In the last decades, few mechanistically novel therapeutic agents have been developed to treat mental and neurodegenerative disorders. Numerous studies suggest that targeting BDNF and its TrkB receptor could be a promising therapeutic strategy for the treatment of brain disorders. However, the development of potent small ligands for the TrkB receptor has proven to be difficult. By using a peptidomimetic approach, we developed a highly potent and selective TrkB inhibitor, cyclotraxin-B, capable of altering TrkB-dependent molecular and physiological processes such as synaptic plasticity, neuronal differentiation and BDNF-induced neurotoxicity. Cyclotraxin-B allosterically alters the conformation of TrkB, which leads to the inhibition of both BDNF-dependent and -independent (basal) activities. Finally, systemic administration of cyclotraxin-B to mice results in TrkB inhibition in the brain with specific anxiolytic-like behavioral effects and no antidepressant-like activity. This study demonstrates that cyclotraxin-B might not only be a powerful tool to investigate the role of BDNF and TrkB in physiology and pathology, but also represents a lead compound for the development of new therapeutic strategies to treat brain disorders.

The synthesis and stereochemistry of a series of new cyclic disulfides containing (poly)spirane 1,2-dithiolane units are reported. Also included is a study of the self-assembled monolayers (SAMs) of these compounds on a gold surface. The characteristics of the resultant SAMs were determined by IR spectroscopy using molecular mechanics calculations.

News aryl thiophenes 4-(thien-3-yl)benzenethiol, 4(thien-3-yl)benzylthiol have been synthesized, electrodeposited and polymerized onto a gold (111) substrate [1]. Optimized oxidative electrodeposition of these thiophenes yields reproducible high coverage monolayers [2,3]. Characterization of the monolayers with cyclic voltammetry and electrochemical impedance spectroscopy experiments indicate that the insertion of a methylene group between the aryl and the thiol anchor function increases the surface coverage (see figure 1). XPS measurements show the expected two types of sulfur (thiol and thiophene) onto the gold surface and suggest that the adsorption of these molecules occurs via the thiol.

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.

The D3 dopamine receptor, a D2-like receptor, is selectively expressed in the ventral striatum, particularly in the shell of nucleus accumbens and islands of Calleja, where it is found in medium sized substance P neurons. The latter co-express the D1 receptor whose interaction with the D3 receptor was studied by treating rats with selective agonists and antagonists. In agreement with the opposite cAMP response, they mediate in cultured neuroblastoma cells, the D1 and D3 receptors exerted opposite influences on c-fos expression in islands of Calleja. However, in agreement with the synergistic influence of cAMP on D3 receptor-mediated mitogenesis on the same cultured cells, D1 and D3 receptor stimulation in vivo synergistically enhanced preprotachykinin mRNA in the shell of accumbens. This indicates that the two receptor subtypes may affect neurons in either synergy or opposition according to the cell or signal generated. Levodopa-induced behavioral sensitization in hemiparkinsonian rats is another example of D1/D3 receptor interaction. Hence repeated levodopa administration induces the ectopic appearance of the D3 receptor in substance P/dynorphin, striatonigral neurons of the dorsal striatum. This induction is secondary to D1 receptor stimulation in neurons of the denervated side and fully accounts for the sensitization, i.e. the increased behavioral responsiveness to levodopa. During brain development, a similar process could operate to control the late appearance of the D3 receptor in D1-receptor bearing neurons of the ventral striatum at a time at which they start to be innervated by dopamine neurons. Finally, taking into account a variety of genetic, developmental, neuroimaging and pharmacological data, we postulate that imbalances between the levels of D1 and D3 receptors in the same neurons could be responsible for schizophrenic disorders.

The classical dopamine (DA) hypothesis of schizophrenia owed its birth to the serendipitous discovery in the early 1950s of the potent antipsychotic properties of chlorpromazine (Delay et al. 1952). The ability of this compound, as well as other related “neuroleptics”, to increase animal brain levels of DA metabolites in rough proportion to their clinical efficacy had been attributed to blockade of, at this time still undiscovered, brain DA receptors (Carlsson and Lindqvist 1963). Conversely, it was hypothesized that overstimulation of these receptors could be involved in the etiology of the disorder. In support were the observations that DA-stimulant drugs such as amphetamine were able to induce psychosis-like manifestations in healthy individuals or to exacerbate psychotic symptoms in schizophrenics. The subsequent discovery that therapeutic potencies of antipsychotics were correlated with their affinity for certain brain DA receptors (Creese et al. 1976) has added arguable value to the hypothesis.

In a NG 108 15 hybrid cell line stably expressing a recombinant dopamine D3 receptor, (+)-UH 232 (cis-(+)-1S,2R)-5-methoxy-1-methyl-2-(di-n- propylamino)tetralin), a partially selective D3 receptor ligand, stimulates mitogenesis, as measured by incorporation of [3H]thymidine, with an EC50 of 7.6 nM and a maximal increase corresponding to 23% of the response elicited by quinpirole, a full agonist. This effect was antagonised by nafadotride, a D3 receptor-selective antagonist. (+)-UH 232 also antagonised quinpirole-induced mitogenesis with a Ki value of 9.4 nM. (+)-UH 232 (1 microM) inhibited by 22% the forskolin-induced accumulation of cAMP, whilst the inhibition by quinpirole (100 nM) was 53%. These results indicate that (+)-UH 232 is a partial agonist at the D3 receptor with an intrinsic activity of 0.2-0.4.

A novel dopamine D3 receptor gene that may be involved in psychiatric diseases has recently been characterized. It has been assigned to chromosome 3 by hybridization with a D3 receptor probe to human sorted chromosomes, and localized to band 3q 13.3 by in situ hybridization.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of Wistar-Furth rats that had been immunized with a Moloney sarcoma virus (Mo-MuSV)-induced tumour, MFU. Two immunization protocols were designed. In the first, the animals received several injections of irradiated (10 000 rad) cells of a tumour cell line established in vitro, MFU-67. The rats received a booster injection 3 days prior to fusion. In the second protocol, immunization was the result of simple tumour growth, and no booster was given. Hybrids were tested by immunofluorescence for the production of immunoglobulins reacting with mouse cells acutely infected with Mo-MuSV. Over 20% of reactive hybrids were observed in the tumour growth protocol, and about 10% in the irradiated cell protocol when the last injection of the series was given 2 weeks before fusion. After 6 months, the proportion fell to 3%. Hybrid lines producing antibody to p30, the major core polypeptide of murine retroviruses, were obtained by cloning. Three of these were selected for closer study and were found to recognize three non-overlapping epitopes on p30. By direct and competitive binding in ELISA tests, the three epitopes were found to have very different distribution patterns among the various strains and isolates of murine retroviruses.

Isoelectric focusing (IEF) analysis of class I endogenous type-C virus induced by iododeoxyuridine treatment of BALB/K-3T3 cells revealed, in addition to the major variant of the p30 polypeptide, which has an isoelectric point of 6.1 (pI 6.1 isop30), a minor isop30 with a pI of 5.6. This value was also found for a prototype BALB/c B-tropic endogenous virus isolate. The pI 5.6 isop30 of the N-tropic isolate was amplified by long-term virus replication in B-type mouse cells, and comparative IEF and XC-assay data suggest that it may correspond to a B-tropic subpopulation which has not yet been detected in vitro in mouse cells of embryonic origin.

The isoelectric point (PI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.

Endogenous ecotropic and xenotropic murine C-type viruses induced in K-Balb-3T3 cells treated with iododeoxyuridine (IdU) were selected by infection of appropriate indicator cells. The isoelectric point (p1) of the major viral polypeptide (p30) was found to be 6.1 for the ecotropic virus (class I), and 5.7 for the xenotropic virus (class II). An isoelectric form (iso p30) of pl 6.5 was observed in the initial induction peak. In addition, the pattern of cellular alteration in NRK cells at its onset varied according to the pseudotype, the class I pseudotype inducing round cell foci while the foci associated with the class II pseudotype consisted of fusiform cells.

We have identified 7-[3Hlhydroxy-N,N-di-npropyl-2-aminotetralin ([3H]7-OH-DPAT) as a selective probe for the recently cloned dopamine D3 receptor and used it to assess the presence of this receptor and establish its distribution and properties in brain. In transfected Chinese hamster ovary (CHO) cells, it binds to D3 receptors with subnanomolar affinity, whereas its affinity is approximately 100-, 1000-, and 10,000-fold lower at D2, D4, and D, receptors, respectively. Specific [3H]7-OH-DPAT binding sites, with a Kd of0.8 nM and a pharmacology similar to those at reference D3 receptors of CHO cells, were identified in rat brain. D3 receptors differ from D2 receptors in brain by their lower abundance (2 orders of magnitude) and distribution, restricted to a few mainly phylogenetically ancient areas-e.g., paleostriatum and archicerebellum-as evidenced by membrane binding and autoradiography studies. Native D3 receptors in brain are characterized by an unusually high nanomolar affinity for dopamine and a low modulatory influence of guanyl nucleotides on agonist binding. These various features suggest that D3 receptors are involved in a peculiar mode of neurotransmission in a restricted subpopulation of dopamine neurons. Dopamine is an important neurotransmitter in brain, being involved physiologically in the control of cognitive, motor, and endocrine processes and pathologically in Parkinson and possibly mental diseases. Until recently, it was largely believed that its various actions were mediated by two receptor subtypes, termed D1 and D2 (1, 2). Molecular biology approaches have led, however, to the identification and cloning of the genes corresponding not only to these two receptor subtypes (3-7) but also to additional and less expected ones, termed D3 (8), D4 (9), and D5 (10, 11). For the latter receptors, the information so far available derives from molecular biology approaches; their pharmacology and signaling system have only been studied in transfected cells, generally fibroblasts, and their distribution has been indirectly approached by studies of mRNA localization. Nevertheless, these otherwise valuable approaches suffer from some limitations. When expressed in fibroblasts, receptors might find a membrane environment, which could modify their pharmacological specificity, and a repertoire of cellular components, particularly GTP-binding proteins (G proteins), which may differ from that found in neurons. Regarding distribution, mRNA detection reveals the sites of receptor synthesis rather than receptor localization, which may be different. Therefore, there is an obvious interest in studying the native receptor protein in brain, particularly in the case ofthe D3 receptor, whose pharmacology in Chinese hamster ovary (CHO) cells and cerebral localization of its mRNA suggest that it may represent an important target for antipsychotics (8, 12, 13). Starting with the idea that designing a selective radioactive probe for the D3 receptor would help to settle these issues, we have screened a series of dopamine agonists and thereby identified 7-hydroxy-N, N-di-n-propyl-2-aminotetralin (7OH-DPAT) (14-16) as fulfilling this purpose. MATERIALS AND METHODS Brain Membranes. Male Wistar rats (IFFA Credo, Orldans, France) weighing 180-200 g were used. They were exposed to an artificial light cycle of 12 h/day and had free access to food and water. After decapitation, brain tissues were rapidly dissected out, frozen on dry ice, and kept at -70°C until use. Tissues were homogenized with a Polytron (setting 7 for 10 sec) in 10 mM Tris-HCl (pH 7.5) containing 1 mM EDTA and centrifuged at 2500 x g for 30 sec; the supernatant was centrifuged at 35,000 x g for 15 min. The pellet was resuspended and centrifuged again, and this washing procedure was repeated twice to eliminate endogenous dopamine. The final pellet was resuspended by sonication in a buffer containing 50 mM NaHepes, 1 mM EDTA, 50 ,uM 8-hydroxyquinoleine, 0.005% ascorbic acid, and 0.1% bovine serum albumin (pH 7.5) (incubation buffer). Membranes from Cell Lines. CHO cell lines expressing rat D2 or D3 dopamine receptors (CHO-D2 or CHO-D3) have been described (12). CHO-D1 were created by transfecting pCD-BS plasmid containing the human D1 receptor gene (6), a generous gift of P. Seeman. COS-7 cells transiently expressing the human D4 receptor were obtained by transfecting pCD-PS plasmid, a generous gift of H. H. M. Van Tol, containing the human D4 receptor gene (9). Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were harvested by trypsin treatment (0.25%) for 4-5 min and centrifugation at 2000 x g for 5 min. They were homogenized with a Polytron in 10 mM Tris HCl (pH 7.5) containing 1 mM EDTA and were centrifuged at 35,000 x g for 15 min. The pellet was then resuspended by sonication in the NaHepes buffer described above. Abbreviations: 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin; p[NH]ppG, 5'-guanylyl imidodiphosphate; G protein, GTPbinding protein. §To whom reprint requests should be addressed. 8155 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8156 Neurobiology: Ldvesque et al.