:9); FXYENE (SEQ ID NO: 10) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 11); and the like. An ER export sequence can have a length of from about 5 amino acids to about 25 amino acids, e.g., from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, or from about 20 amino acids to about 25 amino acids.

[00138] In some embodiments, the signal peptide sequence in the protein can be deleted or substituted with a signal peptide sequence from a different protein.

Light-activated ion channels

[00139] In some aspects, the light-activated polypeptide is an ion channel protein, e.g., a cation channel protein, that can be derived from Chlamydomonas reinhardtii, wherein the cation channel protein can be capable of transporting cations across a cell membrane when the cell is illuminated with light. In another embodiment, the light-activated cation channel protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 12. The light used to activate the light-activated cation channel protein derived from Chlamydomonas reinhardtii can have a wavelength between about 460 and about 495 nm or can have a wavelength of about 480 nm. Additionally, light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can be used to activate the light-activated polypeptide. In some embodiments, activation of the light-activated cation channel derived from Chlamydomonas reinhardtii with light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can cause depolarization-induced synaptic depletion of the neurons expressing the light-activated cation channel. The light-activated cation channel protein can additionally comprise substitutions, deletions, and/or insertions introduced into a native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to particular wavelengths of light, and/or increase or decrease the ability of the light-activated cation channel protein to regulate the polarization state of the plasma membrane of the cell. Additionally, the light- activated cation channel protein can contain one or more conservative amino acid substitutions and/or one or more non-conservative amino acid substitutions. The light-activated cation channel protein comprising substitutions, deletions, and/or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane. In another embodiment, the light-activated cation channel protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the C1C2 amino sequence shown in Figure 19B, and set forth in SEQ ID NO: 19. A crystal structure of C1C2 is presented in Kato et al. (2012) Nature 482:369. In another embodiment, the light-activated cation channel protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the ReaChR amino sequence shown in Figure 19B, and set forth in SEQ ID NO:20.

In some embodiments, the light-activated cation channel comprises a T159C substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light- activated cation channel comprises a L132C substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises an E123T substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises an El 23 A substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises a T159C substitution and an E123T substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises a T159C substitution and an E123A substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises a T159C substitution, an L132C substitution, and an E123T substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises a T159C substitution, an L132C substitution, and an El 23 A substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises an L132C substitution and an E123T substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the light-activated cation channel comprises an L132C substitution and an E123A substitution of the amino acid sequence set forth in SEQ ID NO: 12. In some

embodiments, the light-activated cation channel comprises an H143R amino acid substitution of the amino acid sequence set forth in SEQ ID NO: 12. [00141] In some embodiments, a ChR2 protein comprises at least one (such as one, two, three, or more) amino acid sequence motifs that enhance transport to the plasma membranes of target cells selected from the group consisting of a signal peptide, an ER export signal, and a membrane trafficking signal. In some embodiments, the ChR2 protein comprises an N-terminal signal peptide and a C-terminal ER export signal. In some embodiments, the ChR2 protein comprises an N-terminal signal peptide and a C-terminal trafficking signal. In some

embodiments, the ChR2 protein comprises an N-terminal signal peptide, a C-terminal ER export signal, and a C-terminal trafficking signal. In some embodiments, the ChR2 protein comprises a C-terminal ER export signal and a C-terminal trafficking signal. In some embodiments, the C- terminal ER export signal and the C-terminal trafficking signal are linked by a linker. The linker can comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent protein, for example, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments the ER export signal is more C-terminally located than the trafficking signal. In some embodiments the trafficking signal is more C-terminally located than the ER Export signal.

Step function opsins and stabilized step function opsins

[00142] In other embodiments, the light-activated cation channel protein can be a step function opsin (SFO) protein or a stabilized step function opsin (SSFO) protein that can have specific amino acid substitutions at key positions throughout the retinal binding pocket of the protein. In some embodiments, the SFO protein can have a mutation at amino acid residue C128 of SEQ ID NO: 12. In other embodiments, the SFO protein has a C128A mutation in SEQ ID NO: 12. In other embodiments, the SFO protein has a C128S mutation in SEQ ID NO: 12. In another embodiment, the SFO protein has a C128T mutation in SEQ ID NO: 12. In some embodiments, the SFO protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 13.

[00143] In some embodiments, the SSFO protein can have a mutation at amino acid residue

D156 of SEQ ID NO: 12. In other embodiments, the SSFO protein can have a mutation at both amino acid residues C128 and D156 of SEQ ID NO: 12. In one embodiment, the SSFO protein has an C128S and a D156A mutation in SEQ ID NO: 12. In another embodiment, the SSFO protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 14. In another embodiment, the SSFO protein can comprise a C128T mutation in SEQ ID NO: 12. In some embodiments, the SSFO protein comprises C128T and D156A mutations in SEQ ID NO: 12. [00144] In some embodiments the SFO or SSFO proteins provided herein can be capable of mediating a depolarizing current in the cell when the cell is illuminated with blue light. In other embodiments, the light can have a wavelength of about 445 nm. Additionally, in some embodiments the light can be delivered as a single pulse of light or as spaced pulses of light due to the prolonged stability of SFO and SSFO photocurrents. In some embodiments, activation of the SFO or SSFO protein with single pulses or spaced pulses of light can cause depolarization- induced synaptic depletion of the neurons expressing the SFO or SSFO protein. In some embodiments, each of the disclosed step function opsin and stabilized step function opsin proteins can have specific properties and characteristics for use in depolarizing the membrane of a neuronal cell in response to light.

[00145] Further disclosure related to SFO or SSFO proteins can be found in International Patent

Application Publication No. WO 2010/056970, the disclosure of which is hereby incorporated by reference in its entirety.

ClVl chimeric cation channels

[00146] In other embodiments, the light-activated cation channel protein can be a ClVl chimeric protein derived from the VChRl protein of Volvox carteri and the ChRl protein from

Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChRl having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChRl ; is responsive to light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments, the ClVl protein can further comprise a replacement within the intracellular loop domain located between the second and third transmembrane helices of the chimeric light responsive protein, wherein at least a portion of the intracellular loop domain is replaced by the

corresponding portion from ChRl. In another embodiment, the portion of the intracellular loop domain of the ClVl chimeric protein can be replaced with the corresponding portion from ChRl extending to amino acid residue A 145 of the ChRl. In other embodiments, the ClVl chimeric protein can further comprise a replacement within the third transmembrane helix of the chimeric light responsive protein, wherein at least a portion of the third transmembrane helix is replaced by the corresponding sequence of ChRl. In yet another embodiment, the portion of the intracellular loop domain of the ClVl chimeric protein can be replaced with the corresponding portion from ChRl extending to amino acid residue W 163 of the ChRl. In other embodiments, the ClVl chimeric protein can comprise an amino acid sequence at least about 90%, 91 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 15. [00147] In some embodiments, the ClVl protein can mediate a depolarizing current in the cell when the cell is illuminated with green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 542 nm. In some embodiments, the ClVl chimeric protein is not capable of mediating a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein is not capable of mediating a depolarizing current in the cell when the cell is illuminated with light having a wavelength of about 405 nm. Additionally, in some embodiments, light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can be used to activate the ClVl protein.

ClVl chimeric mutant variants

[00148] In some aspects, the present disclosure provides polypeptides comprising substituted or mutated amino acid sequences, wherein the mutant polypeptide retains the characteristic light- activatable nature of the precursor ClVl chimeric polypeptide but may also possess altered properties in some specific aspects. For example, the mutant light-activated ClVl chimeric proteins described herein can exhibit an increased level of expression both within an animal cell or on the animal cell plasma membrane; an altered responsiveness when exposed to different wavelengths of light, particularly red light; and/or a combination of traits whereby the chimeric ClVl polypeptide possess the properties of low desensitization, fast deactivation, low violet- light activation for minimal cross-activation with other light-activated cation channels, and/or strong expression in animal cells.

[00149] Accordingly, provided herein are ClVl chimeric light-activated opsin proteins that can have specific amino acid substitutions at key positions throughout the retinal binding pocket of the VChRl portion of the chimeric polypeptide. In some embodiments, the ClVl protein can have a mutation at amino acid residue E122 of SEQ ID NO: 15. In some embodiments, the ClVl protein can have a mutation at amino acid residue E162 of SEQ ID NO: 15. In other embodiments, the ClVl protein can have a mutation at both amino acid residues E162 and E122 of SEQ ID NO: 15. In other embodiments, the ClVl protein can comprise an amino acid sequence at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[00150] In some aspects, the C1V1-E122 mutant chimeric protein is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 546 nm. In other embodiments, the C1V1-E122 mutant chimeric protein can mediate a depolarizing current in the cell when the cell is illuminated with red light. In some embodiments, the red light can have a wavelength of about 630 nm. In some embodiments, the C1V1-E122 mutant chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with light having a wavelength of about 405 nm. Additionally, in some embodiments, light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can be used to activate the C1V1-E122 mutant chimeric protein. In some embodiments, activation of the C1V1-E122 mutant chimeric protein with light pulses having a frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can cause depolarization-induced synaptic depletion of the neurons expressing the C1V1-E122 mutant chimeric protein.

[00151] In other aspects, the C1V1-E162 mutant chimeric protein is capable of mediating a

depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 535 nm to about 540 nm. In some embodiments, the light can have a wavelength of about 542 nm. In other embodiments, the light can have a wavelength of about 530 nm. In some embodiments, the CI VI -El 62 mutant chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with light having a wavelength of about 405 nm. Additionally, in some embodiments, light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can be used to activate the C1V1-E162 mutant chimeric protein. In some embodiments, activation of the C1V1-E162 mutant chimeric protein with light pulses having a frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can cause depolarization-induced synaptic depletion of the neurons expressing the CI VI -El 62 mutant chimeric protein.

[00152] In yet other aspects, the C1V1-E122/E162 mutant chimeric protein is capable of

mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments the light can be green light. In other embodiments, the light can have a wavelength of between about 540 nm to about 560 nm. In some embodiments, the light can have a wavelength of about 546 nm. In some embodiments, the C1V1-E122/E162 mutant chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with violet light. In some embodiments, the chimeric protein does not mediate a depolarizing current in the cell when the cell is illuminated with light having a wavelength of about 405 nm. In some embodiments, the C1V1-E122/E162 mutant chimeric protein can exhibit less activation when exposed to violet light relative to CI VI chimeric proteins lacking mutations at E122/E162 or relative to other light-activated cation channel proteins. Additionally, in some embodiments, light pulses having a temporal frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can be used to activate the C1V1-E122/E162 mutant chimeric protein. In some embodiments, activation of the CI VI- E122/E162 mutant chimeric protein with light pulses having a frequency in the range of about 5 Hz to about 100 Hz, e.g., about 10 Hz to about 80 Hz, about 10 Hz to about 60 Hz, including about 10 Hz to about 40 Hz, can cause depolarization-induced synaptic depletion of the neurons expressing the C1V1- E122/E162 mutant chimeric protein.

UTILITY

[00153] The present methods and transgenic animals find use in many applications where it is desired to identify agents, e.g., therapeutic agents, and/or using the agents to treat an individual for a sleeping disorder, e.g., to treat insomnia or narcolepsy. The sleeping disorder may be a disorder that is associated with a neural sleep circuit that includes GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons. In some cases, the sleeping disorder is caused by aberrant activity of neurons in the sleep circuit. In some cases, selective modulation of the sleep circuit that includes GABAergic, TMN-projecting POA neurons, e.g., GAD ^roA→ ™ ^N neurons can compensate for a deficiency the function of another sleep circuit.

EXAMPLES

[00154] 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 : Materials and Methods

[00155] The following materials and methods were used in Examples 2-6.

Animals

[00156] GAD2-IRES -Cre , VGLUT2-IRES-Cre, VGAT-IRES-Cre, GADl-eGFP, CCK-IRES-

Cre, CRH-IRES-Cre and TACl-IRES-Cre mice (Jackson stock no: 010802, 016963, 016962, 007677, 012706, 012704 and 021877 respectively) were obtained from Jackson Laboratory and VGLUT2-eGFP mice were from MMRRC (MMRRC#011835-UCD). GAL-Cre mice were obtained from GENSAT (stock no: KI87). Mice were housed in 12 hr light-dark cycle (lights on 07:00 am and off at 19:00 pm) with free access to food and water. All procedures were approved by Institutional Animal Care and Use Committees of the University of California, Berkeley, University of California, San Francisco (UCSF), Allen Institute for Brain Science and Stanford University and were done in accordance with federal regulations and guidelines on animal experimentation.

Virus Preparation

[00157] AAV ₂ -EFla-DIO-ChR2-eYFP and AAV ₂ -EFla-DIO-eYFP were obtained from the

University of North Carolina (UNC) vector core. The final titer was estimated to be ~10 ¹² gc/mL. AAV _DJ -EFla-DIO-ChR2-eYFP was obtained from Stanford University virus core. Lentivirus rEIAV-DIO-TLoop-ChR2-eYFP or rEIAV-DIO-TLoop-nlsPelican was obtained from Salk virus core. cTRIO reagents: CAV-FLEx ^loxP -Flp, AAV-FLEx^-TVA-mCherry, AAV- FLEx ^ERT -RG and EnvA-pseudotyped, glycoprotein (RG)-deleted, and GFP-expressing rabies viral particles (RVdG) were obtained from Stanford University. HSV-LoxSTOPLox-FlagHA- LlOa was obtained from UCSF.

Surgery

[00158] To implant electroencephalogram (EEG) and electromyogram (EMG) recording

electrodes, adult mice (6-12 weeks old) were anesthetized with 1.5-2% isoflurane and placed on a stereotaxic frame. Two stainless steel screws were inserted into the skull 1.5 mm from midline and 1.5 mm anterior to the bregma, and two others were inserted 3 mm from midline and 3.5 mm posterior to the bregma. Two EMG electrodes were inserted into the neck musculature. Insulated leads from the EEG and EMG electrodes were soldered to a 2 x 3 pin header, which was secured to the skull using dental cement. [00159] For optogenetic activation experiments, a craniotomy was made on top of the POA in the same surgery as for EEG and EMG implant, and 0.1-0.5 μΐ virus was injected into the target region using Nanoject II (Drummond Scientific) via a micro pipette. Optic fibers bilaterally were then implanted into the POA. Dental cement was applied to cover the exposed skull completely and to secure the implants for EEG and EMG recordings to the screws. After surgery, mice were allowed to recover for at least 3 weeks before experiments. For retrograde tracing in Figure 1 A, 0.2-0.3 μΐ red or green RetroBeads (Lumafluor, Inc) was injected into each target region.

[00160] For optrode recording experiments, the optrode assembly was inserted into the POA at a depth of 4.9 mm. Screws were attached to the skull for EEG recordings, and an EMG electrode was inserted into the neck musculature. The optrode assembly, screws and EEG/EMG electrodes were secured to the skull using dental cement.

[00161] For cTRIO experiments, CAV-FLEx ^loxP -Flp (5.0 x 10 ¹² gc/mL) was injected into either the TMN or the PFC, and AAV-FLEx^-TVA-mCherry (2.6 x 10 ¹² gc/mL) and AAV-FLEx ^ERT - RG (1.3 x 10 ¹² gc/mL) were injected into the POA. Two to three weeks later, EnvA- pseudotyped, glycoprotein (RG)-deleted, and GFP-expressing rabies viral particles (KVdG) (5.0 x 10 ^s colony forming units (cfu)/mL) were injected into the POA, and mice were sacrificed 1 week later for histology.

[00162] Stereotaxic coordinates for injections:

TMN: AP -2.45 mm, ML 1 mm, DV 5-5.2 mm from the cortical surface

POA: AP 0 mm, ML 0.7 mm, DV 5.2 mm

CEA: AP -1.5 mm, ML 2.5 mm, DV 3.8-4 mm

PFC: AP +2.0 mm, ML 0.4 mm, DV 2 mm

vlPAG: AP -4.7 mm, ML 0.7 mm, DV 2.3 mm

DMH: AP -1.8 mm, ML 0.4 mm, DV 5.2 mm

Behavior

[00163] Behavioral experiments were carried out in home cages placed in sound-attenuating boxes. Sleep recordings were carried out between 12:00 and 19:00 (light on at 7:00 and off at 19:00). EEG and EMG electrodes were connected to flexible recording cables via a mini- connector. EEG and EMG signals were recorded and amplified using AM Systems, digitally filtered (0.1-1000 Hz and 10-1000 Hz for EEG and EMG recordings respectively), and digitized at 600 Hz using Lab View. Spectral analysis was carried out using fast Fourier transform (FFT), and brain states were classified into NREM, REM and wake states (wake: desynchronized EEG and high EMG activity, NREM: synchronized EEG with high-amplitude, low-frequency (0.5-4 Hz) activity and low EMG activity, REM: high power at theta frequencies (6-9 Hz) and low EMG activity).

Sleep deprivation and sleep rebound experiment

[00164] Sleep deprivation started at the beginning of the light period (7 am) and lasted till 1 pm.

Mice were kept awake by a combination of cage tapping, introduction of foreign objects such as paper towels, cage rotation, and fur stroking with a paintbrush, gentle handling procedures that have been used extensively to induce sleep deprivation (Borbely et al., 1984). After 6 hr of deprivation, sleep-deprived mice were allowed rebound sleep for 4 hr before being euthanized by cervical dislocation and decapitation. c-Fos immunohistochemistry was performed as described below.

Optogenetic manipulation

[00165] Each optic fiber (200 μπι diameter; ThorLabs) was attached through an FC/PC adaptor to a 473-nm blue laser diode (Shanghai laser), and light pulses were generated using a Master 8 (A.M.P.I.). All photostimulation experiments were conducted bilaterally and fiber optic cables were connected at least 2 hr before the experiments for habituation. For photostimulation experiments in ChR2- or eYFP-expressing mice, light pulses (10 ms/pulse, 10 Hz, 4-8 mW) were triggered using Master 8 that provided simultaneous input into two blue lasers.

Optrode Recording

[00166] To identify ChR2-tagged neurons in the POA, custom-made optrodes were used

(Anikeeva et al., 2012). Extracellular signals recorded with a TDT RZ5 amplifier were filtered (0.3-8 kHz) and digitized at 25 kHz.

Optrode recordings

[00167] Custom-made optrodes consisted of an optic fiber (200 μπι in diameter) glued together with 6 pairs of stereotrodes. Two FeNiCr wires (Stablohm 675, California Fine Wire) were twisted together and electroplated to an impedance of ~ 600 kQ using a custom built plating device. The optrode was attached to a driver to allow vertical movement of the optrode assembly. Wires to record cortical EEG and EMG from neck musculatures were also attached for simultaneous recordings. A TDT RZ5 amplifier was used for all the recordings, signals were filtered (0.3-8 kHz) and digitized at 25 kHz. At the end of the experiment, an electrolytic lesion was made by passing a current (100 μ A, 10 s) through one or two electrodes to identify the end of the recording tract.

[00168] Spikes were sorted offline based on the waveform energy and the first three principal components of the spike waveform on each stereotrode channel. For single unit isolation, all channels were separated into groups and spike waveforms were identified either manually using Klusters (Beui-osuite(dot)soi3rceforge{dot ne ) or automatically using the software klustakwik (khistakwikt dot)soLirceforge(dot. net/). The quality of each unit was assessed by the presence of a refractory period and quantified using isolation distance and L-ratio. Units with an isolation distance < 20 and L-ratio > 0.1 were discarded.

[00169] To identify ChR2-tagged neurons, laser pulse trains (10 and/or 20 Hz) were delivered intermittently every min. A unit was identified as ChR2 expressing if spikes were evoked by laser pulses with short first-spike latency (< 10 ms) and the waveforms of the laser-evoked and spontaneous spikes were highly similar (correlation coefficient > 0.9). Mean latency of all identified units was 3.05 ms. Mean correlation coefficient of all identified units was 0.99. To calculate the average firing rate of each unit in each brain state, spikes during the laser pulse trains were excluded.

Histology

[00170] Mice were deeply anesthetized and transcardially perfused using PBS followed by 4% paraformaldehyde in PBS. Brains were post-fixed in fixative, stored in 30% sucrose in PBS overnight for cryoprotection, and further processed for immunohistochemistry and fluorescence in situ hybridization (FISH).

Immunohistochemistry

[00171] Mice were deeply anesthetized and transcardially perfused using PBS followed by 4% paraformaldehyde in PBS. Brains were post-fixed in fixative and stored in 30% sucrose in PBS overnight for cryoprotection. Brains were embedded and mounted with Tissue-Tek OCT compound (Sakura finetek) and 20 μπι sections were cut using a cryostat (Leica). Brain slices were washed using PBS, permeabilized using PBST (0.3% Triton X-100 in PBS) for 30 min and then incubated with blocking solution (5% normal goat serum or normal donkey serum in PBST) for 1 hr followed by primary antibody incubation overnight at 4 °C using following antibodies: anti-GFP antibody (A-11122 or A-11120, Life technologies, 1 :1000)

anti-cFos antibody (sc-52-G and sc-52, Santa cruz biotech, 1 : 1000)

anti-CCK-8 antibody (20078, Immunostar, 1 :500)

anti-Substance P antibody (abl0353, abeam, 1 :500)

anti-CRH antibody (sc-1759, Santa cruz biotech, 1 :500)

anti-HA antibody (C29F4, Cell signaling tech, 1 : 1000)

[00172] The next day, slices were washed with PBS and incubated with appropriate secondary antibodies for 2 hr (1 :500, All from Invitrogen):

A-11008, alexa fluor 488 goat anti-rabbit IgG

A-21206, alexa fluor 488 donkey anti-rabbit IgG

A-11055, alexa fluor 488 donkey anti-goat IgG A-21202, alexa fluor 488 donkey anti-mouse IgG

A-11012, alexa fluor 594 goat anti-rabbit IgG

A-21207, alexa fluor 594 donkey anti-rabbit IgG

A- 11058, alexa fluor 594 donkey anti-goat IgG

[00173] The slices were washed with PBS followed by counterstaining with DAPI or Hoechst and coverslipped. Fluorescence images were taken using a confocal microscope (LSM 710 AxioObserver Inverted 34-Channel Confocal, Zeiss) or Nanozoomer (Hamamatsu).

Fluorescence in situ hybridization (FISH)

[00174] The GAD probe was a kind gift from Dr. Olivier Civelli (University of California,

Irvine). To make TAC1-FISH probes, DNA fragments containing the coding or untranslated sequences were amplified using PCR from mouse whole brain cDNA (Zyagen). A T7 RNA polymerase recognition site was added to the 3' end of the PCR product. The PCR product was purified using a PCR purification kit (QIAGEN). 1 μg of DNA was used for in vitro

transcription by using digoxigenin (DIG) RNA labeling mix (Roche) and T7 RNA polymerase. After DNAse I treatment for 30 min at 37 °C, the RNA probe was purified using probeQuant G- 50 Columns (GE Healthcare). 20 μπι sections were pretreated with proteinase K (0.1 μg/ml), acetylated, dehydrated through ethanol (50, 70, 95, and 100%), and air dried. Pretreated sections were then incubated for 16-20 hr at 60 °C, in a hybridization buffer containing sense or antisense riboprobes. After the sections were hybridized, they were treated with RNase A (20 μg/ml) for 30 minutes at 37 °C and then washed four times in decreasing salinity (from 2x to 0.1 x standard saline citrate [SSC] buffer) and a 30 min wash at 68 °C. Sections were incubated with 3% hydrogen peroxide in PBS for 1 hr and washed using PBS. After incubation in the blocking buffer for 1 hr (TNB buffer, Perkin Elmer), sections were incubated with anti-DIG-POD antibody (1 :500, Roche) in TNB buffer for 2 hr. TSA-plus-Fluorescein reagent was used to visualize the signal. After washing the sections in PBS, they were incubated with blocking buffer for 2 hr followed by incubation with anti-GFP antibody overnight, and finally incubated with a secondary antibody as described above.

cTRIO data analysis

[00175] For analysis of rabies tracing data, consecutive 60 μπι coronal sections were collected and stained using Hoechst. Slides were scanned using Nanozoomer (Hamamatsu). GFP+ input neurons were counted from the forebrain to the posterior brainstem except sections adjacent to the injection sites (1 mm from the injection site), and grouped into 10 regions based on Allen Mouse Brain Atlas (nio ise(dot)brain-niap(dot)org static/atlas) using anatomical landmarks in the sections visualized by Hoechst staining and autofluorescence. The number of neurons were normalized in each region by the total number of input neurons in the entire brain. Single-Cell RNA-Seq

[00176] A previously described procedure was adapted to isolate fluorescently labeled neurons from the mouse brain (Hempel et al., (2007). A manual method for the purification of fluorescently labeled neurons from the mammalian brain. Nat. Protoc. 2, 2924-2929; Sugino et al., (2006). Molecular taxonomy of major neuronal classes in the adult mouse forebrain. Nat. Neurosci. 9, 99-107).

[00177] Single cell isolation. Individual adult male mice (P56+3) were anesthetized in an

isoflurane chamber, decapitated, and the brain was immediately removed and submerged in fresh ice-cold artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 20 mM NaHC0 ₃ , 20 mM dextrose, 3 mM KC1, 1.25 mM NaH ₂ P0 ₄ , 2 mM CaCl ₂ , 2 mM MgCl ₂ , 50 μΜ DL-AP5 sodium salt, 20 μΜ DNQX, and 0.1 μΜ tetrodotoxin, bubbled with a carbogen gas (95% 0 ₂ and 5% C0 ₂ ). The brain was sectioned on a vibratome (Leica VT1000S) on ice, and each slice (300- 400 μπι) was immediately transferred to an ACSF bath at room temperature. After the brain slicing is complete (not more than 15 minutes), individual slices of interest were transferred to a small petri dish containing bubbled room temperature ACSF. The POA was microdissected under a fluorescence dissecting microscope, and the slices before and after dissection were imaged to examine the location of the microdissected tissue and confirm its location. The dissected tissue pieces were transferred to a microcentrifuge tube and treated with 1 mg/ml pronase (Sigma, Cat#P6911-lG) in carbogen-bubbled ACSF for 70 minutes at room temperature without mixing in a closed tube. After incubation, with the tissue pieces sitting at the bottom of the tube, the pronase solution was pipetted out of the tube and exchanged with cold ACSF containing 1 % fetal bovine serum. The tissue pieces were dissociated into single cells by gentle trituration through Pasteur pipettes with polished tips of 600, 300, and 150 μπι diameter. Single cells were isolated by FACS into individual wells of 96-well plates or 8-well PCR strips containing 2.275 μΐ of Dilution Buffer (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936), 0.125 μΐ RNase inhibitor (SMARTer kit), and 0.1 μΐ of 1 : 1,000,000 diluted RNA spike-in RNAs (ERCC RNA Spike-In Mix 1, Life Technologies Cat#4456740). Sorting was performed on a BD FACSAriall SORP using a 130um nozzle, a sheath pressure of 10 psi, and in the single cell sorting mode. To exclude dead cells, DAPI (DAPI*2HC1, Life Technologies Cat#D1306) was added to the single cell suspension to the final concentration of 2 ng/ml. Sorted cells were frozen immediately on dry ice and stored at -80°C.

[00178] cDNA amplification and library construction. The SMARTer kit above was used to reverse transcribe single cell RNA and amplify the cDNA for 19 PCR cycles. To stabilize the RNA after quickly thawing the cells on ice, an additional 0.125 μΐ of RNase inhibitor mixed with SMART CDS Primer II A was immediately added to each sample. All steps downstream were carried out according to the manufacturer's instructions. cDNA concentration was quantified using Agilent Bioanalyzer High Sensitivity DNA chips. For most samples, 1 ng of amplified cDNA was used as input to make sequencing libraries with Nextera XT DNA kit (Illumina Cat#FC-l 31-1096). Individual libraries were quantified using Agilent Bioanalyzer DNA 7500 chips. In order to assess sample quality and adjust the concentrations of libraries for multiplexing on HiSeq, all libraries were sequenced first on Illumina MiSeq to obtain approximately 100,000 reads per library, and then on Illumina HiSeq 2000 or 2500 to generate 100 bp reads.

TRAP

[00179] A previously described procedure was adapted to perform TRAP (Knight et al., (2012).

Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 151, 1126- 1137).

[00180] Mice were sacrificed and the POA was rapidly dissected on ice with a dissection buffer

(lxHBSS, 2.5 mM HEPES [pH 7.4], 4 mM NaHC0 ₃ , 35mM Glucose, 100 ng/ml

Cycloheximide). Brains from 6 mice were then pooled, homogenized in the homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCI, 5 mM MgCl ₂ , 100 nM calyculin A, 2 mM DTT, 100 U/ml RNasin, 100 μg/ml cycloheximide and protease). Homogenates were transferred to a microcentrifuge tube and clarified at 2,000xg for 10 min at 4°C. The supernatant was transferred to a new tube, and 70 μΐ of 10% NP40 and 70 μΐ of l,2-diheptanoyl-57i-glycero-3- phosphocholine (DHPC, 300mM) per 1ml of supernatant were added. This solution was mixed and then clarified at 17,000xg for 10 min at 4°C. The resulting high-speed supernatant was transferred to a new tube. This supernatant served as the input. A small amount (25 μΐ) was added to a new tube containing 350 μΐ of buffer RLT for future input RNA purification.

[00181] Immunoprecipitation was performed with an anti-FLAG antibody loaded beads. The beads were washed four times using 0.15M KCI Wash buffer (10 mM HEPES [pH 7.4], 350 mM KCI, 5 mM MgCl ₂ , 2 mM DTT, 1% NP40, 100 U/ml RNasin, and 100 ng/ml cycloheximide). After the final wash the RNA was eluted by addition of buffer RLT (350 to the beads on ice, the beads removed by a magnet, and the RNA purified using the RNeasy Micro Kit (QIAGEN) and analyzed using an Agilent 2100 Bioanalyzer. cDNA libraries for RNA-seq were prepared with Ovation RNA-Seq System V2 and Ovation Ultralow Library Systems (NuGen) and analyzed on an Illumina HiSeq 2500. Gene classification shown in Table SI was carried out using Panther (http://paiitherdb.org/) (Mi et al., 2013).

Statistics

[00182] Statistical analysis was performed using Matlab, GraphPad Prism or Python. All

statistical tests were two-sided. The 95% confidence intervals (CI) for brain state probabilities were calculated using a bootstrap procedure: For an experimental group of n mice, with mouse i comprising m, trials, the data was repeatedly resampled by randomly drawing for each mouse m, trials (random sampling with replacement). For each of the 10,000 iterations, the mean probabilities was recalculated for each brain state across the n mice. The lower and upper confidence intervals were then extracted from the distribution of the resampled mean values. To test whether a given brain state is significantly modulated by laser stimulation, the difference between the mean probabilities during laser stimulation and the preceding period of identical duration was calculated for each bootstrap iteration.

Example 2: Optogenetic Activation of GAD ^poa→tmn Neurons Promotes Sleep

[00183] To identify the optimal target for retrograde labeling of these neurons, fluorescent latex microspheres (RetroBeads) were injected into the TMN, dorsomedial hypothalamus (DMH), ventrolateral periaqueductal gray (vlPAG), or the prefrontal cortex (PFC). Seven to ten days later, each mouse was sleep deprived for 6 hr, allowed undisturbed rebound sleep for 4 hr, and then sacrificed for c-Fos immunohistochemistry. Elevated c-Fos expression was found in the POA (Figure 1A), consistent with previous studies. Among the POA targets tested, retrobead injection into the TMN caused the highest percentage of labeling among the c-Fos+ neurons (Figure 1A; 64.3 ± 5.1% of c-Fos+ neurons contained RetroBeads from TMN, 33.6 ± 3.5% from DMH, 9.5 ± 1.3% from vlPAG, 0% from PFC, mean ± SEM), consistent with the previous finding (Sherin et al., 1996, 1998). The TMN was thus chosen for retrograde labeling of sleep- active POA neurons.

[00184] To label the TMN-projecting neurons, a lentivirus pseudotyped with the rabies

glycoprotein (RG), which can be taken up by axon terminals and retrogradely transported to the cell body, was used. Since the great majority of c-Fos+ sleep-active neurons in the POA are GABAergic (Figure 8), the pseudotyped lentivirus with Cre -dependent expression of ChR2 and eYFP (rEIAV-DIO-TLoop-ChR2-eYFP) (Cetin and Callaway, 2014) was injected into the TMN of GAD2-Cre mice. Many eYFP-labeled neurons in the POA were found, the majority of which expressed c-Fos+ following sleep rebound (73.6 ± 4.8%, Figure IB). These neurons will be referred to as GAD ^roA→1MN neurons.

[00185] Figures 1A-1B. Retrograde Labeling and c-Fos Staining of Sleep-Active POA

Neurons (Figure 1A) Overlap between c-Fos expression induced by sleep rebound and retrobead (RB) labeling from TMN, DMH, vlPAG or PFC. Coronal diagram was adapted from The Mouse Brain in Stereotaxic Coordinates (Franklin and Paxinos, 2007). Percentage of c-Fos+ cells containing RB differed among target regions (p < 0.0001, one-way ANOVA followed by Dunnett's post-hoc test). Error bar, ± SEM. (Figure IB) Expression of ChR2-eYFP in the POA induced by lentivirus (rEIAV-DIO-TLoop-ChR2-eYFP) injection into the TMN of a GAD2-Cre mouse. Many ChR2-eYFP cells expressed c-Fos following sleep rebound (arrowheads). See also Figure 8.

[00186] Figure 8 c-Fos Staining of Sleep-Active POA Neurons in GAD1-GFP or VGLUT2-

GFP Mice. Overlap between c-Fos expression induced by sleep rebound and GAD1-GFP (top panels) or VGLUT2-GFP (bottom panels). Many c-Fos+ cells contained GAD1-GFP

(arrowheads). Graph shows percentage of c-Fos+ cells containing GAD1-GFP or VGLUT2- GFP. Error bar, ± SEM.

[00187] To test the causal role of Q^r ^POA→ ™ ^N neurons in sleep regulation, they were

optogenetically activated in freely moving mice. Laser stimulation (10 Hz, 2 min/trial, through an optic fiber implanted into the POA) was applied every 15-25 min, and wakefulness, REM, and NREM states were classified based on electroencephalogram (EEG) and electromyogram (EMG) recordings (Figure 2A). Activation of the Q^r ^POA→ ™ ^N neurons caused an immediate increase in NREM sleep and a delayed increase in REM sleep at the expense of wakefulness (Figures 2B and 2C). Analysis of the probability of transitions between each pair of brain states showed that laser stimulation significantly enhanced wake— >NREM and NREM— >REM transitions but decreased NREM— >wake transitions (Figure 10A). In control mice with

GAD ^POA→1MN neurons expressing the fluorescent protein nlsPelican without ChR2, laser stimulation had no effect (Figures 9 A, 9B and 10B), and the laser-induced changes in brain states were significantly different between the ChR2 and control mice (p < 0.001 for wake and REM, two-way ANOVA with bonferroni post-hoc test). Thus, the observed increase in sleep was caused by the activation of QAD ^poa→1mn neurons rather than by the laser light.

[00188] Figures 2A-2C Optogenetic Activation of GAD ^POA→TMN Neurons Induces Sleep.

(Figure 2A) Schematic of optogenetic experiment. Right panel, fluorescence image of POA (box in schematic) in a GAD2-Cre mouse with rEIAV-DIO-TLoop-ChR2-eYFP injected into the TMN. (Figure 2B) Two example trials in an experiment. Shown for each trial are EEG power spectrum, EMG trace, brain states (color coded), and EEG and EMG traces during selected periods (indicated by boxes) on an expanded time scale. Laser stimulation (blue shading, 10 Hz, 120 s) caused both wake— >NREM (upper trial) and NREM— >REM (lower trial) transitions. (Figure 2C) Probability of wake, NREM, or REM states before, during, and after laser stimulation (applied between 0 and 120 s, light blue shading), averaged from 9 mice (p < 0.0001 for change from wake to sleep states, bootstrap). Shading for each trace, 95% confidence interval (CI). See also Figures 9 A, 9B, 1 OA- IOC) [00189] Figures 9A-9B Effect of Laser Stimulation in GAD ^POA→TMN -nlsPelican Control

Mice. (Figure 9A) Schematic of optogenetic experiment. (Figure 9B) Probability of wake, NREM, or REM states before, during, and after laser stimulation (applied between 0 and 120 s, light blue shading), averaged from 4 mice (p = 0.6104, 0.9153 or 0.0548 for REM, NREM or wake respectively, bootstrap). Shading for each trace, 95% confidence interval (CI).

[00190] Figures lOA-lOC Effect of Laser Stimulation on Transition Probability Between

Each Pair of Brain States in GAD ^POA→TMN -ChR2, GAD ^POA→TMN -Ctrl and GAD ^POA -ChR2 mice, Related to Figures 2A-2C, 3A-3F and 9A-9B. (Figure 10A) Transition probability within each 60 s period in GAD ^roA→TMN -ChR2 mice. Error bar, 95% CI (bootstrap). Probability of baseline transition (grey dashed line) was computed after excluding the laser stimulation period. The probability from wake to NREM and NREM to REM during laser stimulation was significantly higher than the baseline, and the probability from NREM to wake transition was significantly lower than the baseline (p < 0.0001, bootstrap). (Figure 10B) Transition probability in control mice. The probability during laser stimulation was not significantly different from baseline (left panel: p = 0.98, >0.99, 0.21 from top to bottom; right panel: p > 0.99, 0.92, 0.93, bootstrap). (Figure IOC) Transition probability in GAD ^POA -ChR2 mice. The probability from NREM to wake during laser stimulation was significantly higher than the baseline, and the probability from wake to NREM was significantly lower than the baseline (p < 0.0001, bootstrap). Red * indicates transition probability that is significantly higher than the baseline. Green # indicates transition probability that is significantly lower than the baseline.

[00191] To test whether the GAD ^{POA→T N} neurons are functionally distinct from other nearby neurons, an AAV with Cre-dependent expression of ChR2-eYFP was injected into the POA of GAD2-Cre mice (Figure 3A), which should infect POA GABAergic neurons irrespective of their projection targets. Optogenetic activation of these neurons caused an immediate and long-lasting increase in wakefulness (Figures 3B and 3C), through an enhanced NREM— >wake and decreased wake— >NREM transition probability (Figure IOC). Thus, while the entire POA GABAergic population contains both sleep- and wake -promoting neurons, the effect of non-selective activation is dominated by an increased wakefulness. To test whether the sleep-promoting effect of TMN-projecting neurons is specific to GABAergic cells, the ChR2-expressing lentivirus was injected into the TMN of VGLUT2-Cre mice (Figure 3D). Optogenetic activation of the VGLUT ^POA→1MN neurons induced an immediate increase in wakefulness (Figures 3E and 3F), indicating that the sleep-promoting effect is specific to neurons that are both GABAergic and TMN projecting. [00192] Figures 3A-3F Optogenetic Activation of GAD ^POA or VGLUT ^POA→TMN Neurons

Promotes Wakefulness (Figure 3 A) Schematic for optogenetic stimulation of GAD ^roA neurons. (Figure 3B) Two example trials of an experiment. Shown for each trial are EEG power spectrum, EMG trace, brain states (color coded), and EEG and EMG traces during selected periods (indicated by boxes) on an expanded time scale. Laser stimulation (blue shading, 10 Hz, 120 s) caused NREM— >wake transitions in both trials. (Figure 3C) Probability of wake, NREM, or REM states before, during, and after laser stimulation (between 0 and 120 s), averaged from 5 mice (p < 0.0001 for increase in wakefulness, bootstrap). Shading for each trace, 95% CI.

(Figure 3D) Schematic for optogenetic stimulation of YQLU ^poa →™ ⁿ neurons. (Figure 3E) Example trials of an experiment. Laser stimulation (blue shading, 10 Hz, 120 s) caused both NREM— >wake (upper trial) and REM— >wake (lower trial) transitions. (Figure 3F) Probability of wake, NREM, or REM states before, during, and after laser stimulation, averaged from 4 mice (p < 0.0001 for increase in wakefulness, bootstrap). See also Figures lOA-lOC

Example 3: GAD ^poa→tmn Neurons Are Sleep Active

[00193] Although the majority of GAD ^roA→1MN neurons labeled with ChR2-eYFP expressed c-

Fos following sleep rebound (Figure IB), suggesting that they are sleep active, c-Fos expression is also modulated by non-activity-related factors. To measure directly the spiking activity of ChR2-labeled Q^r ^POA→ ™ ^N neurons across the sleep-wake cycle, optrode recordings were performed in the POA of freely moving mice. High-frequency laser pulse trains (10 or 20 Hz, 5- 10 ms/pulse, 1 s/train) were applied intermittently, and single units exhibiting reliable laser- evoked spiking with short latencies and low jitter were identified as Q^r ^POA→ ™ ^N neurons (see Example 1, Figures 4A-4C and 11A-11B). For the 17 identified neurons, the mean firing rate was significantly higher during REM (p = 8.5 x 10 ⁴ , Wilcoxon signed rank test) and NREM (p = 0.0014) sleep than during wakefulness. Individually, all of the 17 neurons showed higher firing rates during REM sleep than wakefulness, and 13 of them also showed higher rates during NREM sleep (p < 0.05, Wilcoxon rank sum test; the remaining 4 cells showed no significant difference between NREM and wake states; Figures 4D-4F).

[00194] Figures 4A-4F Optogenetically Identified GAD ^POA→TMN Neurons are Active during

Sleep. (Figure 4A) Example recording of spontaneous and laser-evoked spikes from a

GAD ^POA→1MN neuron. Blue ticks, laser pulses (20 Hz). (Figure 4B) Comparison between laser- evoked (blue) and averaged spontaneous (grey) spike waveforms. (Figure 4C) Spike raster showing multiple trials of laser stimulation at 10 Hz and 20 Hz. (Figure 4D) Firing rates of an example GAD ^POA→TMN neuron. (Figure 4E) Firing rates of 17 identified GAD ^roA→1MN neurons during different brain states. Each line shows firing rates of one unit; grey bar, average across units. Error bar, ± SEM. (Figure 4F) Summary of firing rate modulation of 17 identified (blue) and 51 unidentified (grey) units. W, wake; R, REM; NR, NREM. See also Figures 11 A, l lBand 12.

[00195] Figures 11A-11B Optogenetic Identification of GAD ^POA→TMN Neurons. (Figure 11A)

Distribution of delays in laser-evoked spiking for all identified neurons. Delay is defined as timing of the first spike after each laser pulse. (Figure 11B) Distribution of correlation coefficient between laser-evoked and spontaneous spike waveforms for all identified neurons.

[00196] Compared to these identified Q^r ^POA→ ™ ^N neurons, the 51 unidentified neurons

recorded in the POA showed much greater functional diversity (Figures 4F and 12), including many neurons that were maximally active during wakefulness (Figure 12). Thus, the

GAD ^POA→1MN neurons represent a subset of POA neurons that are sleep active, a property well suited for their sleep-promoting function.

[00197] Figure 12 Firing Rates of Unidentified POA Neurons. Firing rates of unidentified units in the three brain states. Each line represents data from one neuron. Grey bar represents average over units (n = 51). Error bar, ± SEM.

Example 4: Virus-Assisted Mapping of Inputs to GAD ^poa→ ™ ⁿ Neurons

[00198] To explore the circuit mechanisms regulating the activity of GAD ^poa→tmn neurons, monosynaptic inputs to the GAD ^roA→ ™ ^N neurons were mapped using a newly developed cTRIO (cell type-specific tracing the relationship between input and output) method. A retrograde virus CAV-FLEx ^loxP -Flp was injected into the TMN of GAD2-Cre mice to express Flp recombinase specifically in GAD ^roA→TMN neurons, and AAV-FLEx^-TVA-mCherry and AAV-FLEx^-RG were injected into the POA to express TVA (the receptor for the EnvA envelope glycoprotein)- mCherry and RG in the Flp-expressing neurons. Two to three weeks later, EnvA-pseudotyped, G-deleted, and GFP-expressing rabies virus (RVdG) was injected into the POA (Figure 5A) to infect the TVA-expressing QAD ^poa→1mn neurons and transsynaptically label their inputs.

[00199] GFP-labeled neurons were found in multiple brain regions (Figure 5B). Compared to the

PFC-projecting POA GABAergic (GAD ^POA→PFC ) neurons, which are wake promoting (ChR2- mediated activation induced a significant increase in wakefulness, p = 0.0008, bootstrap), GAD ^POA→1MN neurons received significantly more inputs from the hypothalamus and the amygdala, but fewer inputs from the striatum, midbrain and pons (Figure 5B). In particular, the central nucleus of the amygdala (CEA) provided much more GABAergic inputs to Q^r ^POA→ ™ ^N than to GAD ^POA→PFC neurons (Figures 5C and 5D). To test the role of this CEA GABAergic input in modulating brain states, AAV-EFla-DIO-ChR2-eYFP was injected into the CEA of GAD2-Cre mice and stimulated their axonal projection into the POA (Figures 5E and 5F). Laser stimulation significantly suppressed sleep and increased wakefulness, consistent with the expected effect of GABAergic inhibition of the sleep-promoting QAD ^poa→1mn neurons (Figure 5F).

[00200] Figures 5A-5F Mapping of Monosynaptic Inputs to GAD ^POA→TMN Neurons Using cTRIO. (Figure 5A) Schematic of cTRIO to map monosynaptic inputs to GAD ^roA→TMN (left) or G _AD POA→PFC _(RIGHT) neurons. Middle, coronal section of a mouse brain at the POA stained with Hoechst (blue). A region within the square is magnified in the inset. Arrowheads indicate starter cells (yellow) at the injection site (Scale bar in inset, 50 μπι). (Figure 5B) Average fractional inputs in cTRIO ^roA→TMN (purple) or cTRIO ^POA→PFC (grey) tracing (p = 0.0002 for hypothalamus, p = 0.02 for amygdala, p = 0.03 for striatum, p = 0.001 for midbrain, p = 0.003 for pons, t-test). Error bar, ± SEM. (Figure 5C) Comparison of CEA inputs in cTRIO ^POA→1MN and cTRIO ^POA→PFC tracing (p = 0.004, t-test). (Figure 5D) Rabies-labeled presynaptic neurons in the CEA are largely GABAergic. Top, coronal diagram of mouse brain. Middle, coronal section at the CEA (box in coronal diagram) stained with Hoechst. Bottom, rabies-labeled presynaptic neurons and GAD1 FISH signals in the CEA. Arrowheads indicate GFP-labeled neurons expressing GAD1 ; 75.2 ± 4.3% of GFP+ neurons were GAD1+. (Figure 5E) Schematic for optogenetic stimulation of CEA GABAergic projection to the POA. (Figure 5F) Probability of wake, NREM, or REM states before, during, and after laser stimulation (between 0 and 120 s), averaged from 4 mice (p < 0.0001 for increase in wakefulness, bootstrap). Shading for each trace, 95% CI.

Example 5: Gene Expression Profiling of GAD ^POA→TMN Neurons

[00201] Having characterized the function of Q^r ^POA→ ™ ^N neurons in sleep regulation, the molecular markers labeling these neurons were next identified. First, the translating ribosome affinity purification (TRAP) method was used in conjunction with retrograde labeling using herpes simplex virus (HSV). HSV was injected with Cre -dependent expression of the large ribosomal subunit protein RpllOa fused with a FLAG/HA tag (HSV-LoxSTOPLox-FlagHA- LlOa) into the TMN of VG AT -Cre mice. After 30-45 days of expression, the POA was dissected, and ribosome immunoprecipitation was performed using magnetic beads coated with anti-FLAG antibodies to pull down the mRNAs attached to RpllOa for gene profiling (Figures 6 A and 13A).

[00202] Figures 6A-6F. Identification of Molecular Markers for POA Sleep-Promoting

Neurons Using TRAP. (Figure 6A) Schematic of the TRAP method. (Figure 6B) FPKM (fragments per kilobase of transcript per million mapped reads) IP plotted against FPKM input on a log scale. Marker genes enriched in GAD ^→ neurons such as Cck, Crh, Slc32al and RpllOa are highlighted. Red dots indicate genes that are significantly different in IP versus input (p < 0.05, Fisher's exact test), and blue dots indicate non-significant genes. (Figure 6C) Overlap between HA labeling and CCK expression. Shown is a coronal section at the POA stained with HA antibody (red) and Hoechst (blue). Region within the square is magnified in the inset.

Arrowheads indicate HA-labeled neurons stained with CCK antibody (Scale bar in inset, 50 μηι). (Figure 6D) Probability of wake, NREM, or REM states before, during, and after optogenetic stimulation of POA CCK neurons, averaged from 3 mice (p < 0.0001 for all states, bootstrap). Shading for each trace, 95% CI. (Figure 6E) HA-positive POA neurons and their overlap with CRH. Scale bar in inset, 50 μπι. (Figure 6F) Probability of wake, NREM, or REM states before, during, and after optogenetic stimulation of POA CRH neurons, averaged from 4 mice (p = 0.0015 for REM, p < 0.0001 for NREM and wake). See also Figures 13A, 13B, 14A and 14B.

[00203] Figures 13A-13B . Identification of Differentially Expressed Genes in TRAP.

(Figure 13A) Bioanalyzer trace of immunoprecipitated RNA. FU, fluorescence units. (Figure 13B) Histogram display of differentially expressed genes (IP/input).

[00204] RNA-seq revealed multiple genes that were enriched in immunoprecipitated (IP) RNA from the Q^r ^POA→ ™ ^N neurons relative to the input RNA (the remaining lysates after IP, Figures 6B and 13B). Genes encoding neuropeptides were studied further, because they play important roles in interneuronal communication and behavioral regulation along with classical

neurotransmitters and have proved to be very useful markers for specific cell types. Among the highly enriched neuropeptides (Table 1 in Figure 16), corticotropin-releasing hormone (CRH) and cholecystokinin (CCK), both of which have been implicated in brain state regulation, were closely examined. Immunohistochemistry showed that CCK and CRH are expressed in 49.1 ± 7.5 and 6.6 ± 0.9%, respectively, of HA-labeled neurons in the POA (Figures 6C and 6E), indicating that each marker indeed labels a subset of GAD ^POA→TMN neurons. The two markers also showed partial overlap in their POA expression (Figure 14A). To test whether the CCK- or CRH-expressing POA neurons are sleep promoting, AAV-EFla-DIO-ChR2-eYFP was injected into the POA of CCK- or CRH-Cre mice. Laser stimulation significantly increased NREM and REM sleep while suppressing wakefulness in both CCK-Cre and CRH-Cre mice (Figures 6D and 6F), indicating that both markers label sleep-promoting POA neurons.

[00205] Figures 14A-14B Overlap of Identified Molecular Markers in POA. (Figure 14A)

Overlap between CCK and CRH: 55.1 ± 9.4% of CRH+ neurons contained CCK, and 19.8 ± 7.6% of CCK+ neurons contained CRH. (Figure 14B) Overlap between CCK and substance P: 55.4 ± 10.5% of substance P+ neurons contained CCK, and 12.6 ± 1.1% of CCK+ neurons contained substance P.

[00206] Notably, a significant enrichment of galanin (GAL, Table 1 in Figure 16), a

neuropeptide highly expressed in VLPO sleep-active neurons, was also found. However, in GAL-Cre mice (obtained from GENSAT) with AAV-EFla-DIO-ChR2-eYFP injected into the POA, optogenetic activation increased wakefulness and decreased sleep (Figure 15), suggesting that GAL also labels some wake -promoting neurons.

[00207] Figure 15 Optogenetic Stimulation of POA GAL Neurons Promotes Wakefulness.

Probability of wake, NREM, or REM states before, during, and after laser stimulation (applied between 0 and 60 s, light blue shading), averaged from 4 mice (p < 0.0001 for increase in wakefulness, bootstrap). Shading for each trace, 95% confidence interval (CI).

[00208] As a complement to the TRAP method, transcriptome analysis of QAD ^poa→1mn neurons at the single -cell level was also performed. Four weeks after injecting the retrograde lentivirus rEIAV-DIO-TLoop-nlsPelican into the TMN of GAD2-Cre mice, nlsPelican-labeled POA neurons were dissociated for single -cell RNA-seq (Figure 7A). high-level expression of Tacl (Figure 7B), a gene encoding several members of the tachykinin neuropeptide family, some of which have been implicated in sleep regulation, was found. Fluorescent in situ hybridization showed that Tacl was expressed in 48.4 ± 8.4% of nlsPelican-labeled neurons (Figures 7C), but only in -10% of all GAD1+ neurons in the POA (cotmectivity(dot)braiii- mapfdoOorg/transgcmc/experi merit/ 180304064), thus confirming its preferential labeling of the GAD ^POA→TMN subpopulation. In TACl-Cre mice injected with AAV-EFla-DIO-ChR2-eYFP in the POA, optogenetic stimulation significantly increased NREM sleep and decreased wakefulness (Figure 7D), indicating that TAC1 also labels sleep-promoting POA neurons. Notably, TAC1 was not identified by the TRAP method, indicating that using both gene- profiling methods in parallel can significantly enhance the likelihood of identifying useful markers for sleep-promoting neurons.

Example 6: Pdyn-expressing neurons promote non-REM sleep when activated

[00209] Pdyn (encoding prodynorphin) was identified as a gene whose expression is elevated in the GAD ^POA→1MN subpopulation. Similar to TAC1, in Pdyn-Cre mice injected with AAV-EFla- DIO-ChR2-eYFP in the POA, optogenetic stimulation significantly increased NREM sleep and decreased wakefulness. Example 7: Method of identifying an agent that promotes or suppresses sleep

[00210] A pharmaceutical composition that includes a test agent, e.g., a small molecule, nucleic acid, polypeptide, etc., that modulates a functional activity of a gene product encoded by a gene whose expression is upregulated in circuit neurons in a POA of a brain compared to the average expression level of the gene in neurons of the POA, is administered to a non-human mammal, e.g., a mouse. The test agent is administered at a dose sufficient to modulate the functional activity of the gene product in the brain, e.g., in the POA, of the non-human mammal.

[00211] The test agent specifically modulates the functional activity of a gene product encoded by a gene that is upregulated in the GABAergic, TMN -projecting POA neurons, e.g.,

GAD ^POA→1MN neurons, where the gene encodes a polypeptide gene product selected from Table 3, in Figures 18A-18AI.

[00212] After administering the pharmaceutical composition, the sleep circuit-regulated state of the non-human mammal is monitored, e.g., by monitoring a sleep circuit-regulated state (e.g., REM sleep, non-REM sleep, wakefulness) using, e.g., EEG, EMG or a combination thereof.

[00213] If administration of the pharmaceutical composition that contains the test agent increases the probability of REM sleep, and/or non-REM sleep, and reduces the probability of wakefulness in the non-human mammal, the test agent is identified as an agent that promotes sleep. If administration of the pharmaceutical composition that contains the test agent reduces the probability of REM sleep, and/or non-REM sleep, and increases the probability of wakefulness in the non-human mammal, the test agent is identified as an agent that suppresses sleep.

[00214] 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.