WO1994009765A1 - Functional interactions between glial s-100b and central nervous system serotonergic neurons - Google Patents

Abstract

A method for stimulating the production of S-100B in a subject is provided by administering an effective amount of a 5-HT--1A receptor agonist, the agonists including receptor, including an antipeptide antibody to a functional domain of a 5-HT--1A receptor. Additional methods for stimulating the growth of central serotonergic neurons are provided, by contacting such neurons with an effective amount of S-100B, or analog or derivative thereof, and the growth of central serotonergic neurons is inhibited by contacting the neurons with an effective amount of an inhibitor of S-100B production or action. Furthermore, diseases associated with decreased central serotonergic innervation or activity including autism, depression, anxiety, biological rhythm-based sleep disorder, and cortical brain damage may be treated by such methods. Diseases associated with increased central serotonergic innervation but ineffective astroglial S-100B release, such as Down's Syndrome and Alzheimer's Disease may be treated by up-regulating astroglial 5-HT--1A receptors, followed by stimulation of S-100B production or release using a 5-HT--1A receptor agonist, including an anti-peptide antibody to a functional domain of a 5-HT--1A receptor.

Description

FUNCTIONAL INTERACTIONS BETWEEN GLIAL S-100_B AND CENTRAL NERVOUS SYSTEM SEROTONERGIC NEURONS

, BACKGROUND OF THE INVENTION

Field of the Invention

_*. 5 The invention in the field of neuroscience and medicine relates to the discovery and use of a cortical growth factor S-100_B as trophic for cortical and serotonergic neurons in the brain, as well as the use of 5-HT----A agonists, S-100_B and derivatives thereof, to induce corticol or serotonergic growth, 10 stimulation or regeneration, for therapeutic and/or diagnostic applications, in vivo, in si tu and/or in vi tro . Additionally, the present invention relates to the use of 5-H7--_1A antagonists, also including peptides corresponding to functional domains of 5-HT--jA receptors, and antibodies thereto, which up regulate 15 central serontonergic neurons, which subsequent stimulation is enhanced or provided by such up regulation. Description of the Background Art

Serotonergic neurons, which release serotonin (5- hydroxytryptamine, 5-HT) as a neurotransmitter, play a key role 20 in the general maturation of the brain. Changes in the innervation density of serotonergic nerve fiberj would be expected to induce changes in the maturation of "target" neurons with which the serotonergic fibers communicate.

Binding studies with appropriately labelled ligands 25 initially revealed the existence of two major types of serotonin receptors in the brain, termed 5-HT_! and 5-HT₂, and later pointed to the existence of further subtypes, such as 5-χIT_1A and 5-HT,_b (Peroutka, et al., Feder. Proc. 42:212-217 (1983); Pazos et al. , Brain Res. 205:346 (1985)). Other types of 5-HT receptors 30 (e.g., 5-HT₃ and 5-HT₄) have been described more recently (see: Whitaker-Azmitia et al., Eds., Ann . N. Y. Acad. Sci . v600 (1990)).

Serotonin neurons have been shown to regulate their own development, i.e. to "autoregulate" (Whitaker-Azmitia et 35 al., Neuroεci . Lett. 67:307-312 (1986)), due in part to release of growth factors by stimulation of 5-HT_1A recepuors on astrocytes (Whitaker-Azmitia et al., J. Neurochem. 46:1186-91 (1986)). Glial cells, such as astrocytes, have 5-HT receptors (Whitaker et al. , ibid. ) . The conditioned medium from glial cells which had been exposed to a selective agonist for the 5- HT_1A receptor subtype stimulated cultured serotonergic neurons (Whitaker-Azmitia et al., Brain Reε. 497:80-85 (1989)). These results suggest the existence of a soluble hippocampal SGF of glial origin.

A number of proteins have been implicated as neuronal growth factors. Nerve growth factor (NGF) appears to act as a CNS cholinergic growth factor (Hefti, J. Neurosci . 6:2155-2162 (1986)). Epidermal growth factor (EGF) has trophic effects on neuron-like PC-12 cells (Leonard et al. , Mol . Cell . Biol . 7:3156-3167 (1987); Isobe et al. , J^". Neurochem. 43:1494-1496 (1984) ) . Insulin has been shown to mediate growth of cultured fetal neurons (Heindenreich et al. , Endocrinology 125:1451-1457 (1989)). The protein S-100_B/ which is composed of two β- subunits, stimulates neurite extension in cultured chick cortical neurons, hence its designation as a "cortical" growth factor (Kligman et al. , Proc. Natl . Acad. Sci . U. S.A . 82:7136- 7139 (1985) ) . Calmodulin has been shown to have substantial structural homology and a similar Ca²⁺ binding profile to S-100_B (Isobe et al. , Endocrinology 125:1451-1457 (1989)).

A family of proteins named S-100 was first isolated nearly 25 years ago (Moore, Biochem. Biophys . Res. Commun . 19:739-742 (1965)). The member of this family designated S-100_B was previously known to promote neurite extension in vitro, in chick embryo cultures (Kligman et al., Proc. Natl . Acad. Sci . USA 82:7136-39 (1985)). S-100 production was stimulated in cultures of the rat astroglioma line, C6, by dibutyryl cyclic AMP (Labourdette et al., Biochem. Biophys . Res. Comm. 96:1702-09 (1965)). Furthermore, S-100 may be releasable from brain tissue (Shashoua et al. , J^". Neurochem. 42:1536-41 (1984)).

The well-known factor NGF has been found to have no effect of 5-HT sprouting in damaged hippocampal neurons (Kiedrowsk et al. , Eur. J. Neurochem. 43:1494-1496 (1984)), or in cultured dopaminergic neurons (Dreyfus et al., Brain Res. 194: 540-547 (1980) ) . During fetal brain development, S-100_B, as detected by S-100_B specific antibodies, shows an intense yet transient rise in the midline raphe region, where the serotonergic neurons are developing (Van Hartesveldt et al., J. Comp . Neurol . 253:175-184 (1986) ) .

Finally, the human gene for the β subunit of S-100 has been mapped to the distal half of the long arm of chromosome 21 (Allore et al., Science 239:1311-1313 (1988)); this gene was recently shown to contain the cAMP responsive element, CRE, on the promoter region (Allore et al. , J. Biol . Chem. 265:15537- 15543 (1990) ) . Furthermore, recent studies have shown an increase in S-100 immunoreactivity in postmortem Alzheimer's Disease and Down's Syndrome brains (Griffin et al. , Proc . Natl . Acad. Sci . USA 86:7611-7615 (1989)). Therefore, greater understanding of the physiological actions of S-100 and knowledge of which factors regulate its release are believed to be important for the understanding and treatment of both disorders.

A cDNA for the 5-HT_1A receptor from the rat has been cloned and shown to have a coding region consisting of 1266 nucleotides corresponding to the 422 amino acids! of 5-HT_1A (Albert et al, J. Biol . Chem. 265:528 1990). The 5-HT_1A receptor from rat has seven transmembrane domains, a large third cytoplasmic loop and is 89% homologous with the human gene. The rat 5-HT_1A receptor encoding mRNA tissue distribution showed high levels in spectrum, hippocampus, thalamus, amygdala olfactory bulb, mesencephalon, medulla and hypothalamus. Detectable levels were seen in the cortex and basal ganglia, but not in the pineal and the pituitary (Albert et al., 1990). These results are in general agreement with the ³H-8-OH-DPAT binding studies in the rat which showed widespread distribution of receptor labelling except in extrapyramidal areas (substantia nigra, caudate nucleus and the globus pallidus) , cerebellum and habenula were levels were undetectable. (Pazos et al. Brain Res. 346:205-230 (1985); Verge et al. J. Neurosci 6:3474-3482 (1986) ) .

Site-directed antibodies based on synthetic peptides have been used to study many previously uncharacterized proteins (Sutcliff et al. Nature 287:801-805 (1980); Walter et al, Proc. Natl . Acad. Sci . 77:5197-5200 (1980) ; Yu et al. Journal of Cell Biology 114:1217-1232 (1991)). This approach has the advantages of being applicable as soon as the cDNA sequence is known and that the antipeptide antibody can be directed against a specific short region of the molecule. In general, the short sequence is bound to a larger carrier protein to increase its antigenicity (Harlow et al. Antibodies : A Laboratory Manual Cold Spring Harbor Press (1988)). This approach has been applied to the 5- HT_1A receptor against a region of the third cytoplasmic loop. (Raymond et al. Molecular Pharmacology 36:015-0211 (1989)) raised an antibody (JWR 21) against the sequence 242-267 using keyhole limpet hemocyanin (KLH) as the carrier and (El Mestikawy et al. Neurosci . Lett. 118:189-192 (1990)) raised an antipeptide antibodies against a very similar sequence 243-268 using bovine serum albumin as carrier. Anatomical immunoautoradiographic studies with this latter antibody at 1/1000 dilution showed a "striking similar" distribution to that seen with ³H-8-OH-DPAT binding (El Mestikaway et al. Neurosci . Lett. 118:189-192, 1990) . In an immunocytochemical study, labeling of 5-HT perikarya and dendrites membranes was seen in the midbrain raphe nuclei (Sotelo et al. Eur. J. Neurosci . 2:1144-1154, 1990). Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or ai. admission that the cited documu ts is considered material to the patentabilty of the claims of the present application. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

SUMMARY OF THE INVENTION The present invention relates to the discovery that S- 100_B is a protein released upon stimulation of their 5-HT_JA receptors, which stimulation has further been found to promote the growth of central serotonergic neurons and/or cortical neurons.

The present invention also relates to methods for modulating S100_B effects in vi tro, in vivo and in si tu, as well as to diagnostic and/or therapeutic methods involving serotonergic neuronal growth and/or maintenance, through such modulation of S100_B.

The present invention is also directed, in one aspect, to a method for stimulating the production of S-100_B in a subject, comprising administering a S100_B stimulating effective amount of an agonist acting on the 5-HT_1A receptor, by the use of at least one 5-HT--_1A agonist compound or anti-idiotype antibody to 5-HT--_1A receptors which has the in vivo effect of stimulating 5-HT_1A receptors. Non-limiting examples of 5-HT--_1A agonists useful in the present invention may include 5-hydroxytryptamine, 5-methoxytryptamine, buspirone, 8-hydroxy-2- (di-n-propylamino) tetralin (8-OH-B AT) , ipsaspirone, gepirone, SM_^'3997, lysergic acid, ipsapirone, diethylamide, and 5-HT--_1A agonistic antibodies.

The stimulation of 5-HT--_IA receptors may be made subsequent to up regulation of such receptors, which prior up regulation increases the stimulatory effect on cortical and/or serotonergic neurons, preferably serotonergic neurons, which prior up regulation can be accomplished according to known method steps, as known to those skilled in the art, based on the teachings and guidance presented herein.

Stimulation of S-100_B or 5-HT--_1A receptor stimulation in a subject may have the therapeutic or diagnostic effect of causing serotonergic neuron growth and/or stimulation, which may be suitable for treatment and/or diagnosis of at least one diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-sleep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers disease, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotonin production developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction. The present invention is further directed to a method for inducing the growth and/or stimulation of central serotonergic neurons or serotonin release in a subject, comprising administering to the subject an effective amount of S-100_B, a functional derivative or analog thereof, or an agonist acting at the 5-HT_1A receptor, such as those described above and including nti-idiotype antibodies to at least one functional domain of a 5-HT--_1A receptor, from a mammal, preferably a human. The present invention also includes a method for inducing the growth and/or stimulation of central serotonergic neurons or serotonin, in vitro or in vivo by contacting the neurons with S100_B, a 5-HT--_1A agonistic antibody, or a functional derivative or analog thereof. For treatment of diseases associated with dysregulation of serotonergic neurons, in vivo contacting is preferred.

Alternatively, the present invention involves a method for inhibiting the growth of central serotonergic neurons, comprising contacting the neurons with an effective amount of an inhibitor of S-100_B production or action. The inhibitor may be an antibody specific for S-100_B or a 5-HT_1A receptor antagonist, such as the non-limiting examples of spiperone and spiroxatine, or an 5-HT--_1A receptor peptide corresponding to a functional domain of a 5-HT--_1A receptor, or antibodies thereto, such as anti-peptide antibodies. The present invention also relates to a method for treating a disease and/or pathology associated with decreased central serotonergic innervation or activity including decreased serotonin levels in a mammalian, preferably human, subject, by administering a serotonergic stimulating effective amount of S- 100_B, a functional derivative thereof, or a 5-HT_]A agonist including, but not limited to an antibody to a functional domain of a 5-HT--_1A receptor. Diseases for which this method is useful include diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-fileep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotonin production developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction.

The present invention is also directed to a method for treating a disease associated with increased. central serotonergic innervation or activity in a subject, comprising administering an effective amount of an inhibitor of S-100_B production or action. Such inhibitors include an antibody specific for S-100_B 5-HT_1A receptors, and/or a 5-HTι_A receptor antagonist.

In another embodiment, the invention is directed to a method for stimulating serotonergic neuronal growth and/or stimulation in a subject having Alzheimer's disease comprising the steps of: (a) up-regulating the expression of 5-HT--_1A receptors on astroglial cells in the brain of the subject; and then (b) stimulating the induction of the release of S-100_B in the subject according to the methods described above, thereby stimulating the cortical neuronal growth.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a histogram showing the effects of EGF (0.5μg/ml) , NGF [10 μg/ml) , S-100_B (10 μg/ml) ar.tl insulin (100 μg/ml) added daily in 5-fold serial dilutions (most concentrated dose at right) on serotonin uptake. Each bar is the mean + S.E.M. (n=4) of [³H]5-HT uptake as a percent of control. Control uptake was 5038 + 606 cpm for S-100_B/insulin and 6159 + 318 cpm for NGF/EGF. Figure 2 is a graph showing the effects of a single application of S-100_B and calmodulin on [³H]5-HT uptake capacity after 3 days. Final concentration is shown on he abscissa. Each point represents the mean + S.E.M. (n=4) .

Figure 3 is a histogram showing a morpnometric analysis of the total neurite length for individual 5-HT- immunoreactive neurons after 30 h of stimulation. The bars represent the mean + S.E.M. for 10 neurons in each well (number shown under bars) .

Figure 4 is a graph showing effects of native bovine S-100 and of media from astroglial cells stimulated with the selective 5-HTι_A agonist ipsapirone (GCM-IPS)* on the growth of serotonergic neurons in culture as determined by selective uptake of ³H-serotonin. Hatched bars indicate the effects of S- 100 and GCM-IPS in the presence of an antibody o S-100 (final dilution 1/10,000) . Each bar represents the mean ± S.E.M. of 4 cultures, derived from different litters. S-lOϋ, GCM-IPS and the antibody were all added at time of plating and the grpwth assessed 3 days later.

Figure 5 is a pictorial representation of antipeptide 5-HT--_1A receptor antibody binding to a hippocampus in midbrain section of a rat brain at 13500 times wherein tr.3 label is associated with microtubules (MTB) and also bound along the outer plasma membrane (PLMB) neurons.

Figure 6 shows a schematic diagram of the 5-HT--_JA receptor. Figure 7 shows a graphical representation of a purified synthetic peptide corresponding to a portion of a functional domain of a 5-HT--_1A receptor.

Figure 8 shows a graphical representation of radio- labeled anti-goat anti-rabbit Ig fragments to antipeptide antibodies of the present invention wherein labeling radioactivity it various antibody dilutions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the discovery that a protein designated S-100_B or functional derivatives and/or analogs thereof, is a serotonergic specific growth factor (SGF) that is also induced by serotonin. Since this protein has no effect on cholinergic and noradrenergic neurons, nor on cells in the peripheral nervous system, S-100_B, or derivatives or analogs thereof, are specific for modulation of central serotonergic nerves.

The present invention also relates to the discovery that S-100_B is a growth factor released in response to 5-HT_1A receptor stimulation as well as by serotonin. Serotonin neurons have been shown to autoregulate their own development. The present inventors first discovered that this autoregulatory circuit involves the release of a growth factor or factors induced by stimulation of 5-HT_1A receptors in vivo, such as on astrocytes. Therefore, the present invention is directed in one aspect to the use of 5-HT_]A agonists or antagonists as therapeutics, acting via the regulation and/or modulation of S- 100_B production and/or release from astroglial cells and/or serotonergic neurons.

The present invention is also directed to methods involving the use of the S-100_B protein, which is a dimer of two β chains, and is found exclusively in the brain, in contrast to α- oι or ot-β dimers, which are also found outside the brain. Also included within the scope of the present invention are functional derivatives of the S-100_B protein.

An "analog" of S-100_B refers to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.

By "functional derivative" is meant a "fragment, " "variant, " or "chemical derivative" of S-100_B, which terms are defined below. A functional derivative retains at least a portion of the function of the S-100_B which permits its utility in accordance with the present invention, namely serotonergic or cortical growth factor activity.

A "fragment" of the S-100_B refers to any subset of the molecule, or of the β chain, such as a shorter peptide. A "variant" of the S-100_B refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, or by recombinant DNA technology, using well known method steps. Amino acid sequence variants of the S-100_B molecule can also be prepared by mutations in the DNA. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity, without placing the sequence out of reading frame and preferably not creating complementary regions that could produce secondary mRNA structure (see EP Patent Application Publication No. 75,444) . At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis of nucleotides in the DNA encoding the S-100_B molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant ceil culture. The variants typically exhibit the same qualitative biological activity as the naturally occurring analog. Additionally, or alternatively, variants have greater than 80% homology with the corresponding S100_B proteins or fragments, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, while maintaining at least some HT-5_JA receptor modulating activity.

A "chemical derivative" of S-100_B contains additional chemical moieties not normally a part of the protein or peptide. Covalent modifications of the* protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Also included in the scope of the invention are salts of the proteins and peptides of the invention. As used herein, the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein or peptide molecule. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed for example, with amines, such as triethanolamine, arginine, or lysine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. By the term "agonist" is intended any chemical or biological substance capable of binding to a particular receptor, such as the 5-HT_1A receptor or binding site thereof, according to the present invention, which binding stimulates a biological response associated with the receptor. The term is intended to include an endogenous molecule which exerts its physiological action by receptor binding and triggering of a signal to a cell, as well as an exogenous agent which mimics the action of such an endogenous agonist. Thus, if a receptor is linked to a second messenger system that signals a positive response, such as, for example, the increased production and/or secretion of a protein growth factor, such as S100_B, or analog or functional derivative thereof, an agonist will induce production and/or secretion of the growth factor. In cont ^~ast, if a receptor is linked to a second messenger system which signals a negative response, such as a termination of cell growth, an agonist for that receptor will inhibit cell growth.

Agonists for 5-HT_1A receptors include, but are not limited to, 5-hydroxytryptamine (serotonin) , 5- methoxytryptamine, buspirone (U.S. Patent 3,717,634) , 8- hydroxydipropylamineotetralin, ipsaspirone (EPO Publication 129,128A), gepirone (U.S. Patent 4,423,049) , SM23997 (U.S. Patent 4,507,303) , MDL 72832 (U.S. Patent 4,612.312) ipsapirone, lysergic acid diethylamide and anti-idiotype ancibodies to one or more functional domains of a 5-HT--_1A receptor. In addition, newer polycyclic aryl- and heteroarylpipera- zinyl imides with 5-HT_1A-binding and activating properties, such as WY-47,846 (cpd. 34) and other disclosed in Abou-Gharbia et al. , J. Med. Chem. 31:1382-1392 (19?8) , may be useful in the prese.'i invention, which references are hereby entirely incorporated by reference.

In addition, an antibody, preferably a monoclonal antibody (MAb) to the 5-HT_1A receptor which by virtue of its epitope specificity stimulates a response, rather than inhibiting the binding of an agonist, termed an "agonistic antibody, " is also an agonist as intended herein. The present invention is intended to 'encompass additional 5-HT_1A receptor agonists, routinely obtainable according to the present invention, based on the teaching and guidance presented herein without undue experimentation.

By the term "antagonist" is intended a substance which is itself devoid of intrinsic pharmacolog-. activity and stimulates no biological response when bound to a receptor, but has the capacity to bind to a receptor and thereby inhibit binding of, or action of, an agonist. Typically, antagonists act by competing for agonist binding to a receptor.

Antagonists for 5-HTj_A receptors are known in the art and include, but are not limited to, spiperone and spiroxatine. In addition, an antibody specific for the 5-HT,_A receptor which does not have agonist activity, but inhibits bi::ding or action of an agonist, is also an antagonist, as intendRd herein. In addition, newer polycyclic aryl- and heteroaryl-piperazinyl imides with 5-HT_1A-binding properties which would inhibit binding of endogenous agonists (Abou-Gharbia, M. et al. , supra) may be useful as antagonists in the present invention.

In addition, a 5-HT--_1A peptide, as described herein, or a 5-HT--_1A antagonistic antibody to such pept.i.des, preferably a monoclonal antibody (MAb) to the 5-HT_1A receptor which, by virtue of its epitope specificity, inhibits the binding of a 5- HT--_1A agonist, termed an "antagonistic antibody," is also an antagonist as intended herein. The present invention is intended to encompass additional 5-HT_1A receptor antagonists, routinely obtainable according to the present invention, based on the teaching and guidance presented herein without undue experimentation.

Surprisingly, peptides corresponding to portions of functional or other domains, such as transmembrane domains, of 5-HT--_1A receptors have also unexpectedly been discovered to act as 5-HT--_1A receptor antagonists, as do 5-HT--_1A receptor antagonists, as described herein.

The principles of receptors, agonists, and antagonists are αescribed, for example, in Gilm*.v.ι et al., Goodman and Gilman's "The Pharmacological Basis of

Therapeutics", Eighth Edition, Macmillan Publishing Co., New York, 1990, Berkow et al, eds., The Merck Manual , 6th edition, Merck and Co., Rahway, N.J. , 1992; Avery' s Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., WiMiams and Wilkins, Baltimore, MD. (1987) , Katzung, ed. Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. (1992) , which references, and references cited therein, are entirely incorporated herein by reference.

Antibodies of the present invention are those which are specific for, and interact with, S-100_B, funrational derivatives or analogs thereof, or with 5-HT--_1A receptors and modulate the action of the S-100_B-serotonergic neuron autoregulatory system, e.g., as 5-HT--_1A receptor agonists or antagonists.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs) , chimeric antibodies, and anti-idiotypic (anti-Id) antibodies or fragments, analogs or derivatives thereof. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from -he sera of animals immunized with an antigen. In the context of the present invention, a "5-HT--_1A antibody" refers to an antibody, as described herein, which binds or associates with a 5-HT--_1A receptor and has the in vivo biological activity of stimulating or inhibiting the 5-HT--_1A receptor to have the effect of inducing or inhibiting, respectively, serotonergic and/or cortical neuron growth and/or stimulation, which effect may be mediated by the release of S- 100_B, and wherein the modulation of central serontonergic neurons is preferred.

Such antagonistic 5-HT--_1A antibodies of the present invention are preferably generated against a synthetic peptide corresponding to a functional domain of a 5-HT_1A receptor, according to the following criteria.

(1) Functional domain of the receptor: The receptor is homologous to the beta-adrenergic receptor family and many of the various segments of the 5-HT_1A receptor can be inferred from the extensive work with the β-adrenergic receptor. The agonist binding site consists of a least two aspartate (asp) residues within the 2nd and 3rd transmembrane regions (Dohlman et al. Biochemistry 26:2657-2663 (1987). Asp residues (#82 and 116) exist in a similar site in the 5-HT_1A receptor (Figure 6) . A histidine (His) in the 3rd transmembrane site (#126) would provide the needed positive charge for 5-HT binding. The third cytoplasmic loop is believed to be the site of interaction with the G-proteins in the cytoplasm for regulation of the second messenger systems (Kobilka et al. Nature 329:205-230 (1988)). Therefore, peptide sequences can be selected indicate the anatomical location of various segments of the full molecule and determine the cellular distribution of particular functional regions.

As a non-limiting example, domains vere selected in the 2nd external loop (S1A-170) and in the 3rd cytoplasmic loop (S1A-258) (Fig. 6) .

(2) Hydrophilicity regions. The antigenic sites on a peptide can be approximated based on the hydrophilicity score which assumes that the greater the local hydrophilicity, the more antigenic the sequence (Hopp, T.P. Proc. Natl. Acad. Sci. USA 8:3824-3828 (1981)). This measure assigns a numerical value to the various amino-acids; for example K, R, D and E have a value of +3.00, W has a value of -3.4, and G and P have a value of 0. the calculated window average at a residue is calculated across 6 residues. The hydrophilicity score for A1A170 is shown in Table I.

(3) Two dimensional protein structure. There are three states in which a sequence can exist in a secondary structure of a protein molecule. These are betπ sheets, alpha helix and turns (Chou et al. Biochem. 13:222-245 (1974); Chou et al. Adv. Enzymology 47:45-147 (1978)). In the latter state, it is assumed that the amino acids are most exposed.

As a non-limiting example, the above selected peptides both have a significant number of predicted turns (Table I shows results for S1A170) . Table I.

The hydrophilicity scope according to Hopp and Wood (1981) and the secondary structure prediction of Chou and Fasman algorithm are shown below for the nonp.jptide A1A170 (Intelligenetics program) . Notice that a turn structure is predicted for the region showing a high positive hydrophilicity value.

-1 0 +1 +2

***********p

***********p

_*************_M

***********L

*********Q

********W

R

^■p* ***** p******************

E********•_**************

£)********^•*<^■ ************** R*********************** s_{***********************} )*********************** p**************** D********** A*****

* C

(4) Charge Balance of the Protein. The net charge of a peptide sequence should be near neutrality. If the molecule is too highly charged it will present problems during the purification procedure after the peptide is synthesized. If the net charge is highly basic or acidic a cation or anion exchange resin can be used. Bio-rad AG-50 resin has been successfully used for very basic peptides. However, strong deviations from neutrality is also a problem during the attachment to KLB which should proceed at neutral pH (see below) .

As a non-limiting example, S1A-170 has 6 charged residues and a net -2 charge while S1A-258 has 5 charged residues and a net + 1 charge. (5) Amino Acid length. The sequen e for an ideal peptide for antibody formation should have 15-20 amino acids. A strand of 6 amino acids is the lower limit for a recognition site whole more than 20 presents some additional problems with the synthesis and structural considerations (Harlow, E. et al. Antibodies: A Laboratory Manual Cold Spring Harbor Press (1988) ) .

As a non-limiting example, both S1A-170 and S1A-258 have 17 residues.

(6) Phosphorylation and glycosylation sites. A protein molecule has many possible phosphorylation and glycosylation sites. These sites should be avoided in choosing a sequence unless a particular confirmation is sought. Antibodies have ueen raised against phosphoryla*'2d sequences but these antibodies have altered affinity for the un-phosphorylated site (See Czernik et al. Method Enzymol 201:264-283 (1991) . Furthermore, a phosphorylated segment of the molecule often confers allosteric changes in the protein structure which may reduce the affinity for the peptide segment artificially produced. It can be appreciated, that sites adjacent to modified sites may be less desirable for the same reasons. The glycosylation sites are located on the

(ASN, N) residues at positions 10, 11 and 24. Three potr-'.ntial protein kinase C phosphorylation sites are located at 147-152, 227-232 and 341-345 and one additional phosphorylation site 251-253 (El Mestikawy et al, Neurochem. Res. 16:1-10 (1991)).

As a non-limiting example, neither S1A-170 or S1A- 258 have phosphorylation nor glycosylation sites. (7) Position of Cysteine. Cystein-.* (Cys, C) residues are commonly involved in disulfide bridges. For this reason it is advisable to avoid a Cys residue in the middle of a peptide sequence. There are 15 Cysteine residues in the 5-HT_1A receptor, 6 in the transmembrane regions, 3 in the 3rd cytoplasmic loop, four in the three extracellular loop and two in the C-terminal cytoplasmic tail. The cysteines in extracellular loop 1 and 2 have been proposed to form a disulfide link in the /3₂-adrenergic receptor (Dchlman et al. Biochemistry 26: (1987)). Similar cysteines ex:^:.st in the 5-HT_1A receptor (Figure 6) . In designing the sequence, it is advisable to have a terminal cysteine residue in order to bind to KLH protein (Harlow, et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press (1988) . This can be either at the C- or N-terminus of the peptide depending on how the peptide is predicted to be exposed in the molecule. For instance, if the desired sequence is at the N-terminal end of the protein, then the cysteine should be placed at the C-terminal end of the peptide. In this way, the N-terminal end will be exposed after its attachment to the carrier protein.

As a non-limiting example, both peptides have an N- terminal Cys ut S1A-258 also contains a Cys in the center of the peptide at position 266.

(8) Homology with known proteins. In selecting a region of the 5-HT_1A receptor protein comparisons with 5-HT₂, 5- HT_1C, α₂ ^and B₂-adrenergic, muscarinic Ml, and the D₂ receptor were performed (Juluj-^ et al. Proc. Natl. Acad. Sci. USA 87:928-932 (1990); Bunzow et al. Nature 336:783-787 (1988)). Once a segment has been selected it should be compared to all known protein sequences. The sequence data bank searching program (Intelligenetics, Inc., Mt. View, CA- (415) -962-7300) we used is based on the algorithm of Welbur and Lipman. Sequence Homology was searched against the Protein Identification Resource data bank (PIR) and the Protein Sequence Data Bank which contains the translated European Molecular Biology Library (EMBL) . Such a procedure will identify those known structures that could react with the antibody raised. This especially relevant when the protein in question has been identified in the same species chosen for study, and conversely, significant homology to an invertebrate protein is not necessarily a problem. High homology for the selected sequence of the same protein in different specie -. is advantageous. The present invention is also related to the production, by chemical synthesis or recombinant DNA technology, of 5-HT--_1A receptor peptides, preferably as small as possible while still retaining sufficiently high affinity or interaction with G-protein coupled receptors to modulate, such as to inhibit binding to such receptors by 5-HT--_1A receptor ligands.

5-HT--_1A receptor peptides of the present invention may include 5-10 to 50-150 amino acid fragments, consensus sequences or substitution sequences of 5-HT--_1A receptors, including, but not limited to serotonin receptors (5-HT) , cytomegalovirus 5-HT--_IA receptors, endothelial cell 5-HT--_1A receptors, testi 5-HT--_1A receptors, and thoracic aorta 5-HT--_IA receptors, and homologs thereof having a homology of at least

80% with at least one of transmembrane domains 1-7, as described herein. See, e.g., Probst et al DNA and Cell Biology 11:1-20(1992), which is entirely incorporated herein by reference. Accordingly, a "5-HT--_1A receptor peptide" of the present invention includes polypeptides having a "5-HT--_ιA receptor amino acid sequence" which substantially corresponds to at least one 4 to 50 amino acid fragment and/or consensus sequence of a known 5-HT--_1A receptor or group of 5-HT--_1A receptors, wherein the 5-HT--_IA receptor peptide has homology of at least 80%, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology, while maintaining 5-HT--_ιA receptor modulating activity, wherein a 5- HT--_1A receptor peptide of the present inventior. is not naturally occurring or is naturally occurring but is in a purified or isolated form which does not occur in nature. Preferably, a 5- HT--_1A receptor peptide of the present invention substantially corresponds to a transmembrane domain of a 5-HT--_1A receptor or group of 5-HT--_1A receptors as a consensus sequence. Also preferred are 5-HT--_1A receptor peptides wherein the 5-HT--_1A receptor amino acid sequence is 4-10 to 50 amino acids in length, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids, or any range therein.

An amino acid or nucleic acid sequence of a 5-HT--_j*_A receptor peptide of the present invention is said to "substantially correspond" to another amino aci£ or nucleic acid sequence, respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one 5-HT--_1A receptor transmembrane domain, or which may be synergistic when two or more transmembrane domains, consensus sequences or hoirologs thereof are present. Additionally or alternatively, such "substantially corresponding" sequences of 5-HT--_1A receptor peptides include conservative amino acid or nucleotide substitutions, or degenerate nucleotide codon substitutions wherein individual amino acid or nucleotide substitutions are well Known in the art.

Alternatively or additionally, substantially corresponding refers to 5-HT--_1A receptor peptides having amino acid sequences having at least 80% homology or identity to an amino acid sequence of a human 5-HT-~_ιA receptor, such as 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homology or identity.

Accordingly, 5-HT--ι_A receptor peptides of the present invention, or nucleic acid encoding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T.E., Proteins : Structure and Molecular Properties , W.H. Freeman &■ Co., San Francisco, 1983, which are hereby incorporated by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al, supra, at §§ A.l.l-A.1.24, and Sambrook et al, supra, at Appendices C and D.

Conservative substitutions of a 5-HT--_1A receptor peptide of the present invention includes a variant wherein at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid. Such substitutions preferably are made in accordance with the following list as presented in Table IV, which substitutions may be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule, while maintaining the receptor binding, inhibiting or mimicking biological activity, as determined by known 5-HT--_1A receptor receptor activity assays.

Alternatively, another group of substitutions of 5-HT--

_IA receptor peptides of the present invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue inserted in its place according to the following Table V. The types of substitutions which may be made in the protein or peptide molecule of the υresent invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al., supra and Figs. 3-9 of Creighton, supra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: TABLE V

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly) ;

2. Polar, negatively charged residues ar.J their amides: Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, lie, Val (Cys) ; and

5. Large aromatic residues: Phe, Tyr, Trp.

Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. a-helix or β -sheet, as well as changes in physiological activity, e.g. in receptor binding assays.

However, when the exact effect of the substitution, deletion, or insertion is to be confirmed one skilled in the art will appreciate that the effect of the substitution or substitutions will be evaluated by routine screening assays, either immunoassays or bioassays to confirm biological activity, such as receptor binding or modulation of ligand binding to the corresponding 5-HT--_1A receptor. See, e.g., Maranges et al., eds., for example, a substituted polypeptide typically is made by site-speci ic mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a chemically derivatized column or immobilized membranes or hollow fibers (to absorb the mutant by binding to at least one epitope) .

A preferred use of this invention is the production, by chemical or recombinant DNA technology, of 5-HT--_1A receptor peptides, preferably as -small as possible while still retaining sufficiently high affinity for binding to, or association with, 5- HT--_1A receptors. By production of 5-HT--_1A receptor peptides including smaller fragments or variants of such transmembrane domains, one skilled in the art, using known binding and inhibition assays, can readily identify the 5-HT--_IA receptor peptides capable of binding minimizing or modulating G-protein coupled receptors using known methods.

Accordingly, 5-HT--,_A receptor peptides may include consensus sequences and/or fragments of at least one of transmembrane domain 1-7 of one or more 5-HT--_1A receptors, which 5-HT--_1A receptor peptides do not occur naturally, and/or which are provided in an isolated and/or purified form not found in nature.

Consensus peptides of 5-HT--_1A receptor peptides of the present invention may include peptides which are distinct from known 5-HT--_1A receptor sequences in critical structural features, but which are derived from consensus sequences of homologous 5-HT-- _1A receptor transmembrane domains 1-7. Such consensus peptides may be derived by molecular modeling, optionalJy combined with hydrophobicity analysis and/or fitting to model helices, as non-limiting examples. Such modeling can be accomplished according to known method steps using known modeling algorithms, such as, but not limited to, ECEPP, INSIGHT, DISCOVER, CHEM-DRAW, AMBER, FRODO and CHEM-X. Sue¹:, algorithms compare transmembrane domains between related G-protein coupled receptors, determine probable energ - miminized structures and define alternative consensus polypeptide fragments.

Such consensus peptides or fragments of 5-HT--_1A receptors may then be synthesized or produced recombinantly, in order to provide 5-HT--_JA receptor peptides according to the present invention which mimic, modulate or inhibit binding of ligands to G-protein coupled receptors. 5-HT--_1A receptor ligands, in the context of the present invention, refer to biological molecules that bind 5-HT--_1A receptors in vitro, in situ or in vivo, and may include hormones, neurotransmitters, viruses or receptor binding domains, thereof, opsins, rhodopsins, nucleosides, nucleotides, coagulation cascade factors, odorants or pheremones, toxins, colony stimulating factors, platelet activating factors, neuroactive pep■-ides, neurohumors, or any biologically active compounds, such as drugs or synthetic or naturally occurring compounds.

In the context of the present invention, 5-HT--_1A receptor peptides of greater than 15-25 amino acids are preferred such that the 5-HT---_.A receptor peptides are able to span the lipid bilayer.

Additionally, modified amino acids or chemical derivatives of amino acids of consensus or fragments of 5-HT--_1A receptors proteins, according to the present invention may be provided, which polypeptides contain additional chemical moieties or modified amino acids not normally a part of the protein. Covalent modifications of the peptide are thus i*.'eluded within the scope of the present invention. Such modifications may be introduced into a 5-HT--_1A receptor peptide by reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

Preferably, once 5-HT--_1A receptor functional domain peptides meeting the above criteria are determined, then such peptides can be synthesized according to knc-vn method steps, wherein solid phase synthesis is preferred (see. e.g., Harlow and

Lane, supra; Barany et al. The Peptides Analysis Synthesis Biology

2:(E. Gross and J. Meiehofer, Eds.) Academic Press, NY pp. 1-284

(1979)) or p-methyl-benzylhydrylamine polystyrene resin using hydroxybenzotriazole-activated esters of N-CU-BOC protected amino acids on an Applied Biosystems, Inc. Model 430A automated peptide synthesizer

Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens. MAbs may be ob- tained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and/or any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites luid containing high concentrat-ions of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art. See Harlow supra; Ausubel, supra .

Chimeric antibodies are molecules having different portions derived from different animal species, or different Ig subclasses of the same or different species, such as those having variable region derived from a murine ταP.b and a human immunoglobulin constant region. Chimeric antibodies and method steps for their production are known in the am (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al. , Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al. , Nature 312:643-646 (1984); Neuberger et al. , Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 Robinson et al. , International Patent Publication #PCT/US86/02269; Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Better et al., Science 240:1041- 1043 (1988)). These references are hereby entirely incorporated by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immu¬ nizing antibody oy producing an antibody to these idiotypic deter¬ minants (the anti-Id antibody) . For example, anti-Id antibodies to 5-HT _1A receptors are expected to provide 5-HT--_1A agonists as described herein, which would this be included as 5-HT--_1A agonistic antibodies.

An anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so- called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.

Accordingly, mAbs generated against S-100_B, for example, may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs.

Further, the a_"ti-Id mAbs can be coupled to A carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional

BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a S-100_B epitope.

The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as an S-100_B epitope, and possessing biological activity of S-100_B.

The term "antibody" is also meant tr include both in¬ tact molecules as well as fragments thereof, sucn as, for example, Fab and F(ab')₂, which are capable of binding antigen. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1982, ) .

It will be appreciated that Fab and F(ab')₂ and other fragments of the antibodies useful in the present invention may be used according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments).

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term

"epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The preferred animal subject of the present invention is a mammal. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.

By the term "treating" is intended the administering to subjects of S-100_B, a functional derivative thereof, serotonin, or an agonist or antagonist of the 5-HT,_A receptor, for purposes which may include prevention, amelioration, or cure of the diseases discussed below.

For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, trans- dermal, or buccal routes. Preferred routes for administration of substances which do not cross the blood-brain barrier (such as proteins and larger peptides) to subjects with fully developed blood-brain barriers include intracranial and intracerebroventricular (i.e.v.) routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

According to the present invention, 5-HT--_1A receptor agaonists (for serontonergic or cortical neuron stimulation through induction of S-100_B release) or 5-HT--_1A receptor antagonists or anti-S100_B antibodies (for up regulation of serontonergic and/or cortical neurons) , a described herein, or as would be clear to one skilled in the art, based on the teaching and guidance presented herein, may be administered prenatally, neonatally or to an adult. For prenatal administration, the antibody is given systemically to the pregnant female or is introduced in utero, for example, into the amniotic cavity. For neonatal treatment, an antibody is administered systemically, for example, by intravenous or intraperitoneal injection. The antibody can cross the blood-brain barrier and enter the brain from the circulation in a young individual in whom the blood-brain barrier is not completely formed, as is well--known to those of skill in the art. In an individual with a fully formed blood-brain barrier, as in an adult, in order to be effective, the antibody, according to the present invention, must be administered intracranially (i.e.), preferably into the cerebral ventricles (i.e.v.) via a cannula, using methods well-known in the art. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Katzung, supra, which are entirely incorporated herein by reference, including all references cited therein.

In addition to the pharmacologically active compounds, such as the 5-HT_1A receptor agonists and antagonists, S-100_B and functional derivative thereof, or antibodies, the present invention contemplates pharmaceutical preparations whirh may also or alternatively contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active com¬ pound(s), together with the excipient. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Katzung, supra, which are entirely incorporated herein by reference, including all references cited therein.

Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethy...cellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, tal* stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. A number of major developmental disorders involve central serotonergic systems. Autism and Down's Syndrome (DS) are associated with altered serotonergic forebrain innervation, as seen upon postmortem examination of brains from patients with this disorder (Anderson, G.M. et al. , Ann. N.Y. Acad. Sci. 600:331-342 (1990) ) . Hyperserotoninemia has been observed in autistic children and an antibody specific for the human cortical -HT_1A receptor has been identified in an autistic patient (Anderson, G.M. et al. , supra) . The present invention provides a means for treating an individual affected by autism or DS in early developmental stages, for example, while in utero. Thus, for example, S-100_B, a functional derivative thereof, or 5-HT_1A agonist is administered to a pregnant woman carrying a fetus diagnosed as ό.ing autism or DS. Alzheimer's Disease (AD) shares a number of pathophysiological similarities to DS and is also associated with Chromosome 21 (Allore et al., 1988, supra) . Brains of AD patients have increased levels of S-100_B, in particular in astrocytes (Griffin, W.S.T. et al. , supra), which is not normally releasable. AD patients also have increased serotonergic innervation. As a result of the increased presence of 5-HT--, astroglial 5-HT--_1A receptors are down-regulated.

The present invention provides a method for treating AD by manipulating the serotonergic system to cause release of S- 100_B to stimulate growth of cortical and/or serotonergic neurons. The method, and all other therapeutic methods of the present invention, including, but to limited to stimulation of S- 100_B, 5-HT--_1A receptor stimulation in a subject may have the therapeutic or ciagnostic effect of causing so*'.otonergic neuron growth and/or stimulation, which may be suitable for treatment and/or diagnosis of diseases involving serotonergic and/cortical neuronal degeneration, trauma or dysfunction, autism, depression, anxiety, biological rhythm-sleep, disorder, critical brain damage, tryptophan anaboic pathologies, monoamine oxidase pathologies, Down's Syndrome and Alzheimers disease, which may be related to brain immaturity, premature birth, aging, sleep apnea, loss of serotoninproduction developmental disorders, alcoholism, carcinoid syndrome and/or cocaine addiction, is directed to optionally first up-regulating the astroglial 5-HT--_1A receptors in order to render them sensitive to 5-HT--_1A or exogenous agonists, and then to treating with the agonists in order to stimulate S-100_B release.

Up-regulation of the 5-HT--_1A receptors is accomplished by any of a number of means known in the art for depleting central 5-HT--_1A or blocking its action. Such means include depleting stored serotonin from nerve terminals by agents such as reserpine, fenfluramine or methylene deoxymethamenpetamine (MDMA) (Whitaker- Azmitia, P.C. et al., Eds, Ann. N.Y. Acad. Sci., Vol. 600 (1990)), dietary changes which lower central serotonin evels, such as a tryptophan-deficient diet, or drugs which inhibit 5-HT--_1A biosynthesis such as parachlorophenylalanine. Alternatively, since central 5-HT--_1A release is dependent on intact brain corticosteroids, any treatment which blocks brain corticosteroid levels (for example, synthesis inhibitors such as metapyrone or ammoglutethamide) or corticosteroid action (such as corticosteroid antagonists whicn are well-known in the art) wou-d also be useful in up-regulating 5-HT--_1A receptors. Treatment with 5-HT--_ιA receptor antagonists, including 5-HT--_1A receptor peptides, or antipeptide antibodies, or anti-Id mAbs such as those described herein, may also achieve the same or similar effects. Such treatment must be performed for a period of time ranging from about 3 days to about 4 weeks prior to stimulation of central serotonergic neurons, as described herein. Depending on the drug or means chosen to up-regulate the receptors, and the age, weight and health of the subject, one of ordinary skill in the art will be able to determine the appropriate dose and time course of treatment.

Once the 5-HT--_1A receptors have been appropriately up- regulated, resulting in regained sensitivity to agonist action, the treatment method of the present invention involves providing to the brain a 5-HT--_1A receptor agonist as described herein, preferably by the oral or parenteral route, in a dose range of about 1 μg/kg to about 10 mg/kg, for a duration of about 3 days to about 4 weeks, in order to release the S-100_B accumulating in the astroglia. The released S-100_B then acts as a cortical growth factor to stimulate growth of cortical neurons deficient in AD.

In fact, any pathological process associated with loss of cortical neurons and/or serotonergic neurons or their activity, or lack of normal maintenance of serotonergic innervation, can be treated according to the methods of the present invention. For example, such neuronal loss may accompany normal aging. The present invention is thus directed to a method of treating neuronal loss in an aging individual with S-100_B, a functional derivative thereof, or a 5-HT_1A agonist which stimulates S-100_B production by astroglial cells in situ. If necessary, as described for AD, prior treatment may be used to up-regulated 5-HT--_1A receptors in order to allow stimulation of S-100_B release.

Affective disorders, in particular depression, are diseases with an important serotonergic component. In fact, the mode of action of many effective antidepressant drugs is considered to occur via inhibition of 5HT_1A re-uptake, thus prolonging the availability of 5-HT--_1A to act on post-synaptic 5-HT--_1A receptors. Whereas uptake blockade can occur within minutes of treatment, therapeutic benefits are typically seen only after prolonged (e.g. 3 weeks) of antidepressant therapy (see, for example, Gilman et al. , supra). This difference in time courses suggested to the present inventors that depression involves alterations in levels of serotonergic innervation and synapse formation. Thus, according to the present invention, stimulation of seroconergic neuronal growth by treatment with S-100_B, a functional derivative thereof, or a 5-HT_1A agonist which stimulates endogenous S-100_B production, can be used to treat depression. A subject in need of treatment is administered an effective amount of S-100_B, a functional derivative thereof, or a 5-HT_1A agonist. In adults, the treatment with S-100_B, or functional derivatives which do not cross the blood-brain barrier is by i.e.v. infusion. In neonates or immature subjects in whom the blood-brain barrier is not fully formed, the protein or derivative may be administered systemically. 5-HT_1A agonists, most of which readily cross the blood-brain barrier, are administered systemically.

Antidepressant agents have also been useful in the treatment of anxiety and obsessive-compulsive disorders (Gilman et al., supra), indicating the involvement of the serotonergic system in these states. In addition, 5-HT--_1A is involved in synchronization of biological rhythms, and dysregulation may result in sleep disorders (Wauquier, A. et al. , Ann. N.Y. Acad. Sci. 600:447-459 (1990)). According to the present invention, serotonin, S-100_B, a functional derivative thereof, or a 5-HT_1A agonist may be used to treat anxiety and sleep disorders due to its action as an inducer of central serotonergic neuronal growth, as described above for treatment of depression.

Schizophrenia, a major psychiatric illness, is increasingly looked upon as a developmental disorder of the dopaminergic (DA) system wherein central DA activity is increased (Seene, P., Pharmacol. Rev. (1982)). The DA system appears to interact physiologically with serotonergic neurons in a way that stimulation of 5-HT--_1A results in decrease in DA levels. Therefore, stimulation of growth of serotonergic neurons by administration of S-100_B or a functional derivative thereof, or stimulation of S-100_B release, according to the present invention may be useful in preventing or reversing the effects of enhanced dopaminergic activity, and thus the development of schizophrenia. In a preferred embodiment, S-100_B, serotonin or precursor thereof or a 5-HT_1A agonist which stimulates astroglial production and/or secretion of S-100_B is administered to a pregnant female carrying a fetus at risk for schizophrenia, either systemically or by intrauterine introduction. Due to its action as a central cortical and/or serotonergic growth factor, S-100_B promotes the growth and maintenance of cortical neurons, most of which utilize glutamate as their neurotransmitter. According to the present invention, S- 100_B, a functional derivative thereof, or a 5-HT_1A receptor agonist may be used to induce repair of cortical neurons following cortical brain damage, such as that associated with traumatic head injury or stroke. Treatment of a subject in need of repair of cortical neurons is performed as described above for other diseases. According to the methods of the present invention, the amounts and regimens for the administratio.. of S-100_B and functional derivatives thereof, 5-HT_1A agonists and antagonists, and anti-S-100_B agonistic and antagonistic antibodies can be determined readily by those with ordinary skill in the clinical art of treating the particular disease. While individual needs vary, determination of optimal ranges of effective amounts of each component is witnin the skill of the art. Typical dosages comprise about 0.001 to 100 mg/kg body wt such as 0.01-50, 0.05-20, 0.1 to 10 or 1 to 10 mg/kg. The preferred dosages comprise 0.1 to 10 mg/kg body wt.

As an alternative form of treatment to the administration of S-100_B or 5-HT_1A agonists, the present invention also contemplates the implantation or transplantation of an astroglial cell capable of producing S-100_B to a subject having a deficit in such cells or having a genetic lesion rendering such cells non-functional, for example, non-responsive to 5-HT_ιA receptor stimulation. Such implanted cells may be derived from fetal or adult brain or may be a long term cell line, such as the

C6 cell line (Labourdette, G. et al. , supra), which is maintained in culture. Such cells can be implanted in specific target regions in the brain in order to stimulate serotonerg:c cell growth or cortical cell growth, as discussed above. (Se-ci, also: Azmitia, E.C. et al., Eds., Ann. N.Y. Acad. Sci.., vol.495 (1987)).

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified. EXAMPLE I

S-100_B IS A CNS SEROTONERGIC GROWTH FACTOR METHODS

Brains from rat embryos (14 days; Taconia Farms, Germantown, NY) were dissected and the mesencephalic raphe region was removed and dissociated in Ca⁺/Mg²⁺ free Eagles ' minimal essential medium (MEM; Sigma) by repeated trituration. The cells in complete medium (MEM supplemented with non-essential amino acids and 5% fetal calf serum) were plated in 96-well plates (Nunc Labware) coated with poly-L-lysine (25 μg/ml, Sigma) at initial

> plating densities (IPD) of 1 x 10° cells/cm².

After 3 days of incubation the cultures were washed and neuronal growth was assessed by the specific fluoxetine-sensitive high-affinity uptake of 50nM ³H-serotonin for 20 min at 37°C in fresh MEM with 10^"5M pargyline (Azmitia, E.C. et al. , Neuroscience

20:47-63 (1987) ) .

For morphometric analysis, cells were plated on 8-well glass culture chamber slides (Miles Scientific) i.:. r 30h, fixed with ice-cold 4% paraformaldehyde and reacted with a specific 5-HT antibody (1/4000 dilution; Incstar, Stillwater, MI) and an avidin- biotin secondary (Vector Labs) . The 5-HT immunoreactive neurons were analyzed using a Bioquant computer imaging system. The slides were coded and all measures taken blind by a naive observer. On each well, 10 areas (0.5 mm²) were randomly selected and the largest 5-HT-IR neuron in the field measured for somal area and neurite length (Azmitia, E.C, et al., 1987, supra) .

The following protein preparations were dissolved in MEM, sterilized with 0.2 μm Uniflo filter units and added to the cultures at a 1/10 dilution: Human S-100_B, calmodulin (East-Acres Biologicals, Southbridge) , NGF prepared from mouse submaxillary glands (Mobley, W.C. et al. , Biochem. 15:5543 (1979)) , EGF and insulin (Boehringer-Mannheim Biochemicals) . RESULTS

The daily chronic addition of S-100_B produced a dose- dependent increase in 5-HT uptake capacity with maximal effect

(171% of control) at 3.2 ng/ml and a significant increase (150% of control) seen at the lowest concentration tested of 1 pg/ml (Figure 1) . Analysis of variance of the data of Figure 1 yielded the following values:

Comparison df F p

S-100_B/Insulin 23 11.353 < 0.001 NGF/EGF 22 6.148 . < 0.05

A post-hoc Tukey test showed + = p < 0.05 and ++ = p < 0.01.

Neither insulin, NGF nor EGF produrr-d an increase in uptake capacity. A single application of S-100_B at initial cell plating produced, after 3 days of incubation, a dose-related stimulation in uptake capacity (maximal stimulation of 85% at 5 ng/ml) while calmodulin was without effect (Figure 2) . Analysis of variance for the data in Figure 2 showed df=23, F=24.3, and p < 0.0001. Students t-test control comparisons showed + = p < 0.05 and ++ •= P < 0.01. The uptake of [³H]5-HT by the cultured serotonergic neurons is used as an index of total surface area of the neuron, However, it is possible that S-100_B might interact directly with the %-HT protein transporter and produce a change in the uptake of[³H]5-HT independent of a increase in neurite length. Two experiments tested this possibility:

(1) S-100_B (16 ng/ml) added for 15 min. to 3-day-old mesencephalic cultures immediately before [³H]5-.1T uptake produced no change (3140 + 337 vs 3719 + 177, S-100_B and control, respectively) . This indicates that S-100_B is not interacting directly with the 5- HT transporter.

(2) S-100_B was added at initial plating for 30h to mesencephalic cultures and the 5-HT neurons were immunocytochemically stained. The neurite length of 5-HT-IR neurites was increased by 35% and 47% at two doses (16 and 3.2 ng/ml, respectively) of S-100_B (Figure 3; ANOVA showed F = 22.69; df = 20 and p < 0.0001. Post-hoc Tukey test showed ++ = p < 0.01) . No change in somal area was found. This increase in neurite length after 30h is consistent with the increase observed in [³H]5-HT uptake by the cultured mesencephalic neurons after 3 days of incubation. DISCUSSION The results indicate that S-100_B is an SGF. In contrast, EGF, insulin and calmodulin were not found to produce any stimulation, indicating that the SGF activity of S-100_B was not a byproduct of its Ca²⁺ binding potential nor was it due to a general mitogenic potential.

S-100 in the brain is an astroglial specific protein (Isobe, T. et al. , supra) . Recent results have shown that the SGF properties of 5-HT_JA-stimulated, glial cell conditioned medium is blocked by treatment with an anti-S-100 antibody (see Example II, below) .

It also possible that the soluble SGF detected several weeks after a 5,7-DHT lesion in adult hippocampus (Azmitia, E.C, et al., Soc. Neurosci. Abstr. 12 (1986)) and believed to be responsible for collateral sprouting of serotonergic nerves is also S-100_B.

EXAMPLE II

STIMULATION OF ASTROGLIAL 5-HT_1A RECEPTORS RELEASES THE SEROTONERGIC GROWTH FACTOR, S-100_B

To test whether S-100 was the factor released by 5-HT_1A receptor stimulation, astroglial cells were stimulated in primary culture with 100 nM ipsaperone (IPS) , a 5-HT_1A receptor agonist, and collected the conditioned media (GCM-IPS) . vie then added the GCM-IPS to primary cultures of serotonin neurons, with and without the addition of an antibody to S-100, and assessed the effects on neuronal growth. METHODS

Primary astroglial cultures were derived from newborn

(1 to 3 day old) Sprague-Dawley rat pups as previously described

(Whitaker-Azmitia, P.M. et al. , Brain Res. 497:80-85 (1989)) . After one week in culture, cultures were rinsed twice with

Weymouth's medium containing 5 μg/ml insulin and 0.5 mg/ml albumin.

The cells were left to incubate at 37°C for twelve hours before replacing media with fresh serum-free media containing 100 nM ipsapirone, a selective 5-HT_1A receptor agonist. After 24 hr, the media (referred to as GCM-IPS) was collected and stored at -70°C until tested in neuronal cultures. The growth-promoting properties of native bovine S-100

(10-1000 ng/ml and of GCM-IPS (diluted 1 to 500) were compared.

The S-100 was obtained from East Acres Biologicals, Southbridge,

MA (guaranteed >99% homogenous by SDS-PAGE) . S-100 or GCM-IPS, or each of thes.e in the presence of a polyclonal antibody to S-100 (Accurate Chemical, Westbury, NY; final dilution 1/10,000) were added at the time of neuronal plating. The polyclonal antibody had been characterized in our laboratory and shown to be positive for immunocytochemical staining of astrocytes in culture and brain) .

Mesencephalic neuronal cultures were prepared from Sprague-Dawley rat embryos at 13-14 days of gestation (obtained from Hilltop Breeding Laboratories) as previously described Azmitia, E.C. et al. , Neuroscience 20:47-63 (1987). After three days in culture, neuronal growth was assessed by measurement of specific serotonin re-uptake capacity. This indicator has been shown to be a reliable measure of in vivo innervation density and of the maturational state of specific neurons in culture (Azmitia, E.C, 1987, sup-*-a; Currie, D.N. et al. , Brain Res. 199: 473-81 (1980); Dreyfus, C.F. et al. , Brain Res. 128:124-139 (1977)). Briefly, cultures were incubated for 20 minutes with MEM containing 1% glucose and ³H-serotonin (26 Ci/mmole, New England Nuclear; final concentration 50 nM) with or without 50 nM fluoxetine. After removing the radiolabel, the cultures were allowed to dry and 200 microliters of absolute ethanol was added for one hour. Then, 150 μl of the sample was placed into 7 ml of Liquiscint for counting in a Beckman Liquid Scintillation Counter (40% efficiency) . Test cultures were pre-incubated with S-100 or GCM-IPS for one hour before uptake was measured. To visualize astroglial cultures after exposure to serum-free media with or without 100 nM ipsapirone, cultures were rinsed twice with Tris-buffered saline (TBS) at 4°C before incubation with a polyclonal antibody to a specific astroglial marker, glial fibrillary acidic protein (GFAP) (Ac-curate Chemicals; final dilution 1/800 in TBS with 0.2% Triton and 0.1% normal swine serum) for 2 hrs at 37°C After rinsing with TBS, the cultures were stained using the avidin/biotin method prepared as Vectastain (Vector Labs) with final visualization using diaminobenzidine. RESULTS

Both S-100 (500 ng/ml) and GCM-IPS (diluted 1/500) produced an increase in the ³H-serotonin uptake capacity of the cultures after 3 days of exposure, but not when applied acutely. This stimulation was blocked by incubation, at the time of plating, with a polyclonal antibody to S-100 at a 1/10,000 dilution (Figure 8; df=5; f=1497, p < .0001; for individual values, p < .001) . Application of e ^! ther the antibody alone or 0.5 n ipsaperone ( the maximum final concentration in the neuronal cultures after addition of GCM-IPS) was without significant effect. The majority of the activity is thought to reside in the β subunit of S-100 since S- 100_B (β- β dimer) is more active than S-100_A (β-a dimer) .

The morphological alterations were characterized by an increase in process-bearing cells and an increased colonization of the cells. These changes were consistently observed in all eight primary cultures (ie. from eight different litters of animals) used to derive GCM. DISCUSSION It had been previously demonstrated that serotonergic neurons regulate their own growth through activation of a 5-HT_j receptor (Whitaker-Azmitia, P.M. et al. , Neurosci. Lett. 67:307-312 (1986) ) . The present inventors found that astroglial cells contain high levels of -T-HTj receptors in the immature state (Whitaker- Azmitia, P.M. et al. , J. Neurochem. 46:1186-91 (1986)) and that activation of a subtype of these receptors, the 5-HT_jA receptor, leads to secretion into the medium of a factor which can stimulate serotonergic maturation in dissociated tissue culture preparation (Whitaker-Azmitia, P.M. et al. , Brain Res. 497:80-85 (1989)) . Based on the above results, 5-HT_1A receptors on brain astroglial cells appear to be involved in the release of S-100. Therefore, S-100 provides at least one means by which serotonin can autoregulate development of serotonergic nerves .

During fetal brain development, S-lϋϋ, as detected by antibodies, shows an intense yet transient rise in the midline raphe region, where the serotonin cells are developing (Van

Hartesveldt, C.J. et al . , J. Comp. Neurol. 253:175-184 (1986)) . Since in the process of producing and/or releasing S-100, the astroglial cells attain a mature morphology, the present results suggest a functional interaction between astrocytes and neurons during development, whereby both cell types mature through the action of the astroglial 5-HT_1A receptor.

The observed morphological change has also been seen after activation of other receptors linked to generation of cyclic AMP, such as the β-adrenergic receptor, the stimulation of which is linked to the release of nerve growth factor (NGF) (Schwartz, J.P. et al., Naunyn Schmeideberg's Arch. Pharmacol. 300:123-129 (1977) ) . The parallels of these two systems is noteworthy: both involve receptor stimulation coupled to cAMP production which results in astroglial morphological changes and finally release of a growth factor In summary, of the several growth factors tested, only chronic S-100_B showed enhancement (maximal at 3.2 ng/ml is 171%) after 3 days of incubation of the [³H]5-HT uptake capacity by serotonergic neurons. A single application at initial plating of S-100_B (maximal at 5 ng/ml is 185%) , but not calmodulin, increased the development of the [³H]5-HT uptake capacity by the cultured serotonergic neurons. Morphometric analysis of cultured 5-HT immunoreactive (IR) neurons showed an increase (135 and 147%) in neurite length 30 h after S-100_B application of 16 and 3.2 ng/ml (respectively) . The results indicate that S-100_B functions as a serotonergic growth factor in the mammalian brain.

Stimulation of astroglial 5-HT receptor causes astroglial cells to acquire a more mature morphology and to release a factor (or factors) which promotes growth of serotonergic neurons. By uε-ng an antibody- blocking approach, it has been conclusively demonstrated that one of the growth- factors released is the astroglial-specific protein S-100. This may be a particularly important observation, in view of studies implicating S-100 in both Down's Syndrome and Alzheimer's Disease, as discussed above. EXAMPLE III ANTIPEPTIDE ANTIBODIES AGAINST THE 5-HT_1A RECEPTOR

According to the present invention, a method is provided for selecting two new sites for anti-5-HT--_1A receptor antibody recognicion against the 5-HT_1A receptor: S1A-170 (aa 170- 186) and S1A-258 (aa 258-274) . These antibodies recognized a protein brand of approximate molecular weight of 49,000. they showed excellent staining in rat neonatal and adult brain and in adult monkey brain at dilutions as low as 1/10,000. Similarities and differences with the distribution with ³H-8-OH-DPAT binding were seen. Most importantly, clear laminar labelling was seen in those areas known to have high 5-HT,_A binding such as the hippocampus and cortex. In addition, selective cells were labeled in areas thought to have little or no 5-HTj_A receptors, such as the cerebellum and striatum. Light cellular labeling was apparent in many areas. In the hippocampus, polymorphic interneurons were stained. There was less intense labeling in the granule cell layer. The neurons in the rostral dorsal raphe nucleus were clearly stained in the monkey. In the cerebellum, labelling was very high in the neonate but mainly confined to glial cells in the adult. High label was seen in epithelial cells lining the brain and the ventricular system. Tanocytes were labelled in the third ventricle near the median eminence. Staining was seen to astroglial cells in many brain areas and astroglial cultures. Ultrastructural studies revealed heavy staining in occasional primary dendrite shafts in the hippocampus midbrain. The label was associated with the Microtubules (MTB) and in patches along the outer plasma membrane (PLMB) of neurons (Fig. 5 shows dendrite It 13,500 X) . The selection of the peptide domain of the 5-HT_1A receptor against which the antibodies were raised was made the basis of several criteria, as follows:

(1) Functional domain of the receptor: The receptor is homologous to the beta-adrenergic receptor family and many of the various segments of the 5-HT_1A receptor can be inferred from the extensive work with' the β-adrenergic receptor. The agonist binding site consists of a least two aspartate (asp) residues within the 2nd and 3rd transmembrane regions (Dohlman et al.

Biochemistry 26:2657-2663 (1987) . Asp residues (#82 and 116) exist in a similar site in the 5-HT_1A receptor (Figure 6) . A histidine

(His) in the 3rd transmembrane site (#126) Mould provide the needed positive charge for 5-HT binding. The chird cytoplasmic loop is believed to be the site of interaction with the G-proteins in the cytoplasm for regulation of the second messenger systems

(Kobilka et al . Nature 329:205-230 (1988)) . Therefore, peptide sequences can be selected indicate the anatomical location of various segments of the full molecule and deterr.iine the cellular distribution of particular functional regions. We selected domains in the 2nd external loop (S1A-170) and in the 3rd cytoplasmic loop

(S1A-258) (Fig. 6) .

(2) Hydrophilicity regions. The antigenic sites on a peptide can be approximated based on the hydrophilicity score which assumes that the greater the local hydrophilicity, the more antigenic the sequence (Hopp, T.P. Proc. Natl. Acad. Sci. USA 8:3824-3828 (1981)) . This measure assigns a numerical value to the various amino-acids; for example K, R, D and ϊl have a value of +3.00, W has a value of -3.4, and G and P have a value of 0. the calculated window average at a residue is calculated across 6 residues. The hydrophilicity score for A1A170 is shown in Table One.

(3) Two dimensional protein structure. There are three states in wnich a sequence can exist in a secondary structure of a protein molecule. These are beta sheets, alpha helix and turns (Chou et al. Biochem. 13:222-245 (1974); Chou et al. Adv. Enzymology 47:45-147 (1978)) . In the latter state, it is assumed that the amino acids are most exposed. The selected peptides both have a significant number of predicted turns (Table I shows results for S1A170) .

(4) Charge Balance of the Protein. The net charge of a peptide sequence should be near neutrality. If the molecule is too highly charged it will present problems durinj the purification procedure after the peptide is synthesized. If the net charge is highly basic or acidic a cation or anion exchange resin can be used. Bio-rad AG-50 resin has been successfully used for very basic peptides. However, strong deviations from neutrality is also a problem during the attachment to KLB which should proceed at neutral pH (see below) . S1A-170 has 6 charged residues and a net - 2 charge while S1A-258 has 5 charged residues and a net + 1 charge. (5) Amino Acid length. The sequence for an ideal peptide for antibody formation should have 15-20 amino acids. A strand of 6 amino acids is the lower limit for a recognition site whole more than 20 presents some additional problems with the synthesis and structural considerations (Harlow, E. et al. Antibodies: A Laboratory Manual Cold Spring Harbor Press (1988)) . Both S1A-170 and S1A-258 have 17 residues.

(6) Phosphorylation and glycosylation sites. A protein molecule has many possible phosphorylation and glycosylation sites. These sites should be avoided in choosing a sequence unless a particular confirmation is sought. Antibodies have been raised against phosphorylated sequences but these antibodies have altered affinity for the un-phosphorylated site (See Czernik et al. Method Enzymol 201:264-283 (1991). Furthermore, a phosphorylated segment of the molecule often confers allosteric changes in the protein structure which may reduce the affinity for the peptide segment artificially produced. It can be appreciated, that sites adjacent to modified sites may be less desirable for the same reasons. The glycosylation sites are located on the Asparagine (ASN, N) residues at positions 10, 11 and 24. Three potential protein kinase C phosphorylation sites are located at 147-152, 227-232 and 341-345 and one additional phosphorylation site 251-253 (El Mestikawy et al, Neurochem. Res. 16:1-10 (1991)). Neither S1A-170 or S1A-258 have phosphorylation nor glycosylation sites. (7) Position of Cysteine. Cysteine (Cys, C) residues are commonly involved in disulfide bridges. For chis reason it is advisable to avoid a Cys residue in the middle of a peptide sequence. There are 15 Cysteine residues in the 5-HTj_A receptor, 6 in the transmembrane regions, 3 in the 3rd cytoplasmic loop, four in the three extracellular loop and two ■ in the C-terminal cytoplasmic tail. The cysteines in extracellular loop l and 2 have been proposed to form a disulfide link in the /3₂-adrenergic receptor (Dohlman et al. Biochemistry 26: \1987)). Similar cysteines exist in the 5-HT_1A receptor (Figure 6) . In designing the sequence, it is advisable to have a terminal cysteine residue in order to bind to KLH protein (Harlow, et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press (1988) . This can be either at the C- or N-terminus of the peptide depending on how the peptide is predicted to be exposed in the molecule. For instance, if the desired sequence is at the N-terminal end of the protein, then the cysteine should be placed at the C-terminal end of the peptide. In this way, the N-terminal end will be exposed after its attachment to the carrier protein. Both peptides have an N-terminal Cys ut S1A-258 also contains a Cys in the center of the peptide at position 266.

(8) Homology with known proteins. In selecting a region of the 5-HT_1A receptor protein comparisons with 5-HT₂, 5-HT_ιC, α₂ and B₂-adrenergic, muscarinic Ml, and the D, receptor were performed (Juluis et al. Proc. Natl. Acad. Sci. USA 87:928-932 (1990); Bunzow et al. Nature 336:783-787 (1988)). Once a segment has been selected it should be compared to all known protein sequences. The sequence data bank searching program (Intelligenetics Inc., Mt. View, CA- (415) -962 7300) we used is based on the algorithm of Welbur and Lipman. Sequence Homology was searched against the Protein Identification Resource data bank (PIR) and the Protein Sequence Data Bank which contains the translated European Molecular Biology Library (EMBL) . This research will identify those known structures that could react with the antibody raised. This especially relevant when the protein in question has been identified in the same species chosen for study, and conversely, significant homology to an invertebrate protein is not necessarily a problem. High homology for the selected sequence of the same protein in different species is advantageous. Neither selected sequence has any significant homology with any other published receptor or mammalian protein. Interestingly, S1A-170 has a 94% with the human 5-HT_1A receptor, while S1A-258 has only 44% homology with the human 5-HT,_A. In the case S1A170, the methionine (-1,3 hydrophilicity) at position 172 is replaced by an isoleucine (-1.8 hydrophilicity) which should have little consequence to the antigenicity of the sequence. Synthetic Peptides

The peptides were made according to method steps as described in detailed previously (Yu et al., 1991). Briefly, the two sequences selected, CSH 228 (S1A-170-86:H-Pro-Pro-Met-Leu-Gly- Trp-Arg-Thr-Pro-Glu-Asp-Arg-Ser-Asp-Pro-Asp-Ala-Cys- (SEQIDNo:1) ; and CSH229 (S1A-258-274:H-Pro-Gly-Ser-Gly-Asp-Trp-Arg-Arg-Cys-Ala- Glu-Asn-Arg-Ala-Val-Gly-Cys- (SEQ ID No:2) , were synthesized by the solid phase methods (Barany et al. The Peptides Analysis Synthesis Biology 2: (E. Gross and J. Meiehofer, Eds.) Academic Press, NY pp. 1-284 (1979) ) or p-methyl-benzylhydrylamine polystyrene resin using hydroxybenzotriazole-activated esters of N-CU-BO protected amino acids on an Applied Biosystems, Inc. Model 430A automated peptide synthesizer. A modified small-scale (0.1 mmole) rapid-cycle was used. Couplings were done in dimethylformamide and dichloromethane as solvents, and unreacted peptide was capped with acetic anhydride. The side chain-protected amino acids were: Arg (Mts) ; His (BOM); Thr (Bzl) ; Cys (4-CH3-Bzl); Trp (CHO); Ser (Bzl; Glu (OBzl) ; and Asp (cHex) . Double coupling was necessary for several amino acids such as Trp, Leu, Thr, Glu, Ser, Cys and Val.

The peptides were deprotected and cleaved from the resin with liquid FH at -10°C for 2 h in the presence of 5% (v/v) anisole and 5% (v/v) dimethyl sulfide. The peptide was precipitated with ethyl either, and solubilized in 6M Guanidine HC1 @ 10 mg/ml (41 ml) . The formyl group was removed from the indole portion of Trp bv treatment with HF. The sample was cooled to 0°C in a salt ice bath in a round bottom flasκ with stirring.

Ethanolamine was added at a final concentration of IM (2.5 ml) and stirred for 4 hr. at 0°C The temperature was critical with the pH > 8 since a low temperature prevents the cyclization of glutamic acid and aspartic acid. The reaction was quenched by reducing the pH < 7.0 with [HCL] (Baker, HPLC grade). The sample was filtered

(0.45μm pores, Nylon 66, Schleicher &^• Schuell) prior to HPLC purification. The solution was subjected to HPLC using a Waters Delta

Prep 3000 instrument on a column (4.9 x 30 cm) of 300 A, C_ϊ8 silica

(Waters) and elrted with 0.1% (w/v) trifluoroacetic acid with a linear gradient to 60% acetonitrile (Burdick and Jackson) . Both peptides (CSH228 and 229) eluted at approximately 23 min. at approximately 49% acetonitrile. The Mass Spectrometry data showed a [M+H] + ion with no other adducts or deletion products (Figure 7) . Final yields were 50 mg of 228 and 26 mg of 229. Antibody Preparation

The peptides, CSH 228 and 229 were coupled to keyhole limpet hemocyanin (Sigma) via maleimidobenzoyl-N-hyroxysuccinimide

(Pierce Chemical Co., Rockford, IL) as described (Harlow et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press

(1988) . Briefly, the Keyhole limpet hemocyanin (KLH) was dissolved in PBS (10 mg/ml) M-Maleimidobenzoyl-N-hydroxysuccinimide ester

(MBS; Pierce, N22312) was dissolved in dimethylformamide (Burdick and Jackson, Baxter, N.J.) . The MBS solution was very slowly added to the KLH solution, one drop at a time while stirring and the mixture allowed to mix for 30 min at RT. The unbound MBS was removed by filtration on Sephadex G25 in PBS (Pharmacia) . The peptides were dissolved in PBS at 10 mg/ml and added slowly to the

MBS/KLH solution. The pH was adjusted to 7.2 and the mixture is allowed to stir for 3 hr at RT. The solution was extensively dialyzed against PBS in the cold room over night. The protein concentration in the dialysate was determined with the Bio Rad protein microassay and the solution adjusted to 1 mg/ml.

Antisera against the complexes were raised as follows. The complexes (0.5 mg) in 0.5 ml of phosphate-buffered saline (PBS) was mixed with 0.5 ml of Freund's complete adjuvant and 300 μl injected subcutaneously in four spots on the back of adult female

New Nezland white rabbits weighing 8 1/2 and 8 lbs. that were certified specific pathogen free (Hare Maryland, Hewitt, NJ) . Booster injections were give with incomplete adjuvant at 2 week internals with 200μl incomplete adjuvant at 2 spots on the back.

Additional booster injections were given at 2 week intervals until reaching maximum serum titer. The first bleed of 6 ml was made from the central artery of the ear 3 days after the second boost. Once the maximum titer was reached (fourth boost) , full bleed of

15-20 ml were made. The blood was kept at 5°C and spun once at low speed for 10 min and the serum spun a second time in Eppendorf tubes for 10 min before being aliquoted and frozen.

Serum antibody titer was determined by radioimmunoassay. Wells of 96-well polyvinylchloride microtiter plates (Falcon Microtest III) were coated with 50 μl of the appropriate peptide (1 mg/10 ml) for at least 3 hr at RT. The plates were washed 3 times with PBS and unbound sites were saturated with POO μl of 3% (w/v) bovine serum albumin (BSA) . Dilutions of immune and preimmune serum were added to wells at concentration from 1/50 to 1/50,000 for at least lh at RT. The plates were washed 3 times with PBS and 50,000 cpm of ¹²⁵I-labelled goat anti-rabbit IgG F(ab) '² (NEX 167, NEN) added per well and allowed to include at RT for 1 hr. After three washes, the specific activity of each serum sample was determined by counting the radioactivity for 1 min in a gamma radiation counter (Beckman, Gamma 5500 B) . The results for S1A1709 and S1A258 are shown in Fig. 8. Gel Electrophoresis and Immunoblotting

Elec rophoresis was performed on 1mm 12.5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulfate using the buffer system of Laemmli (1977) . 20 and 40 ug of tissue from hippocampus was run in each well along with 5ul of standard (Bio Rad biotinylater SDS-PAGE standards, low range•cat # 161-0306). The hippocampus was removed from a young Long Evans female rat (100-150 gm; Charles River, Kingston, NY) and immediately frozen in liquid nitrogen. A single hippocampus was transferred to lysis buffer (see below) containing 2% SDS. The tissue was immediately homogenized by hand in a 1.5 ml polypropylene eppendorf tube (pellet pestle with disposable tube, #74920, Kontes, N.J.) and allowed to sit for 15 min on ice before spinning in a microfuge Eppendorf Microcentrifuge 5414) at 5°C for 15 min. The supernatant was collected and assayed for protein amount using a commercial dye-binding microassay (Bio Rad) . Routinely, 3ul were added to 1ml of solution and lead on a spectrophotometer at wave length 595 nm. The solution was adjusted to a final concentration of 2 mg/ml. The supernatant was mixed with an equal amount of sample buffer (Laemmli) , β-mercaptoethanol added to a final concentration of 10% (V/v) and the sample boiled for 5 min. The gel ^•••/as run for 5 hr. at 100 volts on a vertical gel electrophoresis apparatus. Proteins were transferred electrophoretically to nitrocellulose using a Bio- Rad Trans Blot Cell overnight at 50 volts in the cold room (Towbin et al. , 1979 & 1984) . The nitrocellulose was incubated with antiserum at dilutions from 1/100 to 1/10,000 in 0.1% (v/v) Tv.*een-20 (Sigma) in Tris buffered (O.lM, pH 7.4) saline (0.9%) solution (TTBS) . It is our experience that the best results were obtained with our antibodies at dilution of 1/1000 to 1/5000. The antibody at lower dilutions (1/250) gave increase background and less sensitivity. The avidin-biotin peroxidase procedure was used to identify the protein band as described by Vector Laboratories. Briefly, the nitrocellulose sheets were cut to include a iitandard and the appropriate rows and washed in small 75mm disposable Petri dishes. The nitrocellulose strips were incubated with the antisera for at least 2 hr at 40°C (the strips would be left with antisera for several days at RT or in the Cold room) . The strips were rinsed three times in TTBS for a total of 10 min on a shaker between biotinylated secondary (30 min incubation) , the ABC reagents (20 min incubation) , and the DAB peroxide reaction. The strips were first incubated with freshly filtered diaminobenτ;idine (5 mg/20 ml TBS; Sigma) and 0.2% Nickel ammonium sulfate for 5 min at RT and the H₂0₂ added at a final concentration of 0.01% (v/v) . Boehringer- Mannheim produces biotinylated reagents that are easier and cheaper to use than Vector Stains products but we have not yet compared their sensitivity. One major band (49.5 kdal) and two minor bands (42.0 and 37.0 kdal) were stained with S1A170 at a dilution of 1/1000.

Lysis Buffer (Draeta et al. Cell 54:17-26 (1988)) was made from highest grade chemicals from Sigma (St. Louis) and Boehringer-Mannheim: 50mM Tris pH 7.4; 150 mM NaCl; 1% NP-40; lOmM EDTA; ImM MgCl₂; 1 mM CaCl₂; 10% Glycerol; 400 μM Sodium Orthovanadate; 50 mM NaFluoride; 50mg/l PMSG (phenylmethane sulfonylfluoride) from lOmg/ml isopropanol; 1 mg/leupeptin; 10 mg/1 Soybean Trypsin Inhibitor; lmg/1 Aprotinin and 10 mg/1 of TPCK (L- l-chiloro-3- [4-tosylamido] -4]phenyl-2-butanone) from 3 mg/ml of ethanol. Immunocytochemistry at the light and ultrastructural level.

Neonatal (1-2 weeks) and adult female rats were perfused with a variety of fixat.* /es and prepared for immunocytocliamistry according to our published procedures (Azmitia et al. J. Neuroscience 3:2083- 2090 (1983) . Rats (Sprague-Dawley, Female, Taconic Breeders, 220 gm) and monkeys (Macaca Fascicularis, female, Charles River Breeding Laboratory, 3.3 kg) were perfused, through the ascending aorta with 4% praformaldehyde, 2% glutaraldehyde, 4% formaldehyde plus 0.1% glutaraldehyde or 3.25% acrolein.with 2% praformladehyde at 20°C and 0.1% MgS0₄ in O.lM phosphate buffer (pH 7.4) at 20°C. The glutaraldehyde and acrolein fixatives were continued after 10 min with the same solution with only paraformaldehyde (total perfusion volume was 100 ml for neonate, 250 ml for the adult rats and 1500 ml in the monkey) for an additional 20 min. The brains were profixed at 5°C for at least 4 hr before being processed for immunocytochemistry. Thirty-micrometer sections of the hippocampus and brainstem were cut on a Vibratome (Oxford) .

The primary anti-serum and the se >ndary sera were diluted in 0.1 M Tris buffered (pH 7.4) saline (0.85%) containing 1% normal sheep serum and 0.1% Triton X-100. The sections were incubated for 18-72 hr at 5°C followed by 2 hr at room temperature (RT) in antipeptide antibody serum at a dilution of 1/1000- 1/10,000. The sections were then processed with the elite Vector stain ABC-kit as directed by the manufacturer. The reaction was run for 2 min at RT in 0.05% 3,3-diaminobenzidine containing 0.2% nickel ammonium sulfate in 0.1 M Tris-buff eret - (pH 7.4) saline (Azmitia et al. J. Neuroscience 3:2083-2090 (1983)) followed for 5 to 10 min at RT in the same solution with the addition of 0.01% hydrogen peroxide. The sections for electron microscopy were viewed after postfixing for 1 hr at 20°C in 2% osmium tetroxide containing 1.5% potassium ferricyanide in 0.1 M phosphate buffer (pH 7.2) and then block stained in 0.5% uranyacetate at 5°C for 30 min. Ultrath .1 sections were taken for the surface of Epon/Araldite-embedded tissue slices and views on the electron microscope without further heavy metal staining.

Advantages Specificity of the antibody raised against synthetic peptides is increased compared to using the ful.^" molecule since a specific region of the structure is targeted. Regions with high homologies with other molecules can be avoided. A functional region (3rd cytoplasmic loop) or a structural portion (second extracellular loop) can be selected for study (Fig. 6) . The antibody peptide can be produced as soon as the primary cDNA structure has been demonstrated.

High titers with the antipeptide antibody can be obtained because the peptide is bound to a carrier protein that provides highly immunogenic sites for T-cell receptor binding. We were able to obtain RIA binding of the native peptide over 100 fold that seen in the preimmune serum at dilution of 1/10,000 (see Fig. 8) . Anatomical and cellular localization of the receptor can be performed in neonatal and adult rats as well as adult monkey. All fixation solutions produced good results. The best staining of CNS was in neonates and this confirms the ligand binding studies which have shown higher values for the 5-HT_1A receptor during early prenatal periods (Bar-Peled et al. Neurosci. Lett. 127:173-176 (1991); Daval et al. Intern J. Neurosci 5:171-180 (1987); Whitaker-Azmitia et al. Develop. Brain Res. 33:285-295 (1987) ) . The most interesting observation «.-as the specific cellular staining observed in neurons, astrocytes, tanocytes, tanocytes and epithelial cells. The complex distribution was not suggested by earlier autoradiographic studies using ligand binding. The great advantage of a receptor antibody lies in its adaption of ultrastructural immunocytochemical localization (see Fig. 5) .

Isolation of the receptor by immunoaffinity purification has been reported previously with an antipeptide antibody to the 5-HTLA receptor (Raymond et al. Molecular Pharmacology 36:015-021 (1989)). The proviJes a rapid and sensitive method to isolate a single protein fraction. The receptor can also be concentrated by the method of immunoprecipitation (EL Mestikawy et al. Neurochem. Lett. 118:189- 192 (1990) ; see also Harlow et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press (1988) ) . Western analysis of the amount of the 5-HT_]A receptor can be performed with both the S1A170 and S1A258 antibodies that we have raised.

Characterization of the antipeptide antibody must establish that the native protein is selectively recognized, such that the following considerations are preferably addressed.

This requires demonstrating a specific cellular staining pattern in immunocytochemical studies. However, as discussed by El Mestikawa et al. Neurochem Res. 16:1-10 (1991) , the distribution of receptor staining with antibodies may not be completely consistent with the radioautographic distribution seen with ligand binding. This is explained by the observation that ³H-

8-OH-DPAT, for . s ance does not recognize the receptor if it is not linked to a G-protein. The antibody, on the other hand, can recognize all the receptor molecules even those in transit from the cell body to the dendritic plasma membrane via microtubules (see

Fig. 5) .

Immunostaining of a single band in Western analysis is usually considered an indication of the general specificity of an antibody. However, this requires careful manipulation of the antibody dilution based and preparation of t.-.e tissue sample. Protein fragments or aggregation of the molecvle can result in several bands on a Western even if the antibody only recognizes a single protein. For this reason, we use a special lysis buffer and treat the tissue with reducing and denaturing conditions (such as B-mercaptoethanol and boiling) .

Immunoprecipitation and immunoaffinity purification of a single protei.- that functions as an active .'=ceptor with the appropriate characteristics is the best criteria that the native protein is labeled by the antibody. In studies with transfected

COS-7 cells, the antibody JWR21 (242-267) was shown to precipitate the [¹²⁵I]N₃-NAPS photoaffinity labelled receptor (Raymond et al. Molecular Pharmacology 36:015-021 (1989)). The preimmune serum, the antigenic peptide block JWR21 antibody or an antibody from a non-overlapping region (268-293) were all unable to precipitate the receptor. In the study 'by El Mestikawy et al. Neurosci. Lett. 118:189-192 (1990), the 5-HT_1A receptor antibody against 243-268 precipitated the binding sites of ³lH-8-OH-DPAT when protein A- sepharose CL-4B was added. No influence of the antiserum alone was seen in the binding.

The synthesis was performed with a 4J-JA ABI sequencer. The alternative is TFMSA cleavage which can be done at the bench.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein^', including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art

(including the contents of the references cited herein) , readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It. is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachi gs and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

References

Albert, P.R. et al. J. Biol. Chem. 265:528-532 (1990) .

Azmitia, E.C. et al. J. Neuroscience, 3:2083-2090 (1983) .

Bar-Peled et al. Neurosci. Lett. 127:173-176- (1991) . Barany, G. et al. The Peptides: Analysis. Synthesis. Biology Vol. 2 (E. Gross and J. Meienhofer, Eds.) Academic Press, NY. pp. 1-284 (1979) .

Bunzow, J.R. et al. Nature 336:783-787 (1988) .

Czernik, A. et al. Methods Enzymol 201:264-283 (1991) . Chou, P. et al. Biochem. 13:222-245 (1974) .

Chou, P. et al. Adv. Enzymology 47:45-147 (1978) .

Daval, G. et al. Intern. J. Neurosci. 5:171-180 (1987) .

Draeta, G. et al . Cell 54:17-26 (1988) .

Dohlman, H.G. Biochemistry 26:2657-2663. El Mestikawy, S. et al. Neurochem. Res. 16:1-10 (1991) .

El Mestikawa, S. et al. Neurosci. Lett. 118:189-192 (1990) .

Harlow, E. et al. Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press (1988) .

Hopp, T.P. Proc. Natl. Acad. Sci. USA 8:3824-3828 (1981) . Julius, D. et al. Proc. Natl. Acad. Sci. USA 87 928-932 (1990) .

Kobilka, B.K et al. Nature 329:75-79 (1987) .

Laemmli, E.K. Nature 227:680-685 (1970) .

Pazos, A. et al. Brain Res. 346:205-230 (1985) .

Raymond, J.R. et al. Molecular Pharmacology 36:015-021 (1989) . Sotelo, C et al. Europ. J. Neurosci. 2:1144-1154 (1990) .

Sutcliffe, J.C. et al. Nature 287:801-805 (1980) .

Towbin, H. et al. Proc. Natl. Acad. Sci. 76:4350-4354 (1979) .

Towbin, H. et al. J. Immunol. Methods 72:313-340 (1986) . Verge, D. et al. J. Neurosci. 6:3474-3482 (1986) . Yu, I.J. et al. Journal of Cell Biology 114:1217-1232 (1991).

Whitaker-Azmitia, P.M. et al. Develop. Brain Res., 33:285-295 (1988) .

Walter, G. et aZ Proc. Natl. Acad. Sci. 77:519.'-5200 (1980).

Claims

WHAT IS CLAIMED IS:

1. A method for stimulating the production or release of S-100_B in an animal, comprising administering to said subject an effective amount of an agonist acting on a central 5-HT_1A receptor.

2. A method according to claim 1, wherein said agonist is selected from the group consisting of 5- hydroxytryptamine, 5-methoxytryptamine, buspirone, 8- hydroxydipropylaminotetralin, ipsaspirone, gepirone, SM23997, lysergic acid diethylamide, and an agonistic antibody.

3. A method for stimulating growth of central serotonergic neurons in an animal, comprising administering to said subject a serotonergic neuron stimulating effective amount of S- 100_B, a functional derivative thereof, or an agonist acting at the 5-HT_1A receptor.

4. A method according to claim 3 or 5, wherein said agonist is selected from the group consisting of 5- hydroxytryptamine, 5-methoxytryptamine, buspirone, 8- hydroxydipropylaminotetralin, ipsaspirone, gepirone, SM23997, lysergic acid diethylamide, and 5-HT--_IA agonistic antibody.

5. A method for stimulating growth of central serotonergic neurons comprising contacting said neurons with an effective amount of S-100_B or a functional derivative thereof.

6. A method according to any of claims 1-5, wherein said contacting is in vi tro.

7. A method according to any of claims 1-5 wherein said contacting is in vivo.

8. A method for inhibiting the growth of central serotonergic neurons comprising contacting said neurons with an effective amount of an inhibitor of S-100_B production or action.

9. A method according to claim 8, wherein said inhibitor is an antibody specific for S-100_B or a 5-HT--_]A receptor antagonistic antibody or peptide.

10. A method according to claim 9, wherein said antibody is a monoclonal antibody.

11. A method according to claim 9, wherein said antibody is a polyclonal antibody.

12. A method according to claim 8, wherein said inhibitor is a 5-HT_jA receptor antagonist.

13. A method according to claim 12, wherein said 5-HT_1A antagonist is selected from the group consisting of spiperone and spiroxatine.

14. A method for treating a disease associated with decreased central serotonergic innervation or activity in a subject, comprising administering to said subject an effective amount of S-100_B, a functional derivative thereof or a 5-HT_JA agonist.

15. A method according to claim 14, wherein said agonist is selected from the group consisting of 5- hydroxytryptamine, 5-methoxytryptamine, buspirone, 8- hydroxydipropylamineotetralin, ipsaspirone, gepirone, SM23997, lysergic acid -iethylamide and a 5-HT--_1A r.: eptor agonistic antibody.

16. A method according to claim 14 wherein said disease is selected from a group consisting of autism, depression, anxiety, biological rhythm-based sleep disorder, and cortical brain damage.

17. A method for treating a disease associated with increased central serotonergic innervation or activity in a subject, comprising administering to said subject an effective amount of an inhibitor of S-100_B production or action.

18. A method according to claim 17 wherein said inhibitor is an antibody specific for S-100_B.

19. A method according to claim 17 wherein said inhibitor is a 5-HT_1A receptor antagonist.

20. A method for stimulating cortical or serotonergic neuronal growth or stimulation in a subject hiving Alzheimer's disease, comprising

(a) up-regulating the expression of 5-HT--_1A receptors on astroglial cells in the brain of said subject; and then (b) stimulating the induction of the release of S-100_B in said subject according to a method according to claim 1, thereby stimulating said serotonergic neuronal growth.

21. A method according to claim 1, wherein said antagonist is a monoclonal 5-HT--_1A receptor antibody or a 5-HT--_1A receptor peptide corresponding to at least a portion of a functional domain of a 5-HT--_1A receptor.

22. A method according to claim 21, vherein said 5-HT- -_1A receptor antibody is a an anti-peptide antibody against a functional domain of a 5-HT--j_A receptor.

23. A method according to claim 3, wherein said antagonist is a monoclonal 5-HT--_1A receptor antibody.

24. A method according to claim 23, wherein said 5-HT- -_1A receptor antibody is a an anti-peptide antibody against a functional domain of a 5-HT--_1A receptor.

25. A method according to claim 14, wherein said antagonist is a monoclonal 5-HT--_1A receptor antibody.

26. A method according to claim 25 wherein said 5-HT-- _1A receptor antibody is a an anti-peptide antibody against a functional domain of a 5-HT--_1A receptor.

27. A method according to claim 20, wherein said antagonist is a monoclonal 5-HT--_1A antibody.

28. A method according to claim 27, therein said 5-HT- -_1A receptor antibody is a an anti-peptide antibody against a functional domain of a 5-HT--_1A receptor.

29. A method according to claim 1, wherein prior to said contacting, the method further comprises the step of up- regulating the expression of 5-HT--_1A receptors on central serotonergic neurons in the brain of said subject.

30. A method according to claim 3, wherein prior to said contacting, the method further comprises the step of up- regulating the expression of 5-HT--_1A receptors on central serotonergic neurons in the brain of said subject.

31. A method according to claim 14, wherein prior to said contacting, the method further comprises the step of up- regulating the expression of 5-HT--_1A receptors on central serotonergic neurons in the brain of said subject.

32. A method according to claim 20, wherein prior to said administering, the method further comprises the step of up- regulating the expression of 5-HT--_1A receptors on central serotonergic neurons in the brain of said subject.

33. A method for stimulating growth of central serontonergic neurons in a subject, comprising administering to said subject a serotonergic neuron stimulating effective amount of a 5-HT--_1A receptor agonist.

34. A method according to claim 33, wherein said 5-HT- -_1A receptor agonist is selected from the group consisting of 5- hydroxytryptamine, 5-methoxytryptamine, buspirone, 8- hydroxydipropylamineotetralin, ipsaspirone, gepirone, SM23997, lysergic acid ύiethylamide and a 5-HT--_1A r?: eptor agonistic antibody.