WO2008127524A2 - Biomarkers of very rapid antidepressant response and of mechanism of very rapid antidepressant response - Google Patents

Abstract

This invention provides molecular markers that are indicators of subjects that are likely to respond to sleep deprivation as a treatment for a psychiatric disorder. In particular, genes are identified whose expression is altered in responders to sleep deprivation for the treatment of a psychiatric disorder (e.g., depression, mania, etc.).

Description

BIOMARKERS OF VERY RAPID ANTIDEPRESSANT RESPONSE AND OF MECHANISM OF VERY RAPID ANTIDEPRESSANT RESPONSE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to USSN 60/893,590, filed on March 7, 2007, which is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This work was supported by Federal Research Grant No: MH74307A. The

Government of the United States of America has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention pertains to the field of psychiatric diagnostics. In particular, molecular markers are provided that are good markers to identify subjects that will respond to sleep deprivation (SD) for the treatment of depression.

BACKGROUND OF THE INVENTION [0004] Sleep deprivation is the only known intervention in depressive illness that has proven antidepressant benefits within 24 hours. More than 61 reports (N= 1,700 patients) have documented that total or partial sleep deprivation leads to significant, albeit transitory, improvement in 30%-50% of severely depressed patients. The antidepressant effects of sleep deprivation have been demonstrated in patients who range in age from adolescence to late life, as well as in patients with diverse clinical and demographic characteristics. The form of depressive symptoms, however, has not generally differentiated responders from nonresponders.

SUMMARY OF THE INVENTION

[0005] In certain embodiments this invention pertains to the discovery that altered expression of certain genes provides a good marker for subjects likely to respond to sleep deprivation for the treatment of certain psychiatric disorders. Without being bound by a particular theory it is also believed that depressed subjects receiving serotonin specific reuptake inhibitor (SSRJs) and/or lithium (LI) that are diagnosed as potential responders to sleep deprivation (as described herein) will have greater antidepressant response and long lasting response with various chronotherapeutic augmentation methods. It is also believed that depressed subjects receiving SSRI and LI CAN show an additive antidepressant effect with each different chronotherapeutic augmentation (i.e. SD + BLT + SPA > SD +BLT > SD > BLT > DRL). It is further believed that depressed patients who have an acute antidepressant response to SD will show significant changes in clock gene function and serotonergic gene function compared to those depressed patients who do not. Further it is believed that depressed patients who have specific genetic polymorphism will have a significantly greater antidepressant response to SD than those who do not.

[0006] The use of the genetic markers described herein facilitates the development of a rapid acting antidepressant treatment. Most current antidepressant treatments take two to eight weeks to work. During this time, some patients commit suicide and most will experience emotional pain from their unresolved depression. Using the methods described herein in conjunction with chronobiological strategies can provide a provide a significantly faster reduction in emotional pain and can result in the reduction of some suicides.

[0007] It is noted that we have shown that circulating lymphocytes have certain resemblance to brain gene expression. Thus, markers of brain tissue are also expressed peripherally permitting the use of peripheral tissues (e.g., lymphocytes) in the assays described herein.

[0008] In certain embodiments, methods are provided for identifying a subject who is likely to respond to sleep deprivation in the treatment of a depressive disorder and/or a manic disorder. The methods typically involve screening a biological sample from the subject for increased or decreased expression of one or more genes listed in Table 1, where upregulation or downregulation (e.g., as indicated herein) of expression of the one or more genes, is an indicator that the subject is likely to respond to sleep deprivation for the treatment of a depressive disorder and/or a manic disorder. In certain embodiments the screening comprises screening the biological sample for increased or decreased expression of two or more, or three or more, or four or more, or five or more, or 8 or more, or 10 or more, or 15 or more or 20 or more, or all of the genes listed in Table 1. In certain embodiments the screening comprises screening genes whose expression is concordant in DLPFC and lymphocytes. In certain embodiments the two or more genes comprises one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK. In certain embodiments the biological sample comprises a lymphocyte. In certain embodiments the biological sample comprises a neurological tissue. In certain embodiments the human is a human undergoing psychiatric evaluation. In certain embodiments the human is a human receiving psychoactive medication. In various embodiments the human is a child, adolescent, or adult. In certain embodiments the screening comprises a nucleic acid hybridization to determine an mRNA level of the two, three, four, five, 8, 10, 15, or 20, or more of the genes listed in Table 1. In various embodiments the determining comprises a method selected from the group consisting of a Northern blot, a Southern blot using DNA derived from an RNA expressed by the two or more genes, an array hybridization, an affinity chromatography, an RT-PCR using an RNA expressed by the two or more genes, and an in situ hybridization. In various embodiments the determining comprises an array hybridization using a high density nucleic acid array. In certain embodiments the determining comprises an array hybridization using a spotted array. In certain embodiments the screening comprises detecting a protein(s) expressed by the two, three, four, five, 8, 10, 15, or 20, or more of the genes listed in Table 1. In certain embodiments the detecting is via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry. In certain embodiments the upregulation or downregulation is with respect to a control or reference value comprising baseline levels of expression determined for a members of a normal healthy population and/or with respect to an established threshold value, and/or with respect to levels of expression determined for the human at an earlier time.

[0009] Also provided are methods of treating a human subject for a psychiatric disorder. The methods typically involve utilizing a biological sample from the human subject to determine whether or not the subject is a likely responder to sleep deprivation in the treatment of the psychiatric disorder according to the methods described above and herein; and subjecting the subject to sleep deprivation alone or in combination with other therapeutic modalities for the treatment of the psychiatric disorder. In certain embodiments the prescribing or providing comprises providing cognitive therapy to the subject and/or psychoactive medication (e.g., Neuroleptics (antipsychotics), antiparkinsonian agents, sedatives and anxiolytics, antidepressants, mood stabilizer(s), an anticonvulsant drugs, etc.). In certain embodiments the medication comprises a neuroleptic selected from the group consisting of trifluoperazine (Stelazine), pimozide (Orap), flupenthixol (Fluanxol), and chlorpromazine (Largactil), flupenthixol (Fluanxol), fluphenazine decanoate (Modecate), pipotiazine (Piportil L4), and haloperidol decanoate (Haldol LA). In certain embodiments the medication comprises an antiparkinsonian agent selected from the group consisting of benztropine mesylate (Cogentin), trihexyphenidyl (Artane), procyclidine (Kemadrin), and amantadine (Symmetrel). In certain embodiments the medication comprises a sedative and/or anxiolytic selected from the group consisting of barbiturates, benzodiazepines, and non-barbiturate sedatives. In certain embodiments the medication comprises an antidepressant selected from the group consisting of a tricyclic (e.g., amitriptyline (Elavil), imipramine (Tofranil), doxepin (Sinequan), clomipramine (Anafranil)), a monoamine oxidase inhibitors (e.g., phenelzine (Nardil) and tranylcypromine (Parnate)), a tetracyclic (e.g. maprotiline (Ludiomil)), trazodone (Desyrel) and fluoxetine (Prozac). In certain embodiments the medication comprises a mood stabilizer selected from the group consisting of lithium and carbamazepine.

[0010] In various embodiments methods are also provided for screening for an agent that mitigates one or more symptoms of a psychiatric disorder. The methods typically involve administering a test agent to a cell and/or a mammal; and detecting altered expression in the cell and/or mammal of one or more, 2 or more, three or more, 4 or more, 5 or more, 8 or more 10 or more, 20 or more, or all of the genes listed in Table 1, where upregulation or downregulation (as indicated herein) of expression of the one or more genes, as compared to a control, is an indicator that the test agent has activity that mediates one or more symptoms of a psychiatric disorder. In certain embodiments the psychiatric illness is a depressive disorder. In certain embodiments the detecting comprises screening the biological sample for increased or decreased expression of one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK. The screening can comprise screening a nucleic acid (e.g., as described above or herein) and/or a protein (e.g., as described above or herein). In certain embodiments the control comprises a cell contacted or mammal not treated with the test agent or treated with the test agent at a lower concentration. In certain embodiments test agent is not an antibody and/or not a protein. In certain embodiments the test agent is a small organic molecule. In certain embodiments the cell is cultured ex vivo.

[0011] In certain embodiments methods are also provided for identifying genes implicated in psychiatric disorders, the method comprising subjecting a mammal to sleep deprivation and determining genes whose expression is altered in response to the sleep deprivation. In various embodiments the mammals are humans diagnosed with depressive disorder where the disorder is mitigated by sleep deprivation. In certain embodiments the determining comprises determining gene expression in peripheral blood cells and/or determining gene expression in neural tissue.

[0012] Methods are also provided for mitigating a psychiatric disorder. The methods typically involve altering expression or activity of one or more genes listed in Table 1. In various embodiments the one or more genes comprise one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.

[0013] In certain embodiments methods are provided of mitigating a psychiatric disorder, where the methods comprise administering the gene product or analogue thereof of one or more genes listed in Table 1. In certain embodiments the one or more genes comprise one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.

DEFINITIONS

[0014] The phrase "dysregulation of the expression of the gene(s) as indicated in Table

XX" or "altered expression of the gene(s) as indicated in Table XX", where XX is the Table number indicates that the expression of the gene(s) is upregulated or downregulated as shown in the table or expression level is not significantly altered as shown in the table. It is not required that the expression levels match those shown in the table, simply when the table shows upregulation of expression of the gene(s) is associated with a particular condition, then measured upregulation of expression of those gene(s) in a subject it taken as an indicator of that condition, and when the table shows that downregulation of expression of the gene(s) is associated with a particular condition, then measured downregulation of expression of those gene(s) in a subject it taken as an indicator of that condition. In various embodiments, the measured upregulation of expression or downregulation of expression is a significant upregulation or downregulation, preferably a statistically significant upregulation or down regulation (e.g., at the 90% or greater, preferably 95% or greater, more preferably 98% or greater or 99% or greater confidence level). In certain embodiments, the upregulation or downregulation is at least 10%, 20%, 25%, or 30%, more preferably at least 50%, 75% or 90%. In certain embodiments, the upregulation is at least 100%, 125% 150%, 200%, 300%, 400%, or 500%. In various embodiments, the change in expression level is at least 1.25 fold, preferably at least 1.5 fold, more preferably at last 2 fold, at least 4, fold, or at least 10 fold. [0015] The phrase "increased or decreased expression" when used with respect to one or more genes indicates increased or decreased levels of mRNA transcript of said genes. This can be produced by increased or decreased regulation of transcription and/or alterations of copy number of the gene(s). Increased or decreased expression is typically with respect to a reference transcription level (e.g., a control). Illustrative controls include, but are not limited to the transcription levels found in a "normal healthy" population (e.g., a healthy population having the same age and/or gender) and/or the same transcription level found in the same subject at a different time (e.g., at a earlier time of life) and/or the transcription level found in one or more "reference" genes. [0016] The term "indicator" when used, e.g. in a diagnostic assay (i.e., when a factor is said to be an indicator of a psychiatric disorder) need not require that the measured factor be dispositive of the presence or absence of the disorder or dispositive of the future occurrence of the disorder . The factor can simply indicate a predisposition to the disorder (e.g., a greater likelihood of presence or future occurrence of the disorder than is found in the absence of the indicator). It will be appreciated that such an indicator can be one of a number of indicators used, typically in a differential diagnosis for the disease/disorder.

[0017] The phrase "significant", when used with respect to upregulation or downregulation of gene expression preferably refers to statistically significant (e.g. at the 90%, preferably 95%, more preferably at least at the 98% or 99% confidence level). [0018] The term "gene product" refers to a molecule that is ultimately derived from a gene. The molecule can be a polypeptide encoded by the gene, an mRNA encoded by a gene, a cDNA reverse transcribed from the mRNA, and so forth.

[0019] The phrase "expression or activity of a gene" refers to the production of a gene product (e.g. the production of an mRNA and/or a protein) or to the activity of a gene product (i.e., the activity of a protein encoded by the gene).

[0020] The term "expression" refers to protein expression, e.g. , mRNA and/or translation into protein. The term "activity" refers to the activity of a protein. Activities include but are not limited to phosphorylation, signaling activity, activation, catalytic activity, protein-protein interaction, transportation, etc. The expression and/or activity can increase, or decrease. Expression and/or activity can be activated directly or indirectly. [0021] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. [0022] The term "antibody", as used herein, includes various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond (Brinkmann et al. (1993) Pr oc. Natl. Acad. Sci. USA, 90: 547-551), an Fab or (Fab)'2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like (Bird et al. (1988) Science 242: 424-426; Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85: 5879- 5883). The antibody may be of animal (especially mouse or rat) or human origin or may be chimeric (Morrison et al. (1984) Proc Nat. Acad. Sci. USA 81 : 6851-6855) or humanized (Jones et al. (1986) Nature 321 : 522-525, and published UK patent application #8707252).

[0023] The terms "binding partner", or "capture agent", or a member of a "binding pair" refers to molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.

[0024] The term "specifically binds", as used herein, when referring to a biomolecule

(e.g., protein, nucleic acid, antibody, etc.), refers to a binding reaction which is determinative of the presence biomolecule in heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g. immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody binds to its particular "target" molecule and does not bind in a significant amount to other molecules present in the sample.

[0025] The terms "nucleic acid" or "oligonucleotide" or grammatical equivalents herein refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10): 1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81 : 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 1 10: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111 :2321, O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al.

(1988) J. Am. Chem. Soc. 1 10:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 1>1:1 '43 (1996)) and non-ribose backbones, including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp 169- 176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. These modifications of the ribose- phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments.

[0026] The terms "hybridizing specifically to" and "specific hybridization" and "selectively hybridize to," as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. Stringent hybridization and stringent hybridization wash conditions in the context of nucleic acid hybridization are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Acid Probes part I, chapt 2, Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, NY ( Tijssen ). Generally, highly stringent hybridization and wash conditions are selected to be about 5⁰C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42°C using standard hybridization solutions {see, e.g., Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65⁰C for 15 minutes {see, e.g., Sambrook supra) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is Ix SSC at 45⁰C for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4x to 6x SSC at 4O⁰C for 15 minutes. [0027] The term "test agent" refers to an agent that is to be screened in one or more of the assays described herein. The agent can be virtually any chemical compound. It can exist as a single isolated compound or can be a member of a chemical {e.g. combinatorial) library. In a particularly preferred embodiment, the test agent will be a small organic molecule.

[0028] The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules {e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0029] The term database refers to a means for recording and retrieving information. In preferred embodiments the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems", mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 schematically illustrates molecular control of the mammalian circadian cycle. [0031] Figure 2 illustrates the time line for treatment interventions.

[0032] Figure 3 illustrates sustained and rapid antidepressant response to sleep deprivation over seven weeks.

[0033] Figure 4 illustrates sustained and rapid antidepressant response to sleep deprivation over seven weeks, a: SDR versus med (p<0.05); b: SDR versus med (p<0.01); c: SDR versus SNDR (p<0.05); d: SDR versus SNDR (p<0.01).

[0034] Figure 5 illustrates changes in CLOCK gene expression between depressed responders and depressed nonresponders to chronotherapeutics.

[0035] Figure 6 illustrates changes in serotonin transporter, SLC6A4, in responders and non-responders to chronotherapeutics. Responders show an increase in gene expression of SLC6A4 after sleep deprivation compared to sleep deprivation nonresponders who show a decrease.

[0036] Figure 7 shows a significant group by genotype by time interaction for GSK3-β in terms of change in HDRS.

DETAILED DESCRIPTION

L Diagnostic/Prognostic methods.

[0037] This invention pertains to the discovery of biomarkers that are strong indicators that a subject will respond to sleep deprivation (SD) for the treatment of a depressive (or manic) disorder. In particular, genes are identified herein whose expression is altered (e.g., upregulated or downregulated) in responders to sleep deprivation. Measurements of the expression level(s) of one, or a plurality, of these genes provides indicates that the subject is a good candidate for sleep deprivation a a component in the treatment of a depressive disorder (or a manic disorder).

[0038] Accordingly, this invention provides biomarkers that can be used by physicians/clinicians/psychotherapists to rapidly identify patients for whom sleep deprivation alone or in combination with other therapeutic modalities, is expected to provide an improved outcome.

[0039] Genes are also identified whose expression is substantially altered in responders to sleep deprivation are shown in Table 1. In certain embodiments particularly preferred genes include, but are not limited to RORA, BHLB2, PERl, and CLOCK.

[0040] Table 1 shows a list of genes whose expression altered in responders to sleep deprivation.

[0041] While, in certain embodiments, the expression level of a single gene identified in Tables 1 , can be used as an indicator for responders to sleep deprivation, in certain embodiments the expression levels of at least 2, 3, 4, or 5 different genes, preferably the expression levels of at least 8, 10, 15, 20, 25, 30, or 40 different genes, more preferably the expression level of at least 50, 60, or 80 different genes is determined. In certain embodiments the expression levels of at least 100, 150, or 200 different genes is determined.

II. Assays for expression of genes that are indicators responders to sleep deprivation.

[0042] This invention identifies a number of genes, altered expression (e.g., upregulation or downregulation) of which provides an indicator of responders to sleep deprivation in the treatment of a psychiatric disorder or the predisposition thereto.

[0043] Expression levels of a gene can be altered by changes in the copy number of the gene and/or transcription of the gene product (i.e., transcription of mRNA), and/or by changes in translation of the gene product (i.e., translation of the protein), and/or by post-translational modification(s) (e.g. protein folding, glycosylation, etc.). Thus, in various embodiments, assays of this invention typically involve assaying for level of transcribed mRNA (or other nucleic acids expressed by the genes identified herein), or level of translated protein, etc. Examples of such approaches are described below.

A) Nucleic-acid based assays.

1. Target molecules. [0044] Changes in expression level can be detected by measuring changes in mRNA and/or a nucleic acid derived from the mRNA (e.g. reverse-transcribed cDNA, etc.). In order to measure gene expression level it is desirable to provide a nucleic acid sample for such analysis. In preferred embodiments the nucleic acid is found in or derived from a biological sample. The term "biological sample", as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Biological samples may also include organs or sections of tissues such as frozen sections taken for histological purposes.

[0045] It was a surprising discovery that nucleic acids derived from tissues other than neurological tissues (e.g., from blood cells) can provide effective diagnostic and/or prognostic indicators of a psychiatric disorder or a predilection to such a disorder. Thus, in certain embodiments, the biological sample is a sample comprising cells of neurological origin and/or non-neurological origin. In certain embodiments, the biological sample comprises blood cells (e.g., peripheral blood lymphocytes and/or lymphoblastic cell lines). [0046] The nucleic acid (e.g., mRNA, or nucleic acid derived from mRNA) is, in certain preferred embodiments, isolated from the sample according to any of a number of methods well known to those of skill in the art. Methods of isolating mRNA are well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in by Tijssen ed., (1993) Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part i. Theory and Nucleic Acid Preparation, Elsevier, N. Y. and Tijssen ed.

[0047] In certain embodiments, the "total" nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and polyA+ mRNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed.), VoIs. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987)).

[0048] Frequently, it is desirable to amplify the nucleic acid sample prior to assaying for expression level. Methods of amplifying nucleic acids are well known to those of skill in the art and include, but are not limited to polymerase chain reaction (PCR, see. e.g, Innis, et al, (1990) PCR Protocols. A guide to Methods and Application. Academic Press, Inc. San Diego,), ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. ScL USA S6: 1173), self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.).

[0049] In certain embodiments, where it is desired to quantify the transcription level

(and thereby expression) of factor(s) of interest in a sample, the nucleic acid sample is one in which the concentration of the nucleic acids in the sample, is proportional to the transcription level (and therefore expression level) of the gene(s) of interest. Similarly, it is preferred that the hybridization signal intensity be proportional to the amount of hybridized nucleic acid. While it is preferred that the proportionality be relatively strict (e.g., a doubling in transcription rate results in a doubling in mRNA transcript in the sample nucleic acid pool and a doubling in hybridization signal), one of skill will appreciate that the proportionality can be more relaxed and even non-linear. Thus, for example, an assay where a 5 fold difference in concentration of the target mRNA results in a 3 to 6 fold difference in hybridization intensity is sufficient for most purposes.

[0050] Where more precise quantification is required, appropriate controls can be run to correct for variations introduced in sample preparation and hybridization as described herein. In addition, serial dilutions of "standard" target nucleic acids (e.g., mRNAs) can be used to prepare calibration curves according to methods well known to those of skill in the art. Of course, where simple detection of the presence or absence of a transcript, or large differences or changes in nucleic acid concentration are desired, no elaborate control or calibration is required.

[0051] In the simplest embodiment, the nucleic acid sample is the total mRNA or a total cDNA isolated and/or otherwise derived from a biological sample (e.g., a sample from a neural cell or tissue). The nucleic acid may be isolated from the sample according to any of a number of methods well known to those of skill in the art as indicated above.

2. Hybridization-based assays.

[0052] Using the known sequence(s) of the various genes identified in Table 1 , detecting and/or quantifying the transcript(s) can be routinely accomplished using nucleic acid hybridization techniques (see, e.g., Sambrook et al. supra). For example, one method for evaluating the presence, absence, or quantity of reverse-transcribed cDNA involves a "Southern Blot". In a Southern Blot, the DNA (e.g., reverse-transcribed mRNA), typically fragmented and separated on an electrophoretic gel, is hybridized to a probe specific for the target nucleic acid. Comparison of the intensity of the hybridization signal from the target specific probe with a "control" probe (e.g. a probe for a "housekeeping gene) provides an estimate of the relative expression level of the target nucleic acid.

[0053] Alternatively, the mRNA transcription level can be directly quantified in a

Northern blot. In brief, the mRNA is isolated from a given cell sample using, for example, an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. As with the Southern blots, labeled probes can be used to identify and/or quantify the target mRNA. Appropriate controls (e.g. probes to housekeeping genes) can provide a reference for evaluating relative expression level.

[0054] An alternative means for determining the gene expression level(s) is in situ hybridization. In situ hybridization assays are well known (e.g., Angerer (1987) Meth.

Enzymol 152: 649). Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the conditions for use can vary depending on the particular application.

[0055] In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block non- specific hybridization.

3_; Amplification-based assays.

[0056] In another embodiment, amplification-based assays can be used to measure transcription level(s) of the various genes identified herein. In such amplification-based assays, the target nucleic acid sequences act as template(s) in amplification reaction(s) (e.g. Polymerase Chain Reaction (PCR) or reverse-transcription PCR (RT-PCR)). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate (e.g. healthy tissue or cells unexposed to the test agent) controls provides a measure of the transcript level.

[0057] Methods of "quantitative" amplification are well known to those of skill in the art are Illustrated in Example 1. For example, quantitative PCR involves simultaneously co- amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y.). One approach, for example, involves simultaneously co-amplifying a known quantity of a control sequence using the same primers as those used to amplify the target. This provides an internal standard that may be used to calibrate the PCR reaction.

[0058] One suitable internal Standard is a synthetic AWl 06 cRNA. The AW 106 cRNA is combined with RNA isolated from the sample according to standard techniques known to those of skill in the art. The RNA is then reverse transcribed using a reverse transcriptase to provide copy DNA. The cDNA sequences are then amplified (e.g., by PCR) using labeled primers. The amplification products are separated, typically by electrophoresis, and the amount of labeled nucleic acid (proportional to the amount of amplified product) is determined. The amount of mRNA in the sample is then calculated by comparison with the signal produced by the known AWl 06 RNA standard. Detailed protocols for quantitative PCR are provided in PCR Protocols, A Guide to Methods and Applications, Innis et al. (1990) Academic Press, Inc. N. Y. The known nucleic acid sequence(s) for the genes identified herein are sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.

4. Hybridization Formats and Optimization of hybridization

a_; Array-based hybridization formats.

[0059] In certain embodiments, the methods of this invention can be utilized in array- based hybridization formats. Arrays typically comprise a multiplicity of different "probe" or "target" nucleic acids (or other compounds) attached to one or more surfaces (e.g., solid, membrane, or gel). In certain embodiments, the multiplicity of nucleic acids (or other moieties) is attached to a single contiguous surface or to a multiplicity of surfaces juxtaposed to each other.

[0060] In an array format a large number of different hybridization reactions can be run essentially "in parallel." This provides rapid, essentially simultaneous, evaluation of a number of hybridizations in a single "experiment". Methods of performing hybridization reactions in array based formats are well known to those of skill in the art (see, e.g., Pastinen (1997) Genome Res. 7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee (1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature Genetics 20: 207-211).

[0061] Arrays, particularly nucleic acid arrays, can be produced according to a wide variety of methods well known to those of skill in the art. For example, in a simple embodiment, "low density" arrays can simply be produced by spotting (e.g. by hand using a pipette) different nucleic acids at different locations on a solid support (e.g. a glass surface, a membrane, etc.).

[0062] The simple spotting, approach has been automated to produce high density spotted arrays (see, e.g., U.S. Patent No: 5,807,522). This patent describes the use of an automated system that taps a microcapillary against a surface to deposit a small volume of a biological sample. The process is repeated to generate high density arrays.

[0063] Arrays can also be produced using oligonucleotide synthesis technology. Thus, for example, U.S. Patent No. 5,143,854 and PCT Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of light-directed combinatorial synthesis of high density oligonucleotide arrays. Synthesis of high density arrays is also described in U.S. Patents 5,744,305, 5,800,992 and 5,445,934. In addition, a number of high density arrays are commercially available.

b. Other hybridization formats. [0064] As indicated above a variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Such assay formats are generally described in Hames and Higgins (1985) Nucleic Acid Hybridization, A Practical Approach, IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. ScL USA 63: 378-383; and John et al. (1969) Nature 223: 582-587. [0065] Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The sample will provide the target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid probe hybridize with the target nucleic acid to form a "sandwich" hybridization complex. To be most effective, the signal nucleic acid should not hybridize with the capture nucleic acid.

[0066] Typically, labeled signal nucleic acids are used to detect hybridization.

Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P- labelled probes or the like. Other labels include ligands that bind to labeled antibodies, fluorophores, chemi-luminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.

[0067] Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand- conjugated probe and an anti-ligand conjugated with a signal.

[0068] The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBAO, Cangene, Mississauga, Ontario), Q Beta Replicase systems, or branched DNA amplifier technology commercialized by Panomics, Inc. (Fremont CA), and the like.

c. Optimization of hybridization conditions. [0069] Nucleic acid hybridization simply involves providing a denatured probe and target nucleic acid under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing. The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label. It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids, or in the addition of chemical agents, or the raising of the pH. Under low stringency conditions (e.g., low temperature and/or high salt and/or high target concentration) hybrid duplexes (e.g., DNA:DNA, RNA:RNA, or RNA:DNA) will form even where the annealed sequences are not perfectly complementary. Thus specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches.

[0070] One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency to ensure hybridization and then subsequent washes are performed at higher stringency to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25 X SSPE at 37°C to 7O⁰C) until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present.

[0071] In general, there is a tradeoff between hybridization specificity (stringency) and signal intensity. Thus, in a preferred embodiment, the wash is performed at the highest stringency that produces consistent results, and that provides a signal intensity greater than approximately 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above which the hybridization pattern is not appreciably altered and which provides adequate signal for the particular probes of interest.

[0072] In a preferred embodiment, background signal is reduced by the use of a blocking reagent (e.g., tRNA, sperm DNA, cot-1 DNA, etc.) during the hybridization to reduce non-specific binding. The use of blocking agents in hybridization is well known to those of skill in the art (see, e.g., Chapter 8 in P. Tijssen, supra.)

[0073] Methods of optimizing hybridization conditions are well known to those of skill in the art (see, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.).

[0074] Optimal conditions are also a function of the sensitivity of label (e.g. , fluorescence) detection for different combinations of substrate type, fluorochrome, excitation and emission bands, spot size and the like. Low fluorescence background surfaces can be used (see, e.g., Chu (1992) Electrophoresis 13:105-114). The sensitivity for detection of spots ("target elements") of various diameters on the candidate surfaces can be readily determined by, e.g., spotting a dilution series of fluorescently end labeled DNA fragments. These spots are then imaged using conventional fluorescence microscopy. The sensitivity, linearity, and dynamic range achievable from the various combinations of fluorochrome and solid surfaces (e.g., glass, fused silica, etc.) can thus be determined. Serial dilutions of pairs of fluorochrome in known relative proportions can also be analyzed. This determines the accuracy with which fluorescence ratio measurements reflect actual fluorochrome ratios over the dynamic range permitted by the detectors and fluorescence of the substrate upon which the probe has been fixed.

d. Labeling and detection of nucleic acids.

[0075] The probes used herein for detection of gene expression levels can be full length or less than the full length of the mRNA(s). Shorter probes are empirically tested for specificity. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 20 bases to the full length of the encoding mRNA, more preferably from about 30 bases to the length of the mRNA, and most preferably from about 40 bases to the length of mRNA. [0076] The probes are typically labeled, with a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the 40 -80 nm diameter size range scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0077] A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. The nucleic acid samples can all be labeled with a single label, e.g., a single fluorescent label. Alternatively, in another embodiment, different nucleic acid samples can be simultaneously hybridized where each nucleic acid sample has a different label. For instance, one target could have a green fluorescent label and a second target could have a red fluorescent label. The scanning step will distinguish sites of binding of the red label from those binding the green fluorescent label. Each nucleic acid sample (target nucleic acid) can be analyzed independently from one another. [0078] Suitable chromogens which can be employed include those molecules and compounds which absorb light in a distinctive range of wavelengths so that a color can be observed or, alternatively, which emit light when irradiated with radiation of a particular wave length or wave length range, e.g., fluoresces. [0079] Desirably, fluorescent labels should absorb light above about 300 nm, preferably about 350 nm, and more preferably above about 400 nm, usually emitting at wavelengths greater than about 10 nm higher than the wavelength of the light absorbed. It should be noted that the absorption and emission characteristics of the bound dye can differ from the unbound dye. Therefore, when referring to the various wavelength ranges and characteristics of the dyes, it is intended to indicate the dyes as employed and not the dye which is unconjugated and characterized in an arbitrary solvent.

[0080] Detectable signal can also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include a compound which becomes electronically excited by a chemical reaction and can then emit light which serves as the detectable signal or donates energy to a fluorescent acceptor. Alternatively, luciferins can be used in conjunction with luciferase or lucigenins to provide bioluminescence.

[0081] Spin labels are provided by reporter molecules with an unpaired electron spin which can be detected by electron spin resonance (ESR) spectroscopy. Exemplary spin labels include organic free radicals, transitional metal complexes, particularly vanadium, copper, iron, and manganese, and the like. Exemplary spin labels include nitroxide free radicals.

[0082] The label can be added to the target (sample) nucleic acid(s) prior to, or after the hybridization. So called "direct labels" are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, so called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N. Y., (1993)). [0083] Fluorescent labels are easily added during an in vitro transcription reaction.

Thus, for example, fluorescein labeled UTP and CTP can be incorporated into the RNA produced in an in vitro transcription.

[0084] The labels can be attached directly or through a linker moiety. In general, the site of label or linker-label attachment is not limited to any specific position. For example, a label may be attached to a nucleoside, nucleotide, or analogue thereof at any position that does not interfere with detection or hybridization as desired. For example, certain Label-ON Reagents from Clontech (Palo Alto, CA) provide for labeling interspersed throughout the phosphate backbone of an oligonucleotide and for terminal labeling at the 3' and 5' ends. As shown for example herein, labels can be attached at positions on the ribose ring or the ribose can be modified and even eliminated as desired. The base moieties of useful labeling reagents can include those that are naturally occurring or modified in a manner that does not interfere with the purpose to which they are put. Modified bases include but are not limited to 7-deaza A and G, 7-deaza-8-aza A and G, and other heterocyclic moieties. [0085] It will be recognized that fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like. Thus, for example, CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013-2016). Similarly, highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281 : 2016-2018).

B) Polvpeptide-based assays. [0086] In various embodiments the peptide(s) encoded by one or more genes listed in Tables 1, can be detected and quantified to provide a measure of expression level. Protein expression can be measured by any of a number of methods well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.

[0087] In one preferred embodiment, the polypeptide(s) are detected/quantified in an electrophoretic protein separation (e.g., a 1- or 2-dimensional electrophoresis). Means of detecting proteins using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N. Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc., N.Y.).

[0088] In another preferred embodiment, Western blot (immunoblot) analysis is used to detect and quantify the presence of polypeptide(s) of this invention in the sample. This technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the target polypeptide(s). [0089] The antibodies specifically bind to the target polypeptide(s) and can be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the a domain of the antibody.

[0090] In preferred embodiments, the polypeptide(s) are detected using an immunoassay. As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte (e.g., the target polypeptide(s)). The immunoassay is thus characterized by detection of specific binding of a polypeptide of this invention to an antibody as opposed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.

[0091] Any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,1 10; 4,517,288; and 4,837,168) are well suited to detection or quantification of the polypeptide(s) identified herein.. For a review of the general immunoassays, see also Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr (1991) Basic and Clinical Immunology 7th Edition. [0092] Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte(s). In preferred embodiments, the capture agent is an antibody.

[0093] Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled polypeptide or a labeled antibody that specifically recognizes the already bound target polypeptide. Alternatively, the labeling agent may be a third moiety, such as another antibody, that specifically binds to the capture agent /polypeptide complex. [0094] Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non- immunogenic reactivity with immunoglobulin constant regions from a variety of species {see, generally Kronval, et al. (1973) J. Immunol, 11 1 : 1401-1406, and Akerstrom (1985) J. Immunol, 135: 2589-2542).

[0095] Preferred immunoassays for detecting the target polypeptide(s) are either competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of captured analyte is directly measured. In one preferred "sandwich" assay, for example, the capture agents (antibodies) can be bound directly to a solid substrate where they are immobilized. These immobilized antibodies then capture the target polypeptide present in the test sample. The target polypeptide thus immobilized is then bound by a labeling agent, such as a second antibody bearing a label.

[0096] In competitive assays, the amount of analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, labeled polypeptide is added to the sample and the sample is then contacted with a capture agent. The amount of labeled polypeptide bound to the antibody is inversely proportional to the concentration of target polypeptide present in the sample. [0097] In one embodiment, the antibody is immobilized on a solid substrate. The amount of target polypeptide bound to the antibody may be determined either by measuring the amount of target polypeptide present in an polypeptide /antibody complex, or alternatively by measuring the amount of remaining uncomplexed polypeptide.

[0098] The immunoassay methods of the present invention include an enzyme immunoassay (EIA) which utilizes, depending on the particular protocol employed, unlabeled or labeled (e.g., enzyme-labeled) derivatives of polyclonal or monoclonal antibodies or antibody fragments or single-chain antibodies that bind the target peptide(s) either alone or in combination. In the case where the antibody that binds the target polypeptide(s) is not labeled, a different detectable marker, for example, an enzyme-labeled antibody capable of binding to the monoclonal antibody which binds the target polypeptide, can be employed. Any of the known modifications of EIA, for example, enzyme-linked immunoabsorbent assay (ELISA), may also be employed. As indicated above, also contemplated by the present invention are immunoblotting immunoassay techniques such as western blotting employing an enzymatic detection system.

[0099] The immunoassay methods of the present invention can also include other known immunoassay methods, for example, fluorescent immunoassays using antibody conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, latex agglutination with antibody-coated or antigen-coated latex particles, haemagglutination with antibody-coated or antigen-coated red blood corpuscles, and immunoassays employing an avidin-biotin or streptavidin-biotin detection systems, and the like.. [0100] The particular parameters employed in the immunoassays of the present invention can vary widely depending on various factors such as the concentration of antigen in the sample, the nature of the sample, the type of immunoassay employed and the like. Optimal conditions can be readily established by those of ordinary skill in the art. In certain embodiments, the amount of antibody that binds the target polypeptide is typically selected to give 50% binding of detectable marker in the absence of sample. If purified antibody is used as the antibody source, the amount of antibody used per assay will generally range from about 1 ng to about 100 ng. Typical assay conditions include a temperature range of about 4°C. to about 45°C, preferably about 25°C to about 37°C, and most preferably about 25°C, a pH value range of about 5 to 9, preferably about 7, and an ionic strength varying from that of distilled water to that of about 0.2M sodium chloride, preferably about that of 0.15M sodium chloride. Times will vary widely depending upon the nature of the assay, and generally range from about

0.1 minute to about 24 hours. A wide variety of buffers, for example PBS, may be employed, and other reagents such as salt to enhance ionic strength, proteins such as serum albumins, stabilizers, biocides and non-ionic detergents can also be included.

[0101] The assays of this invention are scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. The intensity of the band or spot can provide a quantitative measure of target polypeptide concentration.

[0102] Antibodies for use in the various immunoassays described herein, are commercially available or can be produced using standard methods well know to those of skill in the art.

[0103] It will also be recognized that antibodies can be prepared by any of a number of commercial services (e.g., Berkeley antibody laboratories, Bethyl Laboratories, Anawa, Eurogenetec, etc.).

C) Assay Optimization.

[0104] The assays of this invention have immediate utility as prognostic and/or diagnostic assays as described herein, or in screening for agents useful for the treatment of a psychiatric disorder (e.g., schizophrenia and/or bipolar disorder). The assays of this invention can be optimized for use in particular contexts, depending, for example, on the source and/or nature of the biological sample and/or the particular test agents, and/or the analytic facilities available. Thus, for example, optimization can involve determining optimal conditions for binding assays, optimum sample processing conditions (e.g. preferred PCR conditions), hybridization conditions that maximize signal to noise, protocols that improve throughput, etc. In addition, assay formats can be selected and/or optimized according to the availability of equipment and/or reagents. Thus, for example, where commercial antibodies or ELISA kits are available it may be desired to assay protein concentration. Conversely, where it is desired to screen for modulators that alter transcription nucleic acid based assays are preferred. [0105] Routine selection and optimization of assay formats is well known to those of ordinary skill in the art.

D) Assay Scoring.

[0106] In various embodiments, the the assays of this invention level are deemed to show a positive result, when the expression level (e.g., transcription, translation) of the gene(s) is upregulated or downregulated as shown in the tables herein. In certain embodiments this is determined with respect to the level measured or known for a control sample (e.g. either a level known or measured for a normal healthy cell, tissue or organism mammal of the same species and/or sex and/or age), or a "baseline/reference" level determined at a different tissue and/or a different time for the same individual). In a particularly preferred embodiment, the assay is deemed to show a positive result when the difference between sample and "control" is statistically significant (e.g. at the 85% or greater, preferably at the 90% or greater, more preferably at the 95% or greater and most preferably at the 98% or 99% or greater confidence level).

HI. Screening for agents that mitigate one or more symptoms of a psychiatric disorder.

[0107] In certain embodiments this invention provides methods of screening for agents that mitigate one or more symptoms of a psychiatric disorder. The methods typically involve administering one or more test agent to a cell and/or to a mammal; and detecting altered expression in said cell and/or mammal of one or more genes listed in Table 1) where expression of said one or more genes, as compared to a control, is an indicator that said test agent(s) have activity that mediates one or more symptoms of a psychiatric disorder.

[0108] Methods of screening for expression level of one or more gene are known to those of skill in the art and are also described above. [0109] The screening assays are amenable to "high-throughput" modalities.

Conventionally, new chemical entities with useful properties (e.g., modulation of expression of one or more of the genes identified herein) are generated by identifying a chemical compound (called a "lead compound") with the desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. However, the current trend is to shorten the time scale for all aspects of drug discovery. Because of the ability to test large numbers quickly and efficiently, high throughput screening (HTS) methods are replacing conventional lead compound identification methods.

[0110] In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

A) Combinatorial chemical libraries

[0111] In certain embodiments, combinatorial chemical libraries can be used to assist in the generation of new chemical compound leads. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, one commentator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gallop et al. (1994) 37(9): 1233-1250).

[0112] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention. Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et ah, (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara et a (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a Beta- D- Glucose scaffolding (Hirschmann et al.,

(1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic syntheses of small compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho, et al.,

(1993) Science 261 :1303), and/or peptidyl phosphonates (Campbell et ah, (1994) J. Org. Chem. 59: 658). See, generally, Gordon et ah, (1994) J. Med. Chem. 37:1385, nucleic acid libraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083) antibody libraries (see, e.g., Vaughn et a (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522, and U.S. Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33, isoprenoids U.S. Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines U.S. Patents 5,525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like).

[0113] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). [0114] A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.). B) High throughput assays of chemical libraries.

[0115] Any of the assays for agents that modulate expression and/or activity of one or more of the genes described herein are amenable to high throughput screening. As described above, having determined that these components/pathways are associated with the molecular mechanisms underlying addiction, it is believe that modulators can have significant therapeutic value. Certain preferred assays detect increases of transcription (i.e., increases of mRNA production) by the test compound(s), increases of protein expression by the test compound(s), or binding to the gene (e.g., gDNA, or cDNA) or gene product (e.g., mRNA or expressed protein) by the test compound(s). [0116] High throughput assays for the presence, absence, or quantification of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays are similarly well known. Thus, for example, U.S. Patent 5,559,410 discloses high throughput screening methods for proteins, U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Patents 5,576,220 and 5,541 ,061 disclose high throughput methods of screening for ligand/antibody binding.

[0117] In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

IV. Kits.

[0118] In still another embodiment, this invention provides kits for practice of the assays or use of the compositions described herein. In one preferred embodiment, the kits probe nucleic acids (e.g., in a nucleic acid array) to hybridize to the mRNAs described herein. In certain embodiments the kits comprise antibodies that specifically bind to one or more of the proteins encoded by the genes identified herein. The kits can optionally include any reagents and/or apparatus to facilitate practice of the assays described herein. Such reagents include, but are not limited to buffers, labels, labeled antibodies, labeled nucleic acids, filter sets for visualization of fluorescent labels, blotting membranes, and the like.

[0119] In addition, the kits can optionally include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

V. Modulator databases.

[0120] In certain embodiments, the agents that score positively in the assays described herein (e.g. show an ability to alter expression and/or activity of one or more genes as described herein) can be entered into a database of putative modulators for use in a psychiatric disorder. The term database refers to a means for recording and retrieving information. In certain embodiments the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Typical databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to "personal computer systems", mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like. [0121] In certain embodiments this invention also contemplates databases comprising one or more (typically at least 2, 5, or 10 or more, preferably 20, 40, 60, or 80 or more, more preferably 100 or more or even all) of the gene(s) identified herein. The database preferably further includes information regarding the upregulation or downregulation of the expression of the gene(s) in a psychiatric disorder (e.g., schizophrenia, bipolar disorder, etc.). [0122] This invention also contemplates the use of such databases in computer systems and/or chips to provide data upon placement of a query, e.g. in response to a screening assay. EXAMPLES

[0123] The following examples are offered to illustrate, but not to limit the claimed invention.

EXAMPLE 1 Differences in FUNCTIONAL expression (STATE-related) of clock gene and serotonin gene associated with immediate antidepressant response to SD.

[0124] Recent technological developments have enabled psychiatric researcher to use microarray technology [62] to examine over 33,000 well-substantiated human gene expressions of the 15,000 to 20,000 genes on a quantitative basis. We have used this technology to explore newly identified genes in the field of chronobiology to test our hypothesis that alterations of clock gene function are associated with chronotherapeutic response in a small pilot study. We found that four (RORA, CLOCK, BHLB2, PERl) of twelve clock genes (CLOCK, PERl, PER2, NPAS2, CRYl, CRY2, ARNTLlA, BHLB2, BHLB3, CSNKlE, DBP, and RORA) tested showed a significant group by time interaction (See preliminary results for greater details of our findings). As described herein, we have found evidence of a significant increase in gene expression of RORA in sleep deprivation responders (SDR) after chronotherapeutic treatment and a significant decrease in gene expression of RORA in sleep deprivation nonresponders (SDNR).

[0125] RORA was recently identified in 2004 [63] as a key component of the circadian clock which activates BMALl transcription which in turn activates PER/CRY transcription.

RORA appears to be required for the consolidation daily locomotor activity. Depressed patients often show poor consolidation of circadian mediated behaviors such as SWS. Increase in RORA could be associated with greater synchronization of sleep mediated activity.

PER/CRY in mammals and PERIOD/TIMELESS in Drosophila are proteins heterodimers that provide a negative feedback limb of the biological clock feedback loop. The clock genes are self regulating through a series of three interlocking negative and positive feedback loops involving the E-box, the D-box and RRE [54] {see, e.g., Figure 1).

[0126] In summary, the role that clock genes either through differential expression or through association with different markers may play in modulating the circadian rhythm of depressed patients and its potential therapeutic role in the development of new treatments suggests that our findings of significant group by time interactions for genetic expression of clock genes significant implications in laying the foundation for integrating chronobiology with pharmacotherapy to help develop faster antidepressant treatments.

[0127] We have also found evidence for a change in the functional genetic expression of a serotonin gene (SLC6A4, serotonin transporter) associated with an acute antidepressant response to SD.

[0128] Genetic research is beginning to shed some light on why some patients with major depression or bipolar disorder are responders to sleep deprivation and/or serotonergic drug treatment and others are not. Many recent publications [64-67] have found preliminary evidence that response to sleep deprivation is associated with some genetic markers. Baghai et al. 2003a found that the angiotensin 1-converting enzyme polymorphism was associated with response to partial sleep deprivation. However, Benedetti et al. (2003) noted that the DRD2 and DRD3 gene variants were not associated with the antidepressant to sleep deprivation.

[0129] There is preliminary evidence that genetic markers associated with the regulation of the biological clock in the suprachiasmatic nucleus can influence the antidepressant response to sleep deprivation [64]. This suggests a potentially important role of chronobiology in the pathophysiology of mood disorders and its treatment. A single nucleotide polymorphism (SNP) (-50T/C) in the promoter region (nt-171 to +29) of the gene coding for glycogen synthase kinase 3- β (GSK3-β) is associated with a greater response to TSD treatment. In Benedetti 's study, 60 depressed bipolar type I inpatients were given TSD therapy. In addition, those patients with homozygotes for the single nucleotide polymorphism had a later age of onset for bipolar disorder. Benedetti suggests that this chronobiological genotype may play a protective role in bipolar illness in terms of delayed onset and antidepressant responsivity to SD. However, there was a low frequency of the genotype in the patients studied and this finding needs to be replicated. GSK-3 is the mammalian ortholog of shaggy (sgg) in the Drosophila [68]. Shaggy is an enzyme that regulates clock genes in the suprachiasmatic nucleus of the hypothalamus. Reducing SGG/GSK-3 activity can lengthen the circadian period. Conversely, enhancing SGG/GCK-3 can shorten the period of the Drosophila circadian locomotor activity cycle through a premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Premature nuclear translocation by SGG/GSK-3 would provide faster negative feedback to the biological clock and shorten the period. Depressed patients have been reported to have phase advances in body temperature [69] and Cortisol [70] which could be consistent with a shorter circadian periodicity. Martinek and associates report that GSK3-β phosphorylates the protein heterodimers in vitro which could be the mechanism for the premature nuclear translocation of this negative feedback signal for the biological clock.

[0130] The CLOCK gene is an essential component of the circadian rhythm timing system {see, e.g., Figure 1). A polymorphism (T to C nucleotide substitution) in the 3' flanking region at position 3,111 of the DNA sequence of the human CLOCK gene was associated with a substantial 10-14 minute delay in preferences for evening activity compared to morning activity [71]. Patients with major depression usually report their mood worsening in the morning. Morning symptoms are predictive of responsiveness to sleep deprivation therapy as well as to antidepressant drugs [72, 73]. Desan and associates ascertained allele frequency at the T3111 C locus in the human CLOCK gene in European American, African American subjects and those with a history of depression. They found no support for the CLOCK gene alleles at the T311C locus and major depression [74]. Benedetti and associates studied the CLOCK gene allele in 101 patients with a history of bipolar disorder type I or major depression. Genotype groups showed no significant difference in diurnal mood fluctuations. However those with more than 5 years of illness (n=69) showed a significantly higher rate of homozygotes for the C variant. It was almost double that of the other genotype groups [75].

[0131] Benedetti and associates investigated the allelic variation of the serotonin transporter (5 -HTT) linked polymorphic region (5 -HTTLPR) before and after total sleep deprivation followed by light therapy. Benedetti 's study showed that subjects with homozygotes for the long variant of 5 -HTTLPR responded more to TSD and the benefits were sustained more with light therapy compared to those who were heterozygotes and homozygotes for the short variant. Benedetti suggests that genetic polymorphisms influence individual differences in responding to SD and light therapy as well as serotonergic drug treatments [32]. Putzhammer et al. 2004 [65] noted that there was evidence for the 5-HTTLPR genotype in the modulation of the motor response to sleep deprivation. However, Baghai et al. [76] was not able to replicate Benedetti's findings of 5-HTTLPR variants associated response to partial sleep deprivation although Baghai et al. studied unipolar depression whereas Benedetti et al. studied bipolars. Sleep deprivation may exert its antidepressant effect through the serotonergic system [77]. We have investigated the cerebral metabolic correlates of the antidepressant effects of total sleep deprivation [78], [79]. Reduction in function in the anterior cingulate appears to be a key component of a neural circuit for the antidepressant effect of sleep deprivation in our and others studies (e.g. [80]). PET scans of sleep deprivation responders [78, 79] and SSRI medication responders [81] show a common regional cerebral metabolic pattern of reduction in the anterior cingulate. Study of genetic markers related to the serotonergic system in the antidepressant response of sleep deprivation would shed additional light.

EXPERIMENTAL:

[0132] A study was performed to test whether the chronotherapeutic augmentation of antidepressant medication treatment was feasible.

[0133] The research investigation was approved by the Institutional Review Boards

(IRB) of the University of California, Irvine (UCI) and the University of California San Diego (UCSD). A study psychiatrist assessed the subject's decision-making capacity, their ability to understand the risks and benefits of the study, and whether they were competent to decide to volunteer for the study. After receiving a complete study description, written informed consent was obtained in accordance with the Institutional Review Board. Depressed BPD or MDD outpatients were recruited from UCI and UCSD Medical Centers and their affiliated clinics. Exclusion criteria included a history of suicidal behavior, neurological disorders including epilepsy and dementia, substance abuse or alcoholism, use of sleep medication, sleep abnormalities including narcolepsy and apnea, pregnancy, adverse effects to SSRIs, and co- morbid medical disorders that could interfere with compliance. Patients were also excluded if they were unable to arrange their schedules to spend 4 days as inpatients at the UCI or UCSD Medical Centers for the portion of the study involving sleep deprivation, bright light therapy and sleep phase advance treatment.

Subjects

[0134] Fifty-five depressed (49 BPD: 29 males, 20 females and 6 MDD: 1 male, 5 females) outpatients diagnosed with DSM-IV criteria who met the minimum intake inclusion score of 18 on the Hamilton Rating Scale for Depression-24 item version (HRSD-24) were enrolled in the study. Of these, 47 subjects (44 BPD patients (25 males, mean age =41.28 yrs ± 12.21 and 19 females, mean age 40 yrs ± 14.77) and 3 MDD patients (1 male, 21 yrs, 2 females, mean age 37.5 yrs ± 19.09) completed the 8 week study. At the time of enrollment, the subjects were either drug-naive outpatients (N=I 5), patients that were non-responsive to the current medication regimen (N=22) or patients with a history of prior medication (N=IO) and were willing to participate in the research program. Eight subjects did not complete the study for the following reasons: 1 subject relocated out of the area, 3 subjects were unable to tolerate the medications and 4 subjects were non-compliant. }

Assignment to Medication-only or Sleep Deprivation groups: [0135] Upon enrollment, subjects were assigned on a first-come basis either to a sleep deprivation group or to a medication-only comparison group. A larger proportion of patients were assigned to the sleep deprivation group because the research design involved splitting this group into responders and non-responders to sleep deprivation. Results from the literature suggest that approximately 50% of depressed patients respond to sleep deprivation. Therefore, 28 subjects were assigned to the sleep deprivation group while 19 subjects were assigned to the medication-only group. There were no significant statistical differences in depression ratings between the medication-only and the sleep deprivation groups at the start of the study (Day -7) (F= 3.058, p<.087, df=l) or on the baseline sleep deprivation day (Day 0) (F=.729, p<.398, dfM).}

RSD Depression Ratings

[0136] The 24 item version of the HRSD was utilized for the initial diagnosis.

However, since it is not suitable for repeated measures over a 24 hour period in a sleep deprivation experiment due to items such as weight change and hours of sleep, the abbreviated version of the scale (HRSD- 19) was used for analyses . As illustrated in Figure 2, initial baseline HRSD ratings were measured for all subjects on Day -7 and Day -3 twice daily (9am and 6pm) at the initiation of study medications. The HRSD was administered twice daily (9am and 6pm) on the baseline day of sleep deprivation (Day 0); twice daily for a period of one week post-SD (Days 1-6); and twice daily, one day a week, for Weeks 2-7. The ratings were done twice a day so that the diurnal variations in depressive symptoms would not mask the therapeutic effects of the sleep deprivation.

Study Medications

[0137] Medications were administered on an identical schedule for all subjects.

Lithium or other mood stabilizers (as detailed below) were initiated one week prior (Day -7) to the sleep deprivation day. Four days later (Day -3), SSRIs were added to the medical regimen of mood stabilizers. Thus, all subjects were medicated with mood stabilizers and SSRIs for the duration of the study. Since antidepressants including sleep deprivation can precipitate hypomanic or manic reactions in bipolar depression, the risk was minimized in this study with the concurrent administration of lithium or other mood stabilizers prior to treatment with SSRIs and sleep deprivation (including bright light therapy and sleep phase advance). All subjects were carefully monitored. The predominant mood stabilizer in this study was lithium, (n=34, mean daily dose = 724mg). Thirteen subjects with a history of lithium intolerance were administered alternative mood stabilizers. These included: valproic acid, (n=4, mean daily dose= 688 mg); lamotrigine, (n=6, mean daily dose= 192 mg); topiramate, (n=l, mean daily dose= 200 mg); carbamazepine, (n=l mean daily dose 400 mg), and gabapentin (n=l, daily dose, 900 mg.). Three subjects were treated concurrently with lithium (mean daily dose = 724mg) and topiramate (n=2, mean daily dose 333 mg) or lamotrigine (n=l, mean daily dose= 192 mg.)). The predominant SSRI antidepressant treatment used was sertraline, (n=34, mean daily dose 96mg). Other antidepressants were prescribed as an alternative if patients had a negative history with sertraline (n=13). These included: fluoxetine (n=3, mean daily dose 20 mg.); paroxetine (n=3, mean daily dose 40 mg); escitalopram (n=2, mean daily dose 20mg); venlafaxine (n=2, mean daily dose 94 mg; citalopram (n=2, mean daily dose 20 mg.); one subject was treated with sertraline (mean daily dose lOOmg.) and bupropion (mean daily dose 400mg.). There was not a significant over-representation of any of the drugs or doses in any of the patient subgroups. A statistical analysis was performed on patients administered sertraline and lithium versus non-sertraline medications and lithium. Results showed no significant interaction for either group: Sleep deprivation responders (HF, p< .603, F=.747, df= 5.501) and sleep deprivation non-responders (HF, p<.943, F=.187, df=3.954).

Procedure [0138] Medication-only patients (N=I 9) were treated as outpatients throughout the course of the study. Patients in the sleep deprivation protocol (N=28) were studied as inpatients at the UCI Medical Center or the UCSD Medical Center on the morning before the sleep deprivation night and for 3 days post-sleep deprivation, for a total of 4 nights. The sleep deprivation protocol involved additional treatments with morning bright light and sleep phase advance which was administered on each of the three days following the sleep deprivation night (see timeline figure). On the day of sleep deprivation, subjects were kept awake by psychiatric staff from 9am until 6pm on the following day for a total of 33 hours. The patients were checked regularly every 15 minutes by psychiatric staff. During the hours of 11 :00 p.m.- 7:00 a.m. each person was monitored by an overnight nurse, assigned individually to each patient to ensure that they stayed awake. On Day 2, 3 and 4 post sleep deprivation (see timeline figure, Days 1, 2 and 3) morning bright light therapy (5,000 lux for two hours) was administered. In the evening of those same days (Days 1, 2, and 3) subjects were sleep phase advanced. During the entire procedure, subjects were carefully monitored every 15 minutes to ensure that sleep loss did not lead to adverse behavior. All patients in the sleep deprivation group received morning bright light and sleep phase advance independent of whether they responded to the one night of sleep deprivation. [0139] Bright light therapy was administered for three days beginning on the morning after sleep deprivation (Figure 2) for two hours between the hours of 4:00 a.m. -9:00 a.m. The time of the light administration was determined using the Morningness-Eveningness Questionnaire with an algorithm based on research by Terman et al. [82] and Lewy et al. [83]. The algorithm was previously used by Benedetti et al [6] to augment antidepressant action and has been implemented online (Center for Environmental Therapeutics Self Assessment

Instruments: http:/www.dianexus.org). The time of bright light administration was found not to be a significant confounding effect (average time of administration was 6:24am, +54 minutes).

[0140] Sleep phase advance was initiated on the first evening following sleep deprivation. The 3 day schedule for sleep phase advance was as follows: Night 1 : 6:00 p.m. to 1 :00 a.m.; Night 2: 8:00 p.m. to 3:00 a.m. and Night 3: 10:00 p.m.-5:00 a.m.

Data Analyses

[0141] Responses to sleep deprivation were calculated using the mean sleep deprivation baseline HRSD- 19 (Day 0) compared to the mean HRSD- 19 ratings on post sleep deprivation Days 1-2. Using a repeated measures MANOVA analysis and group by time ANOVA analysis, daily mean HRSD- 19 scores were analyzed to test the hypothesis for a significant group by time interaction between the sleep deprivation plus light therapy plus sleep phase advance group versus the medication-only group.

[0142] A second repeated measures MANOVA analysis and group by time ANOVA analysis was done by subdividing the chronotherapeutics package group between sleep deprivation responders, sleep deprivation non-responders and comparing these two chronotherapeutics subgroups to the medication-only group. The median value of HRSD improvement was selected as cutoff in order to divide the sleep deprivation group into two equal halves for analyses. The decrease in depression scores was calculated using the differences in baseline sleep deprivation (Day 0) from the mean HRSD- 19 ratings calculated over 48 hours (Days 1-2) post-sleep deprivation. (Although the majority of sleep deprivation responders improve on Day 1 , a subgroup known as Day 2 responders improve 48 hours later . Thus, it was decided a priori to use a 48 hour sleep deprivation response period in the analyses). Since responders and non-responders were differentiated by mean depression ratings on Days 1 -2 post sleep deprivation, we did not include the data from those time periods in our MANOVA or ANOVA statistical analyses to avoid confounding the data. Post hoc t- tests compared mean daily changes for each day on Day -7, Day -3 and in the first week (Day 0 to Day 7) and weekly (Weeks 2-7) thereafter. One-tailed 't' tests with an alpha p<.05) were used to identify significant changes in depression ratings between the two sleep deprivation groups (responders and non-responders) and the medication-only groups.

RESULTS; [0143] Forty-seven medicated subjects completed the 8 week study {see, e.g., Table 2).

There were no significant differences in depression ratings between the medication-only (n=19; age= 38.4 + 14.5 years ,8 males (M) and 11 females (F), 17 bipolar depressed disorder (BPD), 2 Major Depressive Disorder (MDD)) and the SD group (n=28; age = 44.1 ± 12.3, 18 M and 10 F; 27 BPD, 1 MDD) at the start of the study (Day -7) (F= 3.058, p<.087, df=l) or on the baseline sleep deprivation day (Day 0) (F=.729, p<.398, df=l ).

[0144] Table 2.

Chronotherapeutics package group vs. Medication-only

[0145] Chronotherapeutic package patients showed a significant group by time interaction (Huynh-Feldt, F=3.594, p=.OO5, DF=4.623) compared to the medication-only group. The chronotherapeutic group showed a faster antidepressant response than the medication only group and showed a greater final level of improvement by the end of the study. The chronotherapeutic package showed an approximately fifty percent drop in HDRS by the end of the first week which was sustained to the end of the seven week study {see, e.g., Figure 3). The medication-only group showed some initial improvement of approximately thirty percent improvement by the end of the first week when there was daily contact with staff doing HDRS. However, after the first week, there appeared to be some regression so that by the end of the study, the medication only group showed approximately half the level of improvement of the chronotherapeutic package group. The poor response in the medication only group after seven weeks would be consistent with the patient population at our centers (UCI and UCSD) being comprised of primarily treatment resistant patients. The greater antidepressant response that the chronotherapeutic group had than the medication only group suggests the possibility that chronotherapeutic augmentation may be of particular benefit to treatment resistant patients. The rapidity of the antidepressant response would be consistent with the hypothesis that sleep deprivation accelerates the treatment. The lack of regression seen in the chronotherapeutic group would be consistent with the hypothesis that bright light therapy and sleep phase advance prevented the relapse that has historically been noted in the sleep deprivation literature (see background for details). We had no complications due to manic reactions although several patients were briefly hypomanic after sleep deprivation with a brighter affect and greater psychomotor activity and pressured speech but with no occupational or social problems due to the brief hypomania. Dichotomizing the SD group into acute sleep deprivation responders (SDR) and nonresponders (SDNR) and comparing these two subgroups with the medication- only group

[0146] For the SD group, the median antidepressant response in the 48 hours following

SD was a 35% decrease in HRSD- 19 depression scores calculated as the difference between Day 0 (sleep deprivation baseline) and the mean depression ratings on Days 1 and 2 (post sleep deprivation). Thus, the responder subgroup (n=14; age = 44.1 + 12.6; 8 M 6 F; 14 BPD) showed a decrease of greater than 35% in their HRSD- 19 ratings and the non-responder group (n=14; age = 38.6 + 16.2, 10 M, 4 F; 13 BPD, 1 MDD) was defined by less than a 35% decrease in the HRSD- 19 scores during the 48 period post-sleep deprivation. (Greater improvement in depression in the sleep deprivation responder compared to the non-responder and medication only groups was maintained over the 7 week post-sleep deprivation period (Day 0 -sleep deprivation baseline vs. Week 7 HRSD depression ratings) (see Figure 4 and Table 3). Repeated measures ANOVA (HF, F=3.030, df=9.308, p< 002), MANOVA (RLR, F=2.270, df=13, p<.029).

[0147] Table 3. Mean Hamilton scores (HRDS-19) for all subjects by group over the eight week study. Note that lithium and other mood stabilizers and SSRTs were continued from the start date through week 7. (Mean ± SD) *Phase Adv = Sleep phase advance; BL= bright light therapy in morning hours (detailed in methods). ^ΛHRSD-10 measurements were assessed and the median value (35% decrease in depression scores) was used as a criterion for response.

Acute Sleep Deprivation Responders vs. Non-Responders by Time Interaction:

[0148] Sleep deprivation responders showed significant decreases in HRSD- 19 depression scores compared to non-responders each day during the first week following sleep deprivation. Weekly analyses showed a significant reduction in depression ratings in the sleep deprivation responders vs. non-responders that reached significance during Weeks 2 and 7 (HF, F=2.324, p<.045, DF=5.093).

Acute Sleep Deprivation Responders vs. Medication-Only Group

[0149] Sleep deprivation responders had lower depression ratings than the medication- only group with the exception of Week 5. There was a significant overall decrease in depression in the sleep deprivation responder group over the 7 weeks post-sleep deprivation (HF, F=5.025, p<.000, df= 4.911). Figure 4 shows group differences for each time point

Acute Sleep Deprivation Non-responders vs. Medication-Only Group [0150] There was no significant difference between sleep deprivation non-responders and the medication-only group at any time point in the post-sleep deprivation period although as seen in Figure 4, there was a non-significant trend for lower depression scores in the non- responder group. (HF, F= 1.375, p<.248, df=3.863). Comparisons of change in depression scores at the end of the 7 week study period (Week 7)

[0151] Depression ratings at the end of the study were significantly lower in all groups.

Acute SD responders showed a final mean decrease of 70% (HRDS- 19=6.000 ± 2.50) (Day 0 to Week 7). Acute SD nonresponders had a final 40% decrease (HRDS- 19 = 13.661 ± 2.50) while medication-only patients had a final decrease of approximately 25% (HRDS- 19 =14.224 ± 2.15) over baseline (Day 0) (one-way ANOVA, F= 3.573, p<.036, df=2).}

Functional genomic (state-related) differences in clock genes associated with immediate SD antidepressant response [0152] Four chronotherapeutic responder's samples and four chronotherapeutic nonresponder samples were used to generate the gene expression pilot data obtained in Dr. Vawter' Functional Genomics Laboratory. Total RNA was obtained from fresh lymphocytes [62] and analyzed by microarray analysis using the Affymetrix Human Genome Ul 33 GeneChip. [0153] Gene expression data was analyzed with by a repeated measure on each subject

ANOVA to look at group (SDR vs. SDNR) by time (prechronotherapeutic treatment vs. postchronotherapeutic treatment) interactions using Partek Discovery Suite {see Figure 5).

[0154] Two approaches were used to analyze the 16 GeneChips (8 subjects with pre sleep deprivation and post sleep deprivation samples). The first approach was to use a candidate gene approach by selecting clock genes and other genes hypothesized to play a role in the rapid antidepressant response to chronotherapeutics. Twelve clock genes or genes involved in their immediate regulation were studied (CLOCK, PERl, PER2, NPAS2, CRYl, CRY2, BHLB2, BHLB3, CSNKlE, ARNTL, DBP and RORA). The statistical criterion for the significant group by time interactions in the candidate gene group was p<0.05. There was a significant difference in the change in transcription of four clock genes, RORA, CLOCK, BHLB2 and PERl between depressed responders and depressed nonresponders to chronotherapeutics (See Figure 5). Responders showed increased gene expression for three of the four significant clock genes (RORA, BHLB2 and PERl) after sleep deprivation whereas nonresponders showed decreased gene expression. The one exception to this general pattern was the gene expression for CLOCK which showed a decrease in responders after sleep deprivation whereas nonresponders showed a very slight increase. Increase in gene expression of RORA in responders to sleep deprivation could theoretically be associated with improvement in circadian function of other hormones such as ACTH and Cortisol and could improve physiological function such as the appropriate interaction between REM and SWS. To our knowledge, these findings are the first to show functional changes in genetic expression of clock genes in depression and antidepressant response.

[0155] Of the remaining 172 candidate genes tested, only the serotonin transporter,

SLC6A4, showed a significant group x treatment interaction (p < 0.05). Responders show an increase in gene expression of SLC6A4 after sleep deprivation compared to sleep deprivation nonresponders who show a decrease. This effect is shown in Figure 6. [0156] The second approach used in our functional genomics laboratory with the

Affymetrix gene expression data was a genome wide screening to look for the most significant group by time interactions and group and sleep deprivation main effects. One hundred and fifty genes were significant at p<0.01 for a group by time interaction, 44 genes were significant for main effect of sleep deprivation vs. normal waking and 44 genes were significant for main effect of responders vs. nonresponders. For example, the three most significant genes by p- value criteria that were found on an exploratory basis separating responders from nonresponders were FLJ13056 (NAD kinase), GUCY1A3 (Guanylate cyclase 1, soluble alpha 3), LARS2 (Leucyl-tRNA synthetase2, mitochondrial). These genes are involved with energy regulation or neurotransmitter second messengers. Although these preliminary gene expression findings did not pass a correction for multiple testing, it is intriguing that 4 of 12 master clock genes are dysregulated in responders to chronotherapeutic interventions compared to nonresponders. In addition, one primary target of SSRI treatment, the serotonin transporter was significantly altered in chronotherapeutic treatment responders compared to non responders. These pilot studies were done to generate preliminary findings to use as an a priori hypothesis in a new cohort with this proposed replication.

Genotype (Trait-Related) Differences in Antidepressant Response to SD

[0157] We have also tested a subset of the clinical sample for genetic markers that have been previously associated with sleep deprivation including the HTTLPR polymorphic region and GSK3-β. We have found a significant group by genotype by time interaction for GSK3-β in terms of change in HDRS (see Figure 7). We have not found a significant group by time interaction for HTTLPR short vs. long but we did find a significant difference by t-test for the long, long allele patients having a lower HRDS score at time point 8.

[0158] Figure 7 shows the significant interaction (F=I .925, df = 13, p=.O29) for

Hamilton depression score change over time by SD response by genotype polymorphism for the GSK3-β gene [(TTT, n=6) vs. T/C, n=l 5 vs. C/C, n=l)]. C/C and T/C alleles show a better antidepressant response in terms of sustained effect compared to the T/T for the SDR. This result is consistent with the findings reported by Benedetti et al ([84] who also found that sustained antidepressant response to lithium was significantly better in the C/C compared toT/C. [0159] BLT and SPA is associated with sustaining the rapid antidepressant response to

SD for at least one week and up to seven weeks in the subset of depressed patients who respond in our preliminary study. Within two days after beginning SD and other chronotherapeutic treatment, SDR showed a significant and sustained clinical improvement. The chronotherapeutics treatment sustains the antidepressant response to SD for up to seven weeks. Our seven day findings replicate and extend the findings of Benedetti et al. [40] and [18] who studied patients for one week only. This is the first report of extended seven- week duration of a rapid antidepressant response to sleep deprivation, phase advance of sleep and light therapy which demonstrates that chronotherapeutic intervention can have long lasting therapeutic benefits. Medication plus sleep deprivation and chronotherapeutics appears to significantly shorten the length of time to achieve antidepressant effect in this pilot study. This combination of sleep deprivation and chronotherapeutics and pharmacotherapies could reduce the incidence of suicides occurring before antidepressant action is attained.

[0160] The overall response of the medication only group and the chronotherapeutic nonresponders was relatively poor compared to other medication studies. The fact that our patients included treatment resistant depressed patients may have contributed to this. In our study we did not observe this well-described phenomenon of early relapse to sleep deprivation response due to the combination of other chronotherapeutics to extend the therapeutic benefits of sleep deprivation. Sleep deprivation and chronotherapeutics could affordably accelerate and augment the antidepressant interventions sought by the World Health Organization (WHO) goals for better treatment of depression. Methods:

Functional genetic expression (state- related) studies: Reverse transcription quantitative polymerase chain reaction (Q-PCR)

[0161] RNA integrity after extraction of the total RNA was analyzed by Agilent 2100 Bioanalyzer for 28S and 18S quantitation according to the manufacturers protocol. Total RNA samples are DNAse treated prior to cDNA synthesis. Primers are designed within a 3' exon using the Primer Express program (Applied Biosystems). A nucleotide BLAST (NCBI) is performed to check the specificity of the primers.

[0162] cDNA for quantitative real time PCR was synthesized from human total RNA extracted from lymphocytes. Primers were tested by visualization of the proper amplicon size from cDN A and that a single band on agarose gel was seen. A 1 : 100 dilution of cDN A (5 μl) was added to each Q-PCR reaction containing SYBR Green PCR Master Mix (Applied Biosystems), and amplification is carried out on the 7000 Sequence Detection System (Applied Biosystems). All samples are run in duplicate. The average Ct (amplification cycle threshold) values are used for t-tests after correction for a housekeeping gene (e.g. CRSP9, or SLC9A1) to compare Cohorts. Additional control for genomic DNA contamination of the sample was assessed by including an RT-negative control for each RNA sample and running primers specific to gDNA.

Genotype (trait-related) marker studies: [0163] Genetic studies of the markers 5HTTLPR, and GSK-3-β are performed. Blood samples are used to identify biological factors associated with mood disorders. The blood sample is limited to 50 milliliters or about 3 1/3 tablespoons. Contribution will be a maximum of 4 additional 50 milliliter samples, but no more than a total of 50 milliliters in any 8 hour period. Genotype may vary with ethnicity and ethnicity could vary by chance between treatment arms. Ethnicity is determined in detail (race, country of origin of all 4 grandparents) and will be used as a covariate in analysis.

[0164] Genes relevant to circadian clock function or antidepressant response are selected for genotyping for association with response. These include the twelve core clock genes: CLOCK, PERl, PER2, CRYl, ARNTL (BMALl), DOUBLETIME, TIMELESS, REV- ERB α, and BHLB2. Genes reported to be associated with antidepressant response are also examined such as: serotonin transporter repeat polymorphism (HTTLRP), HTR2a, GSK3b, G beta 2, NTRK2. SNPs are selected first that have been shown to have some functional effect on the gene or associated with treatment response or mood disorder. These genes are supplemented with anonymous snps selected from several databases such as: dbSNP, and HapMap.org, and from Celera/ABI snps using SNPBrowser. Tagging SNPs will be selected from these databases using haplotype block data from the HapMap project and the Tagger SNP selection program. Tagging SNPs are preferable in that they efficiently tag haplotypes from within haplotype blocks in order to obtain maximal information for association with the least number of snps. Inter-block regions will be surveyed using snps of a variety of minor allele frequencies that cover the relevant regions at approximately 5kb density. It is estimated that 150-200 snps will be necessary to optimally cover the genes selected.

[0165] Genotyping is conducted using ABI SNPlex technology. SNPlex allows for 48 multiplexing of snps that can be run in 384 well batches. Hence, the throughput is more than adequate. Accuracy rates in the Kelsoe lab have been measured against Taqman at 99.9%. Genotypes are read blindly in a semi-automated fashion and exported to a custom database.

[0166] A functional repeat in the serotonin receptor promoter is genotyped, HTTLPR, and another gene is examined using a known SNP in the GSK3-β gene (SNP -50 T/C) in the promoter region. The GSK3-β promoter SNP has not been shown to be a functional SNP and may be a marker for a nearby sequence variation of functional significance. Both the HTTLPR and GSK3-β polymorphisms have previously been associated with sleep deprivation response and are tested in patients with mood disorder responding to sleep deprivation and SSRI treatment (n = 75) and patients that do not respond to sleep deprivation treatment and SSRI treatment (n = 75). SNP genotypes assays are selected from Applied Biosystems, and currently include more than 1.8 million TaqMan Pre-Designed SNP Genotyping assays. Genomic DNA is obtained from whole blood using QIAamp (QIAGEN, Inc.) genomic DNA isolation kit and stored at -20 ⁰C.

HTTLPR

[0167] Approximately 20 ng genomic DNA are used as a template for the amplification of the HTTLPR repeat with primer as previously described (Heils et al., 1996), and modified (Durham et al., 2003). Polymerase chain reaction (PCR) products were separated on an Agilent 2100 Bioanalyser using DNA chips. The L and S alleles are determined from the electropherogram and peak height at 528-bp and 484-bp used to establish presence or absence of the alleles.

GSK3-B SNP

[0168] The method of Benedetti et al. (2004) is be used for studying the genotype of the GSK-3-β polymorphism. PCR is performed with these primers: 5'-

G ATTCCC AGACGCCTGTTAC-S' (SEQ ID NO: 1) and 5'-CTCGCTTCCTTCCTTCCTTT- 3' (SEQ ΪD NO:2). An aliquot of PCR product is digested using AIu I (New England Biolabs, USA); fragments were separated on an Agilent Bioanalyzer 2100 (Palo Alto, CA) using DNA lab chips. The electropherograms show unrestricted PCR product (CC genotype) to have a size of 235 bp; while the restricted (TT genotype) produces two bands of 150 and 85 bp.

Heterozygotes will produce 235 bp, and 150 bp, and 85 bp bands. Since there are no known functional coding polymorphisms in GSK-3-β, six additional SNPs spanning the coding and untranslated regions will be purchased from Applied Biosystems to determine the haplotype block. One nearby gene to GSK-3-β (NRl 12, nuclear receptor subfamily 1, Cohort I, member 2) will also be genotyped by SNP assays and correlated with the GSK-3-β haplotype. The NRl 12 gene product belongs to the nuclear receptor superfamily, members of which are transcription factors characterized by a ligand-binding domain and a DNA-binding domain. This receptor is sensitive to dexamethasone. Additional SNP assays can be purchased from Applied Biosystems.

References.

[0169] 1. Wu, J. C. and W. E. Bunney, The biological basis of an antidepressant response to sleep deprivation and relapse: review and hypothesis. Am. J. Psychiatry., 1990. 147: p. 14-21.

[0170] 2. Wirz-Justice, A., et al., Chronotherapeutics (light and wake therapy) in affective disorders. Psychol Med, 2005. 35(7): p. 939-44.

[0171] 3. Wirz-Justice, A., Biological rhythm disturbances in mood disorders. Int

Clin Psychopharmacol, 2006. 21 Suppl 1: p. Sl 1-5.

[0172] 4. Shulte, W., Kombinierte Psycho- und Pharmakotherapiebei

Melancholikern, in Probleme der pharmakopsychiatrischen Kombinations- und Langzeitbehandlung, H. W. Kranz, Editor. 1966, Karger: Basel, p. 150-169. [0173] 5. Wu, J. C. and W. E. Bunney, The biological basis of an antidepressant response to sleep deprivation and relapse: review and hypothesis. Am J Psychiatry, 1990. 147(1): p. 14-21.

[0174] 6. Benedetti, F., et al., Combined total sleep deprivation and light therapy in the treatment of drug-resistant bipolar depression: acute response and long-term remission rates. J Clin Psychiatry, 2005. 66(12): p. 1535-40.

[0175] 7. Holsboer-Trachsler, E., et al., Sleep deprivation and bright light as potential augmenters of antidepressant drug treatment—neurobiological and psychometric assessment of course. J. Psychiatr. Res., 1994. 28(4): p. 381-399. [0176] 8. Benedetti, F., et al., Ongoing lithium treatment prevents relapse after total sleep deprivation. J. CHn. Psychopharmacol., 1999. 19(3): p. 240-245.

[0177] 9. Caliyurt, O. and F. Guducu, Partial sleep deprivation therapy combined with sertraline induces more rapid improvements in quality of life items in major depressive disorder. J Affect Disord, 2005. 88(1): p. 75-8. [0178] 10. Loving, R.T., D.F. Kripke, and S.R. Shuchter, Bright light augments antidepressant effects of medication and wake therapy. Depress Anxiety, 2002. 16(1): p. 1-3.

[0179] 11. Fritzsche, M., et al., Sleep deprivation as a predictor of response to light therapy in major depression. J Affect Disord, 2001. 62(3): p. 207-15.

[0180] 12. Elsenga, S. and R.H. Van den Hoofdakker, Response to total sleep deprivation and clomipramine in endogenous depression. J. Psychiatr. Res., 1987. 21(2): p. 151-161.

[0181] 13. Kuhs, H., et al., Amitriptyline in combination with repeated late sleep deprivation versus amitriptyline alone in major depression. A randomised study. J. Affect. Disord., 1996. 37(1): p. 31-41. [0182] 14. Elsenga, S., D. Beersma, and R.H. Van den Hoofdakker, Total and partial sleep deprivation in clomipramine-treated endogenous depressives. J. Psychiatr. Res., 1990. 24(2): p. 111-119.

[0183] 15. Kuhs, H., et al., Repeated sleep deprivation once versus twice a week in combination with amitriptyline. J. Affect. Disord., 1998. 47(1-3): p. 97-103. [0184] 16. Bump, G. M., et al., Accelerating response in geriatric depression: a pilot study combining sleep deprivation and paroxetine. Depress. Anxiety., 1997. 6(3): p. 1 13- 118.

[0185] 17. Riemann, D., et al., Advanced vs. normal sleep timing: effects on depressed mood after response to sleep deprivation in patients with a major depressive disorder. J. Affect. Disord., 1996. 37(2-3): p. 121-128.

[0186] 18. Voderholzer, U., et al., Is the antidepressive effect of sleep deprivation stabilized by a three day phase advance of the sleep period? A pilot study. Eur Arch Psychiatry Clin Neurosci, 2003. 253(2): p. 68-72. [0187] 19. Baxter, L.R., Jr., Can lithium carbonate prolong the antidepressant effect of sleep deprivation? Arch Gen Psychiatry, 1985. 42(6): p. 635.

[0188] 20. Baxter, L.R., Jr., et al., Prolongation of the antidepressant response to partial sleep deprivation by lithium. Psychiatry. Res., 1986. 19(1): p. 17-23.

[0189] 21. Colombo, C₅ et al., Total sleep deprivation combined with lithium and light therapy in the treatment of bipolar depression: replication of main effects and interaction. Psychiatry. Res., 2000. 95(1): p. 43-53.

[0190] 22. Souetre, E., et al., Antidepressant effects of the sleep/wake cycle phase advance. Preliminary report. J. Affect. Disord., 1987. 12(1): p. 41-46.

[0191] 23. Grube, M. and P. Hartwich, Maintenance of antidepressant effect of sleep deprivation with the help of lithium. Eur. Arch. Psychiatry. Neurol. Sci., 1990. 240(1): p. 60-61.

[0192] 24. Szuba, M.P., et al., Lithium sustains the acute antidepressant effects of sleep deprivation: preliminary findings from a controlled study. Psychiatry. Res., 1994. 51(3): p. 283-295. [0193] 25. Telger, K., R. Tolle, and H. Fischer, [Repeating antidepressive sleep deprivation therapy (partial sleep deprivation)] . Psychiatr. Prax., 1990. 17(3): p. 121-125.

[0194] 26. Wiegand, M.H., CJ. Lauer, and W. Schreiber, Patterns of response to repeated total sleep deprivations in depression. J Affect Disord, 2001. 64(2-3): p. 257-60. [0195] 27. Berger, M., et al., [Chronotherapeutic approaches in depressive disorders]. Wien. Med. Wochenschr., 1995. 145(17-18): p. 418-422.

[0196] 28. Riemann, D., et al., How to preserve the antidepressive effect of sleep deprivation: A comparison of sleep phase advance and sleep phase delay. Eur. Arch. Psychiatry. Clin. Neurosci., 1999. 249(5): p. 231-237.

[0197] 29. Smeraldi, E., et al., Sustained antidepressant effect of sleep deprivation combined with pindolol in bipolar depression. A placebo-controlled trial. Neuropsychopharmacology., 1999. 20(4): p. 380-385.

[0198] 30. Shelton, R.C. and P.T. Loosen, Sleep deprivation accelerates the response to nortriptyline. Prog. Neuropsychopharmacol. Biol. Psychiatry., 1993. 17(1): p. 1 13- 123.

[0199] 31. Elsenga, S. and d.H.R.H. van, Clinical effects of sleep deprivation and clomipramine in endogenous depression. J Psychiatr Res, 1982. 17(4): p. 361-74.

[0200] 32. Benedetti, F., et al., Antidepressant effects of light therapy combined with sleep deprivation are influenced by a functional polymorphism within the promoter of the serotonin transporter gene. Biol Psychiatry, 2003. 54(7): p. 687-92.

[0201] 33. Wehr, T.A., D.A. Sack, and N.E. Rosenthal, Antidepressant effects of sleep deprivation and phototherapy. Acta. Psychiatr. BeIg., 1985. 85(5): p. 593-602.

[0202] 34. Wehr, T.A., et al., Phase advance of the circadian sleep-wake cycle as an antidepressant. Science, 1979. 206(4419): p. 710-3.

[0203] 35. van, d.B.W., et al., Sleep deprivation in bright and dim light: antidepressant effects on major depressive disorder. J Affect Disord, 1990. 19(2): p. 109-17.

[0204] 36. Sack, D. A., et al., Potentiation of antidepressant medications by phase advance of the sleep-wake cycle. Am. J. Psychiatry., 1985. 142(5): p. 606-608. [0205] 37. Riemann, D., et al., [Treatment of depression with sleep deprivation and sleep phase advancement] . Fortschr. Neurol. Psychiatr., 1995. 63(7): p. 270-276.

[0206] 38. Albert, R., et al., [Sleep deprivation and subsequent sleep phase advance stabilizes the positive effect of sleep deprivation in depressive episodes]. Nervenarzt, 1998. 69(1): p. 66-69. [0207] 39. Berger, M., et al., Sleep deprivation combined with consecutive sleep phase advance as a fast-acting therapy in depression: an open pilot trial in medicated and unmedicated patients [see comments]. Am. J. Psychiatry., 1997. 154(6): p. 870-872.

[0208] 40. Benedetti, F., et al., Sleep phase advance and lithium to sustain the antidepressant effect of total sleep deprivation in bipolar depression: new findings supporting the internal coincidence model? J Psychiatr Res, 2001. 35(6): p. 323-9.

[0209] 41. Praschak-Rieder, N., et al., [Therapeutic sleep deprivation and phototherapy]. Wien. Med. Wochenschr., 1999. 149(18): p. 520-524.

[0210] 42. Neumeister, A., et al., Bright light therapy stabilizes the antidepressant effect of partial sleep deprivation. Biol. Psychiatry., 1996. 39(1): p. 16-21.

[0211] 43. Benedetti, F., et al., Sleep deprivation hastens the antidepressant action of fluoxetine. Eur. Arch. Psychiatry. Clin. Neurosci., 1997. 247(2): p. 100-103.

[0212] 44. de Montigny, C, Lithium addition in treatment-resistant depression: evidence fo the involvement of the serotonin system, in Biological Psychiatry, G. Ragcagni, N. Brunello, and T. Fukuda, Editors. 1991, Elsevier Science Publishers: Amsterdam, p. 243-244.

[0213] 45. McCance-Katz, E., et al., Serotonergic function during lithium augmentation of refractory depression. Psychopharmacology. (Berl). 1992. 108(1-2): p. 93-97.

[0214] 46. Bschor, T., et al., Lithium augmentation in treatment-resistant depression: clinical evidence, serotonergic and endocrine mechanisms. Pharmacopsychiatry, 2003. 36 Suppl 3: p. S230-4.

[0215] 47. Iwahana, E., et al., Effect of lithium on the circadian rhythms of locomotor activity and glycogen synthase kinase-3 protein expression in the mouse suprachiasmatic nuclei. Eur J Neurosci, 2004. 19(8): p. 2281-7.

[0216] 48. Berger, M., CD. van, and D. Riemann, Sleep and manipulations of the sleep-wake rhythm in depression. Acta Psychiatr Scand Suppl, 2003(418): p. 83-91.

[0217] 49. Wirz- Justice, A., et al., Brightening depression. Science, 2004.

303(5657): p. 467-9. [0218] 50. Bunney, W.E. and B. G. Bunney, Molecular clock genes in man and lower animals: possible implications for circadian abnormalities in depression. Neuropsychopharmacology, 2000. 22(4): p. 335-45.

[0219] 51. Bunney, B.G., S.G. Potkin, and W.E. Bunney, Dysregulation of circadian rhythms in mood disorders: molecular mechanisms, in Biology of Depression, J. Lucinio, Editor. 2005, Wiley: Weineim. p. 467-492.

[0220] 52. Mansour, H. A., T.H. Monk, and V. L. Nimgaonkar, Circadian genes and bipolar disorder. Ann Med, 2005. 37(3): p. 196-205.

[0221] 53. Mansour, H. A., et al., Association study of eight circadian genes with bipolar I disorder, schizoaffective disorder and schizophrenia. Genes Brain Behav, 2006. 5(2): p. 150-7.

[0222] 54. Ueda, H. R., et al., System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nat Genet, 2005. 37(2): p. 187-92.

[0223] 55. Brainard, G.C. and J.P. Hanifin, Photons, clocks, and consciousness. J Biol Rhythms, 2005. 20(4): p. 314-25.

[0224] 56. Cermakian, N., et al., On the communication pathways between the central pacemaker and peripheral oscillators. Novartis Found Symp, 2003. 253: p. 126-36.

[0225] 57. Malek, Z.S., et al., Tissue-specific expression of tryptophan hydroxylase mRNAs in the rat midbrain: anatomical evidence and daily profiles. Eur J Neurosci, 2005. 22(4): p. 895-901.

[0226] 58. Grossman, G. H., et al., Sleep deprivation stimulates serotonin release in the suprachiasmatic nucleus. Neuroreport., 2000. 11(9): p. 1929-1932.

[0227] 59. Sprouse, J., J. Braselton, and L. Reynolds, Fluoxetine Modulates the

Circadian Biological Clock via Phase Advances of Suprachiasmatic Nucleus Neuronal Firing. Biol Psychiatry, 2006.

[0228] 60. Challet, E., et al., Sleep deprivation decreases phase-shift responses of circadian rhythms to light in the mouse: role of serotonergic and metabolic signals. Brain Res, 2001. 909(1-2): p. 81-91. [0229] 61. Yuan, Q., et al., Serotonin modulates circadian entrainment in

Drosophila. Neuron, 2005. 47(1): p. 115-27.

[0230] 62. Vawter, M.P., et al., Microarray screening of lymphocyte gene expression differences in a multiplex schizophrenia pedigree. Schizophr Res, 2004. 67(1): p. 41-52.

[0231] 63. Sato, T.K., et al., Afunctional genomics strategy reveals Rora as a component of the mammalian circadian clock. Neuron, 2004. 43(4): p. 527-37.

[0232] 64. Benedetti, F., et al., A glycogen synthase kinase 3 -beta promoter gene single nucleotide polymorphism is associated with age at onset and response to total sleep deprivation in bipolar depression. Neurosci Lett, 2004. 368(2): p. 123-6.

[0233] 65. Putzhammer, A., et al., Evidence of a role for the 5-HTTLPR genotype in the modulation of motor response to antidepressant treatment. Psychopharmacology (Berl), 2004.

[0234] 66. Baghai, T. C, et al., Influence of a functional polymorphism within the angiotensin I-converting enzyme gene on partial sleep deprivation in patients with major depression. Neurosci Lett, 2003. 339(3): p. 223-6.

[0235] 67. Benedetti, F., et al., Dopamine receptor D2 and D3 gene variants are not associated with the antidepressant effect of total sleep deprivation in bipolar depression. Psychiatry Res, 2003. 118(3): p. 241-7. [0236] 68. Martinek, S., et al., A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock. Cell, 2001. 105(6): p. 769-79.

[0237] 69. Wehr, T.A., et al., Circadian rhythm disturbances in manic-depressive illness. Fed. Proc, 1983. 42(11): p. 2809-2814.

[0238] 70. Carpenter, W.T. and W.E. Bunney, Adrenal cortical activity in depressive illness. Am J Psychiatry, 1971. 128: p. 63-72.

[0239] 71. Katzenberg, D., et al., A CLOCK polymorphism associated with human diurnal preference. Sleep, 1998. 21(6): p. 569-76.

[0240] 72. Reinink, E., et al., Prediction of the antidepressant response to total sleep deprivation by diurnal variation of mood. Psychiatry. Res., 1990. 32(2): p. 113-124. [0241] 73. Fahndrich, E., Biological predictors of success of antidepressant drug therapy. Psychiatr Dev, 1987. 5(2): p. 151-71.

[0242] 74. Desan, P. H., et al., Genetic polymorphism at the CLOCK gene locus and major depression. Am J Med Genet, 2000. 96(3): p. 418-21. [0243] 75. Benedetti, F., et al., Influence of CLOCK gene polymorphism on circadian mood fluctuation and illness recurrence in bipolar depression. Am J Med Genet, 2003. 123B(I): p. 23-6.

[0244] 76. Baghai, T.C., et al., No Influence of a Functional Polymorphism within the Serotonin Transporter Gene on Partial Sleep Deprivation in Major Depression. World J Biol Psychiatry, 2003. 4(3): p. 111-4.

[0245] 77. Wu, J. C, M. Buchsbaum, and W.E. Bunney, Jr., Clinical neurochemical implications of sleep deprivation's effects on the anterior cingulate of depressed responders. Neuropsychopharmacology, 2001. 25(5 Suppl): p. S74-8.

[0246] 78. Wu, J. C, et al., Effect of sleep deprivation on brain metabolism of depressed patients. Am. J. Psychiatry., 1992. 149(4): p. 538-543.

[0247] 79. Wu, J., et al., Prediction of antidepressant effects of sleep deprivation by metabolic rates in the ventral anterior cingulate and medial prefrontal cortex. Am. J. Psychiatry., 1999. 156(8): p. 1149-1158.

[0248] 80. Smith, G. S., et al., Cerebral glucose metabolic response to combined total sleep deprivation and antidepressant treatment in geriatric depression. Am. J. Psychiatry., 1999. 156(5): p. 683-689.

[0249] 81. Mayberg, H. S., et al., Cingulate function in depression: a potential predictor of treatment response. Neuroreport., 1997. 8(4): p. 1057-1061.

[0250] 82. Terman, M., et al., Predictors of response and nonresponse to light treatment for winter depression. Am J Psychiatry, 1996. 153(11): p. 1423-9.

[0251] 83. Lewy, A.J., et al., Antidepressant and circadian phase-shifting effects of light. Science, 1987. 235(4786): p. 352-4. [0252] 84. Benedetti, F., et al., Long-term response to lithium salts in bipolar illness is influenced by the glycogen synthase kinase 3-beta -50 T/C SNP. Neurosci Lett, 2005. 376(1): p. 51-5.

[0253] 85. Posternak, M. A., et al., Assessing past treatment history: test-retest reliability of the Treatment Response to Antidepressant Questionnaire. J Nerv Ment Dis, 2004. 192(2): p. 95-102.

[0254] 86. Golden, R.N., et al., The efficacy of light therapy in the treatment of mood disorders: a review and meta-analysis of the evidence. Am J Psychiatry, 2005. 162(4): p. 656-62. [0255] 87. Hedeker, D. and R.D. Gibbons, A random-effects ordinal regression model for multilevel analysis. Biometrics, 1994. 50(4): p. 933-44.

[0256] 88. Gibbons, R.D., et al., Some conceptual and statistical issues in analysis of longitudinal psychiatric data. Application to the NIMH treatment of Depression Collaborative Research Program dataset. Arch Gen Psychiatry, 1993. 50(9): p. 739-50. [0257] 89. Gibbons, R.D. , et al., Random regression models: a comprehensive approach to the analysis of longitudinal psychiatric data. Psychopharmacol Bull, 1988. 24(3): p. 438-43.

[0258] 90. Verbeke, G. and G. Molenberghs, eds. Linear Models in Practice: a

SAS-Oriented Approach. 1997, Springer: New York. [0259] 91. Hosmer, D.W. and S. Lemeshow, Applied Logistic Regression. 1989,

New York: Wiley.

[0260] 92. Ten Have, T.R., A mixed effects model for multivariate ordinal resopnse data including correlated discrete failure times with ordinal responses. Biometrics, 1996. 52(2): p. 473-91. [0261] 93. Cohen, J., Statistical power analysis for the behavioral sciences. Second ed. 1988, Hillsdale, NJ: Erlbaum.

[0262] 94. Hedeker, D., R. Gibbons, and C. Waternaux, Sample size estimation for longitudinal designs with attrition: comparing time-related contrasts between two groups. Journal of Educational and Behavioral Statistics, 1999. 24: p. 70-93. [0263] 95. Diggle, P. J., Analysis of Longitudinal Data. Second Edition ed. Oxford

Statistical Science Series. 2002, New York: Oxford University Press.

[0264] 96. Colombo, C, et al., Rate of switch from depression into mania after therapeutic sleep deprivation in bipolar depression. Psychiatry. Res., 1999. 86(3): p. 267-270. [0265] 97. Post, R.M., et al., Rate of switch in bipolar patients prospectively treated with second-generation antidepressants as augmentation to mood stabilizers. Bipolar Disord, 2001. 3(5): p. 259-65.

[0266] 98. Little, R.J.A. and D.B. Rubin, Statistical Analysis with Missing Data.

1987, New York: Wiley. [0267] 99. Little, R. J. A., Modeling the drop-out mechanism in repeated-measure studies. J American Statistical Assoc, 1995. 90: p. 1112-1121.

[0268] 100. Scharfstein, D., A. Rotnizky, and J. Bobbins, Adjusting for nonignoralbe drop-out using semiparametric nonresponse models. J American Statistical Assoc, 1999. 94: p. 1096-1120. [0269] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a subject who is likely to respond to sleep deprivation in the treatment of a depressive disorder and/or a manic disorder, said method comprising: screening a biological sample from said subject for increased or decreased expression of one or more genes listed in Table 1 , where upregulation or downregulation (as indicated herein) of expression of said one or more genes, is an indicator that the subject is likely to respond to sleep deprivation for the treatment of a depressive disorder and/or a manic disorder.

2. The method of claim 1, wherein said screening comprises screening said biological sample for increased or decreased expression of three or more genes listed in Table 1.

3. The method of claim 1, wherein said screening comprises screening said biological sample for increased or decreased expression of five or more genes listed in Table 1.

4. The method of claim 1 , wherein said screening comprises screening said biological sample for increased or decreased expression often or more genes listed in Table 1.

5. The method of claim 1, wherein said screening comprises screening genes whose expression is concordant in DLPFC and lymphocytes.

6. The method of claim 1, wherein said two or more genes comprises one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.

7. The method of claim 1, wherein said biological sample comprises a lymphocyte.

8. The method of claim 1, wherein said biological sample comprises a neurological tissue.

9. The method of claim 1, wherein said human is a human undergoing psychiatric evaluation.

10. The method of claim 1 , wherein said human is a human receiving psychoactive medication.

11. The method of claim 1 , wherein said human is a child or an adolescent.

12. The method of claim 1, wherein said human is an adult.

13. The method of claim 1, wherein said screening comprises a nucleic acid hybridization to determine an mRNA level of said two or more genes.

14. The method of claim 13, wherein said determining comprises a method selected from the group consisting of a Northern blot, a Southern blot using DNA derived from an RNA expressed by said two or more genes, an array hybridization, an affinity chromatography, an RT-PCR using an RNA expressed by said two or more genes, and an in situ hybridization.

15. The method of claim 13, wherein said determining comprises an array hybridization using a high density nucleic acid array.

16. The method of claim 13, wherein said determining comprises an array hybridization using a spotted array.

17. The method of claim 1, wherein said screening comprises detecting a protein(s) expressed by said two or more genes.

18. The method of claim 17, wherein said detecting is via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry.

19. The method of claim 1, wherein said upregulation or downregulation is with respect to a control comprising baseline levels of expression determined for a members of a normal healthy population.

20. The method of claim 1, wherein said upregulation or downregulation is with respect to a control comprising levels of expression determined for said human at an earlier time.

21. A method of treating a human subject for a psychiatric disorder, said method comprising: utilizing a biological sample from said human subject to determine whether or not the subject is a likely responder to sleep deprivation in the treatment of said psychiatric disorder according to the methods of any of claims 1-20; and subjecting said subject to sleep deprivation alone or in combination with other therapeutic modalities for the treatment of said psychiatric disorder.

22. The method of claim 21 , wherein said prescribing or providing comprises providing cognitive therapy to said subject.

23. The method of claim 21 , wherein said prescribing or providing comprises prescribing psychoactive medication for said subject.

24. The method of claim 23, wherein said prescribing or providing comprises prescribing psychoactive medication for said subject where said psychoactive medication is selected from the group consisting of Neuroleptics (antipsychotics), antiparkinsonian agents, sedatives and anxiolytics, antidepressants, a mood stabilizer, and an anticonvulsant drug.

25. The method of claim 24, wherein said medication comprises a neuroleptic selected from the group consisting of trifluoperazine (Stelazine), pimozide (Orap), flupenthixol (Fluanxol), and chlorpromazine (Largactil), flupenthixol (Fluanxol), fluphenazine decanoate (Modecate), pipotiazine (Piportil L4), and haloperidol decanoate (Haldol LA).

26. The method of claim 24, wherein said medication comprises an antiparkinsonian agent selected from the group consisting of benztropine mesylate (Cogentin), trihexyphenidyl (Artane), procyclidine (Kemadrin), and amantadine (Symmetrel).

27. The method of claim 24, wherein said medication comprises a sedatives and/or anxiolytics selected from the group consisting of barbiturates, benzodiazepines, and non-barbiturate sedatives.

28. The method of claim 24, wherein said medication comprises an antidepressant selected from the group consisting of a tricyclic (e.g., amitriptyline (Elavil), imipramine (Tofranil), doxepin (Sinequan), clomipramine (Anafranil)), a monoamine oxidase inhibitors (e.g., phenelzine (Nardil) and tranylcypromine (Parnate)), a tetracyclic (e.g. maprotiline (Ludiomil)), trazodone (Desyrel) and fluoxetine (Prozac).

29. The method of claim 24, wherein said medication comprises a mood stabilizer selected from the group consisting of lithium and carbamazepine.

30. A method of screening for an agent that mitigates one or more symptoms of a psychiatric disorder, said method comprising: administering a test agent to a cell and/or a mammal; and detecting altered expression in said cell and/or mammal of one or more genes listed in Table 1 , where upregulation or downregulation (as indicated herein) of expression of said one or more genes, as compared to a control, is an indicator that said test agent has activity that mediates one or more symptoms of a psychiatric disorder.

31. The method of claim 30, wherein said psychiatric illness is a depressive disorder.

32. The method of claim 30, wherein said detecting comprises screening said biological sample for increased or decreased expression of three or more genes listed in Table 1.

33. The method of claim 30, wherein said detecting comprises screening said biological sample for increased or decreased expression of five or more genes listed in Table 1.

34. The method of claim 30, wherein said detecting comprises screening said biological sample for increased or decreased expression often or more genes listed in Tables 1.

35. The method of claim 30, wherein said detecting comprises screening said biological sample for increased or decreased expression of one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.

36. The method of claim 30, wherein said detecting comprises a nucleic acid hybridization to determine an mRNA level.

37. The method of claim 36, wherein said detecting comprises a method selected from the group consisting of a Northern blot, a Southern blot using DNA derived from said two or more genes, an array hybridization using probes that bind to RNAs from said two or more genes, an affinity chromatography, an RT-PCR using an RNA derived from said two or more genes, and an in situ hybridization.

38. The method of claim30, wherein said detecting comprises detecting protein(s) expressed by said two or more genes.

39. The method of claim 38, wherein said detecting is via a method selected from the group consisting of capillary electrophoresis, a Western blot, mass spectroscopy, ELISA, immunochromatography, and immunohistochemistry.

40. The method of claim 30, wherein said control comprises a cell contacted or mammal not treated with said test agent or treated with said test agent at a lower concentration.

41. The method of claim 30, wherein said test agent is not an antibody.

42. The method of claim 30, wherein said test agent is not a protein.

43. The method of claim 30, wherein said test agent is a small organic molecule.

44. The method of claim 30, wherein said cell is cultured ex vivo.

45. A method of identifying genes implicated in psychiatric disorders, said method comprising subjecting a mammal to sleep deprivation and determining genes whose expression is altered in response to said sleep deprivation.

46. The method of claim 45, wherein said mammals are humans diagnosed with depressive disorder where said disorder is mitigated by sleep deprivation.

47. The method of claim 45, wherein said determining comprises determining gene expression in peripheral blood cells.

48. The method of claim 45, wherein said determining comprises determining gene expression in neural tissue.

49. A method of mitigating a psychiatric disorder, said method comprising altering expression or activity of one or more genes listed in Table 1.

50. The method of claim 49, wherein said one or more genes comprise one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.

51. A method of mitigating a psychiatric disorder, said method comprising administering the gene product or analogue thereof of one or more genes listed in Table 1.

52. The method of claim 52, wherein said one or more genes comprise one or more genes selected from the group consisting of RORA, BHLB2, PERl, and CLOCK.