COMPOSITIONS AND METHODS FOR TREATING NEUROLOGICAL DISORDERS

FIELD OF THE INVENTION The invention is generally directed to methods and compositions for treating one or more symptoms of a neurological disorder, in particular epilepsy, depression and anxiety, insomnia, stroke, pain, bipolar, autism and combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of and priority to U.S. Provisional

Patent Application No. 60/931,419 filed on May 23, 2007, and where permissible is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Epilepsy is the most prevalent chronic neurologic condition. In developed countries, its incidence is 30™50 per 100 000 population per year and the prevalence is approximately 5-8 cases per 1 000 population. The rapid growth of health care expenditures has led to increased interest in economic evaluation of health care programs.

Gamma-aminobutyric acid (GABA) and glutamic acid are major neurotransmitters which are involved in the regulation of brain neuronal activity. GABA is a major inhibitory neurotransmitter in the mammalian central nervous system. Meythaler et al., Arch. Phys. Med. Rehabil.; 80:13-9 (1999). Imbalances in the levels of GABA in the central nervous system can lead to conditions such as spastic disorders, convulsions, and epileptic seizures. As described in U.S. Pat, No. 5,710,304, when GABA levels rise in the brain during convulsions, seizures terminate.

Because of the inhibitory activity of GABA and its effect on convulsive states and other motor dysfunctions, the administration of GABA to subjects to increase the GABA activity in the brain has been tried. Because it is difficult to develop and administer a GABA compound which is able to cross the blood brain barrier utilizing systemic administration of GABA compounds, different approaches have been undertaken including making GABA lipophilic by conversion to hydrophobic GABA amides or

GABA esters, and by administering activators of L-glutamic acid decarboxylase (GAD) whose levels vary in parallel with increases or decreases of brain GABA concentration, and which have been reported to increase GABA levels. Additional therapies for modulating GABA concentrations in vivo are needed.

Thus, it is an object of the invention to provide methods and compositions for treating one or more symptoms of a neurological disorder. It is another object to provide methods and compositions to enhance or promote GABAergic transmission in a subject in need thereof. It is still another object of the invention to provide methods and compositions for inhibiting or reducing GABAergic transmission in a subject.

SUMMARY OF THE INVENTION

Methods and compositions for modulating GABA release in a subject are provided. A preferred embodiment provides a composition containing an effective amount of an ErbB4 Hgand to enhance or promote GABA release, i.e., GABAergic transmission. The ErbB4 ligand can be an agonist ligand or an antagonist ligand depending on the disorder to be treated. Representative disorders that can be treated include, but are not limited to epilepsy, depression and anxiety, insomnia, stroke, pain, bipolar, autism, or a combination thereof.

Exemplary agonist ligands include NRGl, variants thereof, antibodies to ErbB4, and antibody fragments that bind to ErbB4. Exemplary antagonist ligands include the extracellular domain of ErbB4 and fusion proteins thereof, and antibodies or antibody fragments that bind to NRGl . The extracellular domain of ErbB4 binds to endogenous NRGl and thereby reduces or inhibits GABA release.

Methods for treating neurological disorders are also provided. Preferred methods include administering an effective amount of an ErbB4 agonist Hgand to a subject in need thereof to promote or enhance GABA release in the subject. By increasing GABA release a sedative effect can be induced in the subject.

Methods for inducing a stimulatory effect in a subject are also provided. In these methods, an effective amount of an ErbB4 antagonist

Hgand is administered to subject to reduce or inhibit GABA release in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is a bar graph of percent ErbB4 positives for puncta rings and neuropils in coronal sections of prefrontal cortex. +/- SEM, n = 60 for puncta-rings and n ^~ 10 for neutrophils of 20 independent sections. Figures IB and 1C are bar graphs of percent cluster colocalization of coronal sections of prefrontal cortex stained with anti-ErbB4 antibody and anti- GAD65 (Gl 166) (Figure IB) and anti-VGAT (131003) antibodies (Figure 1C).

Figure 2 A is a line graph of [ ³ H]GABA release faction/total fraction versus time (mins). Cortical slices were preloaded with [ ³ H]GABA for 30 min in the presence of b-alanine (1 mM), an inhibitor of [ ³ H]GABA uptake by glial cells, aminooxyacetic acid (0.1 mM), an inhibitor of GABA degradation, and nipecotic acid (1 mM), an inhibitor of the GABA transporter in neurons. Basal and depolarization (20 mM KCl)-evoked release of [ ³ H]GABA were monitored sequentially. Controls (open circles) and NRGl (closed circles). Figure 2B is a line graph of percent [ H]GABA release versus NRGl concentration (nM). Basal (open circles) K+ evoked (closed circles). Figure 2C shows representative traces of mIPSCs in pyramidal neurons in prefrontal cortical slices. Figure 2D is a line graph of cumulative counts versus mIPSC amplitude (pA). Controls (open circles) and NRGl (closed circles). Figure 2E is a line graph of cumulative counts versus mIPSC interevent interval (ms). Controls (open circles) and NRGl (closed circles). Figure 2F is a bar graph of miPSC (percent) in control and NRGl treated pyramidal neurons in prefrontal cortical slices (n = 12). Amplitude (clear) and Frequencies (hatched). Figure 2G is a bar graph of percent eIPSC amplitude in prefrontal cortical slices treated with NRGl versus control or washout n = 12, *p < 0.01. Representative eIPSCs of control, NRGl-treated, or NRGl -treated/washed slices are shown on top. Figure 2H is a line graph of percent eIPSC amplitude versus NRGl (nM). n - 6, *p < 0.05, **p < 0.01. Figure 21 is a bar graph of K ⁺ evoked [ ³ H]GABA release (percent) (left axis, clear rectangles) and eIPSC amplitude (percent) (right axis, solid rectangles)

in prefrontal cortical slices treated with NRGl, denature NRGl, or BDNF. n = 8 for [ ³ H]GABA release; for eIPSCs, n - 6 for control, NRGl , and denatured NRGl , and n = 4 for BDNF. *p < 0.05, #p < 0.05; **p < 0.05, ##p < 0.01. Figure 3A is a line graph of [ H]GABA release (percent) versus

NRGl (nM). Basal (open circles) and K ⁺ -evoked (closed circles). [ ³ H]GABA-loaded cortical synaptosomes were treated with 5 nM NRGl with (evoked) or without (basal) 20 mM KCl. [ ³ H]GABA release was assayed 10 min after NRGl stimulation. Shown are means ± SEM of six individual experiments in triplicate. *p < 0.05, **p < 0.01. Figure 3B is a line graph (right) with a series of recordings induced by paired stimulus (10s apart) separated by indicated interpulse intervals (shown at the left). The line graph is IPSC2/ IPSCl versus interspike intervals (ms) of GABAergin transimission in prefrontal cortex treated with NRGl (solid circles) or controls (open circles). Inset shows the amplitudes of the first and second IPSCs. n=6, *p < 0.05.

Figure 4 A is a bar graph showing quantitative analysis of phospho- ErbB4 (p-ErbB4) in GAD65~positive cortical neurons treated with ecto- ErbB4 for 10 min prior to the addition of NRGl (5 nM, final concentration) for another 10 min. n - 7, *p < 0.05. Figure 4B is a line graph of eIPSC amplitude (nA) versus time (min) for cortical slices treated with sequential addition of NRGl (5 nM) and ecto-ErbB4 (1 mg/ml and 2 mg/ml) (all final concentrations). On the top are averaged traces before (a) and after (b) NRGl, and after different dosages of ecto-ErbB4 ([c] and [d], 1 and 2 mg/ml, respectively). Figure 4C is a bar graph of K ⁺ -evoked [ ³ H]GABA release (percent, left axis) or eIPSC amplitude (percent, right axis) in cortical slices treated with 1 or 2 mg/ml ecto-ErbB4 with or without NRGl (1 μg/ml). for 10 min prior to assays of [ ³ H]GABA and eIPSCs. n = 5 for [ ³ H]GABA release, n = 6 for eIPSCs. *p < 0.01 and #p < 0.01 for [ ³ H]GABA release and eIPSCs, respectively.

Figure 5 A is a bar graph of phospho-ErbB4 (p-ErbB4) in cortical neurons treated with 5 mM AG1478, an inhibitor of ErbB4, or AG879, an inhibitor of ErbB2, for 10 min prior to the addition of NRGl (5 nM, final concentration). Neurons were fixed and stained with phospho-ErbB4 and

GAD65 antibodies, and visualized with Alexa 594 and FITC-coupled secondary antibodies respectively, and quantified. Figure 5 B is a bar graph of K ⁺ -evoked [ ³ H]GABA release (percent) in cortical slices treated with 5 raM AG1478 or AG879 for 10 mm prior to assay of [ ³ H]GABA or eIPSC recording, n = 5 for [ ³ H]GABA release, n = 6 for eIPSCs. *p < 0.05, #p < 0.05; **p < 0.01 , ^m p < 0.01. K ⁺ -evoked [ ³ H]GABA release (clear rectangles), eIPSC amplitude (solid rectangles).

Figure 6 A is a line graph of K ⁺ -evoked [ ³ H]GABA release (percent) versus NRGl (nM) in ErbB4 ^v" ht+ cortical slices( A) and ErbB^ht (o). Figure 6B is bar graph of eIPSC amplitude (percent) in cortical slices of control

(ErbB4 ^+/+ h( ⁺ ) and ErbB4 ^~/" kt mice. Shown are normalized eIPSC amplitudes, n = 6 ₅ *p < 0.05. The eIPSC amplitudes in ErbB4 ^+/+ ht ⁺ and ErbB4 ^"A ht ⁺ were 1014 ± 170 and 598 ± 160 pA, respectively, n = 17, p < 0.01.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, a "variant" polypeptide contains at least one amino acid sequence alteration as compared to the amino acid sequence of the corresponding wild-type polypeptide.

As used herein, an "amino acid sequence alteration" can be, for example, a substitution, a deletion, or an insertion of one or more amino acids.

As used herein, "conservative" amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties.

As used herein, "non-conservative" amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered. Non-conservative substitutions typically alter the function of the protein.

The terms "individual", "host", "subject", and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, murines, simians, humans, mammalian farm animals, mammalian sport animals, and mammalian pets.

As used herein the term "effective amount" or "therapeutically effective amount" means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of the disorder being treated or to otherwise provide a desired pharmacologic and/or physiologic effect The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, etc.), the disease, and the treatment being effected.

The term "soluble ErbB4" or "ecto-ErbB4" are used interchangeably and refer to the extracellular domain or ErbB4 or a fusion protein thereof. IL Compositions for Treating Neurological Disorders

It has been discovered that ErbB4, a receptor for NRGl, is present in GABAergic terminals of the prefrontal cortex, and that NRGl facilitates evoked release of GABA from slices of the prefrontal cortex, but has no effect on basal GABA release. The potentiation effect of NRGl requires ErbB4 because it was blocked by the ErbB4 inhibitor AG 1478 and was abolished in cortical slices of ErbB4 mutant mice. In addition, evoked GABA release and eIPSCs in the absence of exogenous NRGl were blocked by inhibitors of NRGl signaling, suggesting a role of endogenous NRGl in regulating GABA neurotransmission. Together, these results identify a novel function of NRG 1 - regulation of GABAergic transmission via presynaptic ErbB4 receptors.

Therefore, one embodiment provides compositions and methods for treating one or more symptoms of a neurological disorder by modulating GABAergic transmission via NRGl to induce a sedative or stimulatory outcome. A preferred embodiment provides compositions and methods for treating one or more symptoms of epilepsy, depression and anxiety, insomnia, stroke, pain, bipolar, autism by administering an effective amount of a ErbB4 ligand, for example NRGl or a variant thereof. Ligand agonists of ErB4 such as NRGl induce a sedative effect in a subject by potentiating GABAergic transmission.

Another embodiment provides compositions and methods for inducing a stimulatory effect in a subject. Exemplary compositions include ErbB4 ligand antagonists such as ecto-Erb4r or soluble ErbB4. Ligand antagonists inhibit or reduce ErbB4 activity and thus reduce GABAergic

transmission. Reduction in GABAergic transmission induces a stimulatory effect.

A. NRGl and Neurotransmission at Excitatory and Inhibitory Synapses NRGl (NRGl), a family of polypeptides that plays an important role in neural development, is implicated in nerve cell differentiation, neuron migration, neurite outgrowth, and synapse formation (Buonanno and Fischbach, 2001; Corfas et al., 2004; Mei and Xiong, 2008) (L. Mei and W. C. Xiong. Neuregulin 1 signaling in neural development, synaptic plasticity and schizophrenia. Nature Rev, Neuroscl 9:437-452, 2008). NRGl and its receptor ErbB tyrosine kinases are expressed not only in the developing nervous system, but also in adult brain. In the adult, ErbB receptors are concentrated at the postsynaptic density (PSD), presumably via interaction with PDZ domain containing proteins including PSD-95 and erbin (Garcia et al, 2000; Huang et al., 2000, 2001; Ma et al., 2003). NRGl suppresses induction of LTP at Schaffer collateral-CAl synapses in the hippocampus without affecting basal synaptic transmission (Huang et al,, 2000; Ma et al., 2003). Subsequently, NRGl was shown to reverse LTP and reduce whole-cell NMDA receptor currents in pyramidal neurons of prefrontal cortex, and was also shown to decrease NMDA receptor-mediated EPSCs in prefrontal cortex slices (Gu et al., 2005; Kwon et al., 2005). Interestingly, the NRGl gene is strongly associated with schizophrenia in diverse populations in Iceland, Scotland, China, Japan, and Korea (Fukui et al., 2006; Kim et at, 2006; Stefansson et al., 2002, 2003; Yang et al, 2003). ErbB4 mRNA is enriched in regions where interneurons are clustered in adult brains (Lai and Lemke, 1991). GAD-positive neurons from the embryonic hippocampus express ErbB4 (Huang et al., 2000). During development, loss of NRGl/ErbB4 signaling alters tangential migration of cortical interneurons, leading to a reduction in the number of GABAergic interneurons in the cortex (Anton et al., 2004; Flames et al., 2004). In adult mice, deletion of ErbB4 in the central nervous system (CNS) resulted in lower levels of spontaneous motor activity, reduced grip strength, and altered cue use in performing a maze task (Golub et al., 2004). The ErbB4 gene is

also associated with schizophrenia (Law et al., 2006; Nicodemus et al., 2006). γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian forebrain. GABAergic inhibitory interneurons are essential to the proper functioning of the CNS (McBain and Fisahn, 2001). GABAergic dysfunction is implicated in several neurological disorders, including Huntington's chorea, Parkinson's disease, and epilepsy, and in psychiatric disorders such as anxiety, depression, and schizophrenia (Coyle, 2004). NRGl has been shown to regulate differentiation of neural cells, neuronal navigation, and neuron survival in developing CNS (Buonarmo and Fischbach, 2001; Corfas et al., 2004). In the peripheral nervous system, NRGl signaling is implicated in Schwann cell differentiation and myelination, muscle spindle development, and synapse- specific expression of AChR subunit genes (Adlkofer and Lai, 2000; Fischbach and Rosen, 1997; Hippenmeyer et al., 2002; Si et al., 1996). Interestingly, NRGl and its receptor ErbB kinases are continuously expressed in various brain regions, including the prefrontal cortex, hippocampus, cerebellum, oculomotor nucleus, superior colHculus, red nucleus, substantia nigra, and pars compacta (Lai and Lemke, 1991 ; Law et al., 2004; Yau et al., 2003). Moreover, ErbB4 colocalizes with PSD-95 and NMDA receptors in hippocampal neurons (Garcia et al., 2000; Huang et al., 2000). Furthermore, NRGl signaling may be increased by the interaction of ErbB4 with PSD-95 (Huang et al., 2000). These observations suggest that NRGl may play a role in synaptic plasticity, maintenance or regulation of synaptic structure, or some combination thereof in adult brain. Indeed, we found that NRGl blocks induction of long-term potentiation (LTP) at Schaffer collateral-CAl synapses (Huang et at, 2000). NRGl can depotentiate LTP at hippocampal CAl synapses and reduce whole cell NMDA receptor, but not AMPA receptor, currents in prefrontal cortex pyramidal neurons (Gu et al., 2005; Kwon et al., 2005). Recently, ErbB4 has been shown to play a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function (Li et al., 2007).

B. NRGl, ErbB4, and Neurological and Psychiatric Disorders

Schizophrenia exhibits familial characteristics, which suggests a strong genetic component. Disturbances in GABAergic neurotransmission have been thought to be a pathologic mechanism of schizophrenia.

Postmortem studies of patient brains reveal decreased levels of the mRNA encoding GAD67 (Hashimoto et al., 2003) and the GABA transporter GAT- 1 (Ohnuma et al., 1999). On the other hand, GABA-A receptor mRNA was shown to be increased in the prefrontal cortex (Ohnuma et al., 1999). Furthermore, treatment of schizophrenia with antiepileptic drugs that target GABAergic transmission has shown positive results (Hosak and Libiger, 2002). However, the date provided herein is the first to disclose or suggest the treatment of neurological disorders such as epilepsy, depression and anxiety, insomnia, stroke, pain, bipolar, autism by modulating the NRG 1 /ErbB4 signal transduction pathway .

C. Ligands of ErbB4

Compositions for treating one or more symptoms of a neurological disorder containing an ErbB4 ligand are provided. The ErbB4 ligand can be an agonist ligand or an antagonist ligand. An ErbB4 agonist ligand induces or promotes ErbB4 activity and thereby induces or promotes GABAergic transmission which increases local concentrations of GABA. Because GABA is an inhibitory neurotransmitter, increased concentrations of GABA induce or promote a sedative effect in a subject. Representative ErbB4 agonist ligands include but are not limited to NRGl and variants thereof. ErbB4 antagonist ligands include but are not limited to ecto-ErbB4 or soluble ErbB4 or variants thereof. The antagonist ligand induces or promotes a stimulatory response by reducing the amount of GABAergic transmission for example by binding endogenous NRGl.

In certain embodiment, the ErbB4 ligand is a small molecule, for example a molecule of about 500 Daltons. The small molecules can be obtained by screening a library of compounds for binding to ErbB4. Such screening techniques are routine a known in the art.

1. Variants of ErbB4 Ligands

Exemplary variants of ErbB4 ligands include, but are not limited to NRGl or ecto-ErbB4 polypeptides that are mutated to contain a deletion, substitution, insertion, or rearrangement of one ore more amino acids. In one embodiment the variant ErbB4 ligand has the same activity, substantially the same activity, or different activity as a reference NRGl or ecto-ErbB4 polypeptide, for example a non-mutated NRGl or ecto-ErbB4 polypeptide.

A variant NRGl or ecto-ErbB4 polypeptide can have any combination of amino acid substitutions, deletions or insertions. In one embodiment, isolated NRGl or ecto-ErbB4 variant polypeptides have an integer number of amino acid alterations such that their amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with an amino acid sequence of a wild type NRGl or ecto-ErbB4 polypeptide. In a preferred embodiment, NRGl or ecto-ErbB4 variant polypeptides have an amino acid sequence sharing at least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the amino acid sequence of a wild type murine or wild type human NRGl or ecto-ErbB4 polypeptide (GenBank Accession Number NRGl: L12261 ; ErBB4: L07868].

Percent sequence identity can be calculated using computer programs or direct sequence comparison. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D. W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTP and TBLASTN programs are publicly available from NCBI and other sources. The well-known Smith Waterman algorithm may also be used to determine identity.

Exemplary parameters for amino acid sequence comparison include the following: 1) algorithm from Needleman and Wunsch (J MoI. Biol, 48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoff and Hentikoff (Proc. Natl Acad. ScL U.S.A., 89:10915-10919 (1992)) 3) gap penalty = 12; and 4) gap length penalty - 4. A program useful with these parameters is publicly available as the "gap" program (Genetics Computer Group, Madison, Wis.). The aforementioned parameters are the default

parameters for polypeptide comparisons (with no penalty for end gaps).

Alternatively, polypeptide sequence identity can be calculated using the following equation: % identity = (the number of identical residues)/(alignment length in amino acid residues)* 100. For this calculation, alignment length includes internal gaps but does not include terminal gaps.

Amino acid substitutions in NRGl polypeptides may be "conservative" or "non-conservative". As used herein, "conservative" amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties, and "non-conservative" amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered. Non-conservative substitutions will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

Examples of conservative amino acid substitutions include those in which the substitution is within one of the five following groups: 1) small aliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro, GIy); 2) polar, negatively charged residues and their amides (Asp, Asn, GIu, GIn); polar, positively charged residues (His, Arg, Lys); large aliphatic, nonpolar residues (Met, Leu, He, VaI, Cys); and large aromatic resides (Phe, Tyr, Trp). Examples of non-conservative amino acid substitutions are those where 1) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteine or proline is substituted for (or by) any other residue; 3) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or 4) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) a residue that does not have a side chain, e.g., glycine.

2. Fusion Proteins

Fusion proteins that contain an ErbB4 binding domain operably linked to a second polypeptide, in particular a heterologous polypeptide. The

fusion protein optionally includes peptide or polypeptide linker domain that separates the ErbB4 binding domain from the second polypeptide.

Optionally, the second polypeptide contains a domain that functions to dimerize or multimerize two or more fusion proteins. Dimerization or multimerization can occur between or among two or more fusion proteins through dimerization or multimerization domains. Alternatively, dimerization or multimerization of fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric/homomultimeric or heterodimeric/heteromultimeric. Typcially, the second polypeptide contains an Fc domain. III. Formulations

Pharmaceutical compositions including Hgands of ErbB4 are provided. The pharmaceutical compositions may be for administration by oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in unit dosage forms appropriate for each route of administration. The preferred route is oral. The one or more active agents can be administered as the free acid or base or as a pharmaceutically acceptable acid addition or base addition salt.

Examples of pharmaceutically acceptable salts include but are not limited to mineral or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Such conventional nontoxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; and the salts prepared from organic acids such as acetic, propionic, succinic, gly colic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, furnaric, tolunesulfonic, naphthalenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic salts.

The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and "Handbook of Pharmaceutical Salts: Properties, Selection, and Use," P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley- VCH, Weinheim, 2002.

1. Formulations for Enteral Administration In a preferred embodiment the compositions are formulated for oral delivery. Oral solid dosage forms are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules or incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder (e.g., lyophilized) form. Liposomal or proteϊnoid encapsulation may be used to formulate the compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Patent No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulation will include the peptide (or chemically modified forms thereof) and inert ingredients which protect peptide in the

stomach environment, and release of the biologically active material in the intestine.

The ErbB4 ligands may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where the moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. PEGylation is a preferred chemical modification for pharmaceutical usage. Other moieties that may be used include: propylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyproline, poly-l,3-dioxolane and ρoly-l,3,6-tioxocane [see, e.g., Abuchowski and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, N. Y.) pp. 367-383; andNewmark, et al. (1982) J. Appl. Biochem. 4:185-189].

Another embodiment provides liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and sweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. The ErbB4 ligands can be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Another form of a controlled release is based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the peptide (or derivative) or by release of the peptide (or derivative) beyond the stomach environment,

such as in the intestine. To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT) ₅ hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP) ₅ Eudragit L30D™, Aquateric™, cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, and Shellac™. These coatings may be used as mixed films.

3. Topical or Mucosal Delivery Formulations Compositions can be applied topically. The compositions can be delivered to the lungs while inhaling and traverses across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent™ nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II™ nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin™ metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler™ powder inhaler (Fisons Corp., Bedford, Mass.).

Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator. Oral formulations may be in the form of chewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations will require the inclusion of penetration enhancers. 4. Controlled Delivery Polymeric Matrices

Controlled release polymeric devices can be made for long term release systemically following implantation of a polymeric device (rod, cylinder, film, disk) or injection (microparticles). The matrix can be in the form of microparticles such as microspheres, where peptides are dispersed

within a solid polymeric matrix or microcapsules, where the core is of a different material than the polymeric shell, and the peptide is dispersed or suspended in the core, which may be liquid or solid in nature. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used for delivery of disclosed compounds, although biodegradable matrices are preferred. These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release profiles. The polymer is selected based on the period over which release is desired. In some cases linear release may be most useful, although in others a pulse release or "bulk release" may provide more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90% by weight of water), and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres can be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathϊowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); and Mathiowitz, et at, J. Appl. Polymer Sci. 35, 755-774 (1988).

The devices can be formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or swallowed.

IV. Methods of Treatment

Methods for treating one or more symptoms of a neurological disorder are provided. Exemplary neurological disorders that can be treated with the disclosed compositions include, but are not limited to epilepsy,

depression and anxiety, insomnia, stroke, pain, bipolar, autism, or a combination thereof. One embodiment provides administering to subject in need thereof an effective amount of an ErbB4 ligand to increase or decrease GABAergic transmission in the subject. The ErbB4 ligand can be an agonist ligand or an antagonist ligand depending on the disorder to be treated.

Exemplary ErbB4 ligands include, but are not limited to antibodies to ErbB4. The antibodies can be polyclonal, monoclonal, chimeric, humanized, single-chain, or fragments of these antibodies that bind to ErbB4.

The term "ErbB4 ligand" includes agonist and antagonist ligands. Agonist ligands include, but are not limited to NRGl , variants thereof, and fragments of NRGl or variants thereof that bind ErbB4 and induce or inhibit GABAergic transmission relative a control. A control can be GABAergic transmission in the absence of the ErbB4 ligand. Antagonist ligands include the extracellular domain of ErbB4 (also referred to as soluble ErbB4 and fusion proteins thereof. Methods for producing fusion proteins are known in the art.

One embodiment provides a method for increasing GABAergic transmission in a subject by administering to the subject an effective amount of an ErbB4 agonist ligand, for example NRGl, a variant thereof, or an ErbB4 binding fragment thereof. The agonist ligand binds to ErbB4 and promotes or enhances GABA release i.e, GABAerginc transmission. The increase in the inhibitory transmitter GABA induces a sedative effect in the host.

Another embodiment includes administering to a subject in need thereof an effective amount of an ErbB4 antagonist ligand, for example soluble ErbB4 or a fragment thereof that binds to ErbB4 or a fusion protein thereof. The antagonist ligand binds to ErbB4 and inhibits or reduces GABA release, i.e., GABAergic transmission.

For all of the disclosed compounds, as further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the

duration of the treatment desired. Generally dosage levels of 0.001 to 100 mg/kg of body weight daily are administered to mammals.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Examples Example 1: Localization of ErbB4 in GABAergic presynaptic terminals.

Reagents and Animals

The NRGl used is a recombinant polypeptide containing the entire EGF domain of the b-type NRGl (rHRG bl77-244) (Holmes et al., 1992). It was prepared in 1% bovine serum albumin (BSA). BDNF was a gift from Regeneron Pharmaceuticals. The ectodomain of ErbB4 (aa 1-659, ecto- ErbB4) was subcloned into pC4DNA/Fc to generate pErbB4ex/Fc. Stable HEK293 cells expressing ecto-ErbB4 were generated and cultured in IgG- low medium for condition media collection. ErbB4ex/Fc was purified by a HiTrap column (Amersham). AG 1478 and AG879 were from Calbiochem; poly-L~lysine, nipecotic acid, b-alanine and TMPH (2,2,6,6,-

Tetramethylpiperidin-4-yl heptanoate) from Sigma; DL-AP 5, CNQX, TTX, bicuculline, LY341495, ipratropium, nicergoline, sotalol, metergoline, MDL 72222, RS 23597-190, and L-741742 from Tocris Bioscience; and aminooxyacetic acid from Chemika. When necessary, chemicals were dissolved in dimethylsulfoxide (DMSO, Sigma); the final concentration of DMSO was 0.001% or less when applied to brain slices. Antibodies were from Sigma (GAD65, Gl 166); Cell Signaling Technology [ErbB4, #4795; p- ErbB4 (Y1284), #4757]; Transduction Labs (phosphotyrosine, 610024); NeoMarkers (ErbB2, MS-303-PO; ErbB3, MS-229-PO); Santa Cruz

Biotechnology (ErbB4, sc-283); and Synaptic Systems (VGAT, 131003). ErbB4 ^"A ht ⁺ mice were kindly provided by Martin Gassmann (Tidcombe et at, 2003). GAD-GFP mice were from the Jackson Lab.

In Situ Hybridization In situ hybridization was performed essentially as previously described (Simmons et al. ₅ 1989), with minor modifications. Adult Sprague- Dawley rats were perfused for 20 min with 4% paraformaldehyde in 0.1 M sodium borate buffer (pH 9.5). Sagittal sections (30 mm) were cut on a sliding microtome and mounted on gelatin and poly-L-lysine-coated slides. Tissue sections were fixed for 30 min in 10% buffered formalin and washed in 50 mM KPBS prior to prehybridization. ErbB4 sequence #1009-1931 (accession # NM-021687), NRGl type I/II sequence #345-845 (accession # NM-031588), and NRGl type IO sequence #555-1321 (accession #AF194438) were subcloned in pCRScript. Plasmids were digested with Notl, Spel, and EcoRI, respectively, for the production of individual antisense RNAs using T7 RNA polymerase. Transcriptions were performed using 125 μCi ³³ P-UTP (2000-4000 Ci/mmole, NEN). After hybridization, the sections were defatted in xylene, rinsed in 100% ethanol and then 95% ethanol, air dried, and dipped in NTB2 emulsion (Kodak) diluted 1:1 with water. The slides were exposed for 2-5 weeks and developed in Kodak D- 19 developer. All images were captured with a Hamamatsu Orca ER CCD camera using dark-field microscopy on an Olympus BX-51 microscope at 1.25 3 magnification.

Immunostaining Immunostaining of rat cortical neurons (E 17, DIV 14) was performed as previously described (Huang et al., 2000). Briefly, neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 20 min, and permeabϋized by incubation in PBS containing 1% BSA and 0.1% Triton X- 100 for 30 min at room temperature. After washing, neurons were incubated in the buffer containing antibodies against phospho-ErbB4 (1 :200), GAD65 (1:200), or both for 1 hr at room temperature. Brain sections (20 mm) were fixed with 10% formaldehyde and blocked in 5% BSA/1% normal goat serum (Ren et al., 2004). Sections were incubated overnight at 4 ⁰ C in PBS containing rabbit anti-ErbB4 with or without anti-GAD65 or VGAT.

Fluorochrome-conjugated secondary antibodies were used to visualize the immunoreactivity with a confocal microscope. Results

ErbB4 transcripts were expressed. throughout cortical layers 2-6b (Lai and Lemke, 1991; Yau et al., 2003). In addition, ErbB4 transcripts were identified at high levels in the medial habenula, the reticular nucleus of the thalamus and in the intercalated masses of the amygdala. These observations are consistent with the notion that ErbB4 is expressed in interneruons. In agreement, ErbB4 was shown to be present in GAD-positive neurons isolated from the hippocampus (Huang et al., 2000). To determine in vivo subcellular localization of ErbB4 in GAD-positive neurons, prefrontal sections of GIN (GFP-expressing Inhibitory Neurons) mice were stained. They express GFP under the control of the gadl promoter that directs specific expression in GABA interneurons, especially those that are somatostatin positive, in the hippocampus (Oliva et al., 2000). Presynaptic terminals of GABAergic neurons appear as discrete puncta rings in the prefrontal cortex, surrounding soma of postsynaptic neurons in cortical layers II- VI (Pillai-Nair et al., 2005). The anti-ErbB4 antibody 0618 and sc~ 283 specifically recognized ErbB4 because their immunoreactivity was diminished in ErbB4 mutant mice. ErbB4 was detected in puncta rings and neuropils, colocalizing with GFP. Quantitatively, about 90% of puncta rings and neuropils in the prefrontal cortex expressed ErbB4 (Figure IA). These results suggest that ErbB4 is present at terminals of GABAergic neurons including somatostatin neurons. To test this hypothesis further, we determined whether ErbB4 colocalizes with GAD65 and vesicular GABA transporter (VGAT), both well-characterized markers of GABAergic terminals (Tafoya et al., 2006). The ErbB4 immunoreactivity co-localized with GAD65 and VGAT in puncta-ring like structures (Figures IB and 1C). 32 % of GAD-65 clusters and 59% of VGAT clusters were ErbB4-positive, suggesting ErbB4 localization at specific subsets of GABA terminals (Figure IB and 1C). On the other hand, 32% and 49% of ErbB4 clusters colocalized with GAD-65 and VGAT ₅ respectively, in agreement with the notion that ErbB4 is also localized at non-GABAergic synapses (Huang et al., 2000).

Taken together, these results indicate that ErbB4 is present at groups of presynaptic terminals of GABAergic neurons in the cerebral cortex.

Example 2: Increase in depolarization-evoked GABA release by NRGl. Electrophysiological Recordings in Slices

Transverse prefrontal cortical slices (0.3 mm) were prepared from P28-P36 mice using a VibrosHce (Leica VT 1000S) in the ice-cold solution, which contained 2.5 mM KCl, 1.25 niM NaH ₂ PO4, 10 mM MgSO ₄ , 0.SmMCaCl ₂ , 26 mM NaHCO ₃ , 10 mM glucose, and 230 mM sucrose. Slices were allowed to recover for at least 2 hr in ACSF (1 hr at 34 ⁰ C followed by 1 hr at 22°C) in a solution containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH ₂ PO ₄ , 2 mM MgSO ₄₅ 2 mM CaCl ₂ , 26 mM NaHCO ₃ , and 10 mM glucose. Slices were placed in the recording chamber and superfused (1.5 ml/min) with ACSF at 34°C. AU solutions were saturated with 95% O ₂ /5% CO ₂ . Neurons were visualized with an IR-sensitive CCD camera with a 403 water-immersion lens (Zeiss, Axioskop2 Fsplus) and recorded using whole-cell voltage-clamp techniques (MultiClamp 700B Amplifier, Digidata 1320A analog-to-digital converter) and pClamp 9.2 software (Axon Instruments). Glass pipettes were filled with the solution containing 125 mM Cs-gluconate, 10 mM CsCl, 1 mM MgCl ₂ , 10 mM HEPES, 1 mM EGTA, 0.1 mM CaCl ₂ , 10 mM sodium phosphocreatine, 4 mM Mg-ATP, 0.3 mM GTP, 0.2 mM leupeptin, and 5 mM lidocaine N-ethylchloride (QX314) (pH 7.2, with the osmolarity adjusted to 280 mOsm with sucrose). The resistance of pipettes was 2-3 Mω. For mIPSC recording, QX314 was omitted in the pipette filling solution, whereas 1 mM TTX was included in the superfusing solution, eIPSCs were generated with a two-concentric bipolar stimulating electrode (25 mm pole separation; FHC, ME) positioned about 100 mm from the neuron under recording. Single or paired pulses of 0.2 ms were delivered at 0.1Hz and synchronized using a Mater- 8 stimulator (A.M.P.I). The holding potential for both mIPSCs and eIPSCs was -65 mV. All experiments were done at 34 ⁰ C in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 mM) and AP-5 (50 mM) to block AMPATNMDA receptors. Data were collected when series resistance fluctuated within 15% of initial

values (8-15 MU), they were filtered at 2 kHz, and they were sampled at 10 kHz.

Results

NRGl may regulate GABAergic neurotransmission. To test this hypothesis, the effects of NRGl on GABA release in cortical slices were determined by both biochemical and electrophysiological approaches. Basal [ ³ H]GABA release was low, at a rate of 3.75 ± 035% (n - 8) of total radioactivity per 10 mm (Figure 2A). Treatment of slices with 20 mM KCl, a condition known to depolarize neurons, increased [ ³ H]GABA release by 2.5- 3.5 folds within 10 min (Figure 2 A). NRG 1 had no effect on basal

[ ³ H]GABA release; by contrast, it increased depolarization-evoked GABA release in a dose-dependent manner (Figures 2B). This effect was not inhibited by antagonists of glutamate receptors, suggesting that the increase in GABA release does not require glutamatergic signaling. To demonstrate that NRGl regulates physiological function of GABA transmission, inhibitory postsynaptic currents (IPSCs) were recorded from prefrontal cortical slices. As shown in Figure 2C-2F, NRGl did not appear to affect the frequency, amplitude, and decay times of miniature IPSCs (mIPSCs) that were blockable by bicuculline, a GABA receptor antagonist. These results are in agreement with observations above that basal GABA release was not affected. By contrast, as shown in Figure 2G, it enhanced evoked IPSCs (eIPSCs) that were sensitive to bicuculline (Supplemental Figure SlD). The increase in eIPSCs had a similar dose-response curve to evoked [ ³ H]GABA release (Figure 2H) and was abolished when NRGl was heat-denatured (Figure 21). Furthermore, the NRG regulation remained unchanged in the presence of antagonists of metabotropic glutamate receptors, cholinergic receptors, serotonin receptors, adrenergic receptors and/or dopamine receptors. As a control, BDNF decreased depolarization-evoked GABA release and eIPSCs in cortical slices, in agreement with earlier studies (Canas et al., 2004; Frerking et al., 1998). These results indicate that NRGl increases evoked GABA release, without affecting basal release, likely via direct effect on GABAergic presynaptic terminals.

Example 3: NRGl effects on GABAergϊc presynaptic terminals.

To further determine whether NRGl regulates GABA release directly at presynaptic terminals, we performed the following two experiments. First, we investigated whether NRGl is able to regulate [ ³ H]GABA release from synaptosomes, free of neural circuit. As shown in Figure 3 A, NRGl increased depolarization-evoked GABA release from synaptosomes while it had no effect on basal GABA release. Moreover, this effect was concentration-dependent, with a maximal response of 28 ± 1.5% (n = 6) similar to that observed in cortical slices (Figure 3A). Second, we characterized the paired-pulse ratios (PPRs) of control and NRG 1 -affected eIPSCs in response to two stimulations. At inhibitory synapses, second stimulation generates smaller eIPSC because of depletion of vesicles in the releasable pool by the first stimulation (Lambert and Wilson, 1994). Shown in Figure 3B (left panel) were averaged traces of eight consecutive eIPSCs induced by paired stimuli at different interpulse intervals. The PPRs at 25 ms intervals were reduced from 0.86 + 0.07 in control to 0.68 ± 0.05 in NRGl- treated slices (n = 6, P < 0.01). The reduction in PPRs was not recovered even at 200 ms intervals. The depression effect of NRGl on the amplitudes of the second eIPSCs provides further evidence that NRGl regulates evoked GABA release by a presynaptic mechanism. In addition, these results also suggest that NRGl may increase the probability of GABA release in response to depolarization.

Example 4: Endogenous NRG is necessary to maintain activity- dependent GABA release. Cell Culture

Primary cortical neurons were cultured as described previously (Huang et al., 2000). Briefly, cerebral cortex was dissected out of Sprague- Dawley rat embryos (El 8) and dissociated by gentle trituration in PBS (Cellgro). Cells were seeded on poly-L-lysϊne-coated 12-well plates and cultured in Neurobasal media (Gibco). Experiments were performed 14 days after seeding (DIV 14). C2C12 cells were obtained from E. S. Ralston (NIH) and cultured as previously described (Siet al., 1996). To generate ecto- ErbB4, HEK293 cells were cotransfected with pC4-B4Ex/Fc, which expresses the entire ectodomain fused with the Fc fragment, and pEGFP-Cl,

which contains the neomycin resistance gene at a ratio of 10:1. Cells resistant to G418 (0.4 mg/ml) were cloned. Cells were cultured in 2% low Ig fetal bovine serum to collect condition medium. Ecto~ErbB4 was purified by chromatography using HiTrap protein G beads (Amersham). Results

NRGl is expressed in various regions in the brain. (Law et al., 2004). NRGl -type I/II transcripts were detected prominently in cortical layer 6b and at lower levels in layers 2 and 3. In comparison, NRGl -type III transcripts were primarily detected in cortical layer 5. Hybridization of NRGl -type I/II was also observed in the reticular nucleus of the thalamus and in cholinergic interneurons in the globus pallidus. NRGl type III was expressed in the reticular nucleus of the thalamus. Both NRGl isoforms were also observed in the piriform cortex and throughout the hippocampus. Notably, the distinct isoforms of NRGl appear to be expressed in a laminar-specific, and largely non-overlapping manner in the cortex. These observations indicate that

NRGl is available in various areas in the brain including the cerebral cortex. To determine whether endogenous NRGl regulates GABA release, we generated ecto-ErbB4 that contains the entire extracellular region of ErbB4 fused to the FC fragment. Ecto-ErbB4 binds to and thus prevents NRG from interacting with ErbB receptor kinases. As shown in Figure 4A ₅ treatment with ecto-ErbB4 inhibited NRGl activation of ErbB4 in GAD-positive neurons. Such treatment blocked NRGl potentiation of eIPSCs in a dose- dependent manner (Figures 4B and 4C), demonstrating the neutralizing ability of ecto-ErbB4. NRGl -enhanced evoked GABA release was also inhibited by ecto-ErbB4 (Figure 4C). Remarkably, treatment with ecto-

ErbB4 alone reduced both evoked GABA release and eIPSCs in the absence of exogenous NRGl (Figure 4C). These observations and results from studies of inhibitors of ErbB4 suggest a role of endogenous NRG in regulating evoked GABA release. Example 5: ErbB4 is necessary for NRGl -enhancement of evoked GABA release. lmmunoprecipitation and Western Blotting

Immunoprecipitation was carried out as previously described (Huang et al., 2000). Briefly, cell lysates (1 mg of protein) were incubated with

indicated antibodies (1-2 mg) at 4 ⁰ C for 1 hr with constant rocking in 1 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium-deoxycholate, 1 mM PMSF, 1 mM EDTA, 1 mg/ml aprotinin, leupeptin, and pepstatin protease inhibitors). Samples were then incubated at 4 ⁰ C for 1 hr with agarose beads (1:1 slurry, 50 ml) conjugated with protein A (for rabbit antibodies) or G (for mouse antibodies). Bound proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane, which was blocked with TBS containing 5% nonfat dry milk and 0.05% Tween 20 for 1 hr. The membrane was then incubated overnight at 4 ⁰ C with primary antibodies and developed by horseradish peroxidase- conjugated secondary antibodies and enhanced chemiluminescence system (Amersham Pharmacia). Results Of the three ErbB kinases, ErbB2 and ErbB4, but not ErbB 3, are catalytically active (Citri and Yarden _> 2006). To determine which ErbB is involved in NRGl regulation of evoked GABA release, cortical neurons were treated with AG879 and AG1478, specific inhibitors of ErbB2 and ErbB4, respectively (Fukazawa et al., 2003). Cell culture is described in Example 4. ErbB4 tyrosine phosphorylation in response to NRGl was blocked in neurons pretreated with AG1478, but not AG879 (Figures 5A). Treatment with AG 1478 prevented NRGl from increasing evoked GABA release and eIPSCs in cortical slices (Figure 5B). These results suggest a role of ErbB4 in NRGl regulation of GABAergic transmission. As observed with ecto-ErbB4, AGl 478 alone decreased depolarization-evoked [ H]GABA release and eIPSCs (Figure 5B), providing further evidence that endogenous NRG activity may be necessary to maintain GABA release elicited by neuronal activation. As control treatment with AG879 had no detectable effect on evoked GABA release and eIPSCs in the presence or absence of exogenous NRGl (Figure 5B). Taken together, these observations demonstrate that activation of ErbB4 _? but not ErbB2, is required for NRGl's effect. To investigate the involvement of ErbB4 further, evoked GABA release in ErbB4 mutant mice was characterized. ErbB4 null mutant mice die around embryonic day 11. The embryonic lethality can be genetically rescued by expressing ErbB4 under a cardiac-specific myosin promoter

(Tidcombe et al., 2003). This line of mice <ErbB4-/-ht ⁺ ), however, do not express ErbB4 in the brain or other non-cardiac tissues (data not shown). Ablation of the ErbB4 gene had no effect on basal and depolarization-evoked [ ³ H]GABA release (Figure 6A). However, unlike control slices, NRGl was unable to increase evoked [ ³ H]GABA release and eϊPSCs in ErbB4-/-ht ⁺ slices (Figures 6A and 6B). These observations identify an important role of ErbB4 in NRGl regulation of evoked GABA release.

The data presented herein provide evidence that ErbB4 is present at GABAergic terminals in the prefrontal cortex. The identification of the subtype or subtypes of GABA interneurons that express ErbB4 will require further investigation. Interestingly, ErbB4 colocalizes with GAD-GFP in GIN mice. An earlier study demonstrated that hippocampal GAD-GFP- labeled neurons of these mice are mostly somatostatin positive (Oliva et al., 2000). Whether GFP-labeled neurons in the prefrontal cortex are somatostatin positive was not characterized in detail. Nevertheless, the data show that NRGl activates ErbB4 and regulates GABAergic transmission. This trophic factor has no effect on basal GABA release but increases GABA release evoked by neuronal activation. Because glutamatergic neurotransmission can be regulated by NRGl (Gu et al., 2005; Li et al., 2007) and because glutamatergic activity is known to increase GABAergic transmission (Belan and Kostyuk, 2002), it is possible that NRGl regulation of evoked GABA release may be mediated by a glutamatergic mechanism. The results provided herein, however, suggest otherwise; NRGl enhancement of evoked [ ³ H]GABA release was not attenuated by inhibitors of NMDA and AMPA receptors. Moreover, NRGl enhanced eIPSCs in the presence of these inhibitors. Therefore, it is likely that NRGl regulates GABA release by directly activating ErbB4 receptors on presynaptic terminals. The presence of ErbB4 in GAD-GFP-positive puncta-ring-like structures and the colocalization with GAD65 and VGAT provide anatomical evidence in support of this notion. Moreover, NRGl was able to increase depolarization-evoked GABA release from synaptosomes that were free of interneural network, suggesting that the regulatory machinery for NRGl was present in presynaptic terminals. Furthermore, NRGl decreases PPRs of

eIPSCs in response to two consecutive stimulations, suggesting that it may facilitate vesicle release evoked by neuronal activation of interneurons.

The data provided here provides evidence that endogenous NRGl plays a role in maintaining evoked GABA release. First, treatment with ecto- ErbB4 alone attenuated evoked GABA release, presumably by neutralizing endogenous NRGl. Second, inhibition of ErbB4 reduced evoked GABA release in the absence of exogenous NRGl. In light of the fact that interneuron activity in vivo could be high (Mountcastle et al, 1969), it is likely that NRGl plays an important role in controlling neuronal activity in the brain. These data are consistent with expression of NRGl by cortical pyramidal neurons and ErbB4 by interneurons. While ErbB4 is expressed in interneurons throughout the cortex, distinct isoforms of NRGl appear to be expressed in a lamina-specific and largely non-overlapping manner in the cortex. The readily available NRGl may maintain basal activity-dependent GABAergic transmission. Interestingly, NRGl or ErbB4 heterozygotes show hyperactivity in an open field (Gerlai et al., 2000; Stefansson et al., 2002). Statistical Analysis

Data were presented as mean ± SEM of three or more independent experiments. For multiple group comparisons, statistical differences were calculated by one-way ANOVA followed by Dunnett's test. For comparison of means from the same group of cells, Student's paired t test was used. mIPSCs were analyzed by the Kolmogorov-Srnirnov (K-S) test. Values of p < 0.05 were considered significant.