B. Grimaldi

Magnetic field desensitizes 5-HT1B receptor in brain: pharmacological and functional studies

It was previously suggested that exposure to magnetic fields (MFs) could generate dysfunction of the CNS. The physiological manifestations described lead us to postulate that these symptoms might be related to a dysfunction of the serotonergic system and particularly of the 5-HT(1B) receptors. Accordingly, MFs could modify the conformation of these receptors altering their functional activities. In rat brain membrane preparations, we showed that the affinity constant of 5-HT for 5-HT(1B) receptors was modified under exposure to MFs since K(d) varied from 4.7+/-0.5 to 12+/-3 nM in control and exposed (2.5 mT) membranes, respectively. This effect was intensity-dependent (the sigmoidal dose-response curve was characterized by an EI(50) of 662+/-69 microT and a maximal increase of 321+/-13% of the control K(d)), reversible, temperature-dependent and specific to the 5-HT(1B) receptors. Similar results have also been obtained with the human 5-HT(1B) receptors. In parallel assays, the functional activity of 5-HT(1B) receptors was investigated. The capacity of a 5-HT(1B) agonist to inhibit the cAMP production was reduced by 37% (53.7+/-3.5% to 33.7+/-4.1%) following exposure to MFs and the cellular activity of the receptors (inhibition of the synaptosomal release of 5-HT) also was markedly reduced (66.5+/-3.2% to 28.5+/-4.2%). These results clearly show that in in vitro assays, MF specifically interacts with 5-HT(1B) receptors, inducing structural changes of the protein that result in a functional desensitization of the receptors. Thus, in vivo, exposure to MFs may lead to physiological changes, particularly in the field of mood disorders where the 5-HT system is strongly involved.

Immunocytochemical localization of neurons expressing 5-HT-moduline in the mouse brain

The localization of 5-HT-moduline, an endogenous cerebral tetrapeptide (LSAL) which specifically interacts with 5-HT1B receptors (Rousselle et al., 1996; Massot et al., 1996) was examined in mouse brain using an immunocytochemistry technique with a polyclonal anti-peptide antibody highly specific for this tetrapeptide. Highest levels of 5-HT-moduline immunoreactivity were observed in the cerebral cortex including cingulate, retrosplenial, parietal and pyriform cortical areas and in the basal ganglia. Intense immunoreactivity also occurred in the hippocampus, subiculum, various hypothalamic and thalamic nuclei and in some midbrain regions such as the substantia nigra and the superior colliculi. Immunoreactive neurons generally showed intense and extensive labelling of the perikarya and dendritic arborizations with moderate to heavy characteristic deposits of reaction product along plasma membranes and within cytoplasm while the nuclei were devoid of reaction product. The results obtained indicated that 5-HT-moduline immunoreactivity was heterogenously distributed in neuronal structures of mouse brain. The distribution of 5-HT-moduline immunoreactivity closely correlated with that of 5-HT-moduline specific binding sites as visualized by autoradiography (Massot et al., 1996). Moreover, it seems to overlap with the distribution of serotonergic innervation and also with that of 5-HT1B receptors in mouse brain (Boschert et al., 1994; Bruinvels et al., 1994; Chopin et al., 1994; Langlois et al., 1995). These data provide evidence that 5-HT-moduline immunoreactivity is located in cells with the morphological appearance of neurones. Its localization in brain areas which also contain 5-HT1B receptors, is in good agreement with previous demonstrations that this peptide specifically interacts with 5-HT1B receptors to regulate their functional activity and accordingly controls the modulatory activity of the serotoninergic system on various CNS functions.

Characterization of 5-ht6 receptor and expression of 5-ht6 mRNA in the rat brain during ontogenetic development

We have determined the pharmacological characteristics of the rat 5-ht6 receptor stably expressed in CHO cells. Moreover, using RT-PCR experiments the in vivo expression of the gene encoding this receptor was studied in rat at various embryonic days (ED) starting from ED10 to birth (PN0) and at post-natal days (PN) up to PN36. The pharmacological analysis of the [3H]5-HT binding in stably transfected CHO cells expressing rat 5-ht6 receptors revealed the presence of a single class of high affinity saturable binding sites for 5-HT corresponding to an affinity constant: Kd = 27.2±3.4 nM. This receptor also exhibited a high affinity for a number of typical and atypical antipsychotics, tricyclic antidepressant drugs and ergot alkaloïds. In stably transfected CHO cells, serotonin elicited a potent stimulation of adenylyl cyclase activity which was blocked by antipsychotic and antidepressant drugs. These results confirm the hypothesis that 5-ht6 receptors may correspond to an important target for atypical antipsychotics and reveal an original pharmacological profile for this receptor. The study of the ontogeny of the 5-ht6 mRNA in rat developing brain showed that 5-ht6 mRNA were first detectable with a high level on ED12, slighly decreased up to ED17 and then remained stable at high level until the adult age. The ontogenetic pattern of 5-ht6 mRNA expression appeared to correlate with the occurence of the first cell bodies of serotonergic neurons; the early expression of 5-ht6 mRNA and the fact that this receptor is positively coupled to the production of cAMP may suggest a role for 5-ht6 receptor in the early growth process involving the serotonergic system.

5-Hydroxytryptamine-moduline: a novel endogenous peptide involved in the control of anxiety.

The serotonergic system is considered as a neuromodulatory system interacting with other neurotransmissions in the brain and participating in the elaboration of an adapted response of the central nervous system to external stimuli. Indeed, serotonin is involved in a large number of physiological events, such as temperature regulation, sleep, learning and memory, behaviour, sexual function, hormonal secretions and immune activity, and in parallel, it is also implicated in pathological disorders particularly in stress, anxiety, aggressivity and depression. At least 14 different types of serotonin receptors mediate serotonergic activity and among them, serotonin-1B receptors play an important role in the control of the serotonergic function. Serotonin-1B receptors are autoreceptors localized on serotonergic neuron terminals (varicosities) where they inhibit the evoked release of serotonin and its biosynthesis; they are also heteroreceptors located on non-serotonergic terminals, where they inhibit the release of the corresponding neurotransmitters (acetylcholine, GABA, noradrenaline, etc.). 5-Hydroxytryptamine-moduline, an endogenous tetrapeptide (Leu-Ser-Ala-Leu) recently isolated and characterized from rat and bovine brain extracts, was shown to specifically interact with serotonin1B receptors as an allosteric modulator having antagonistic properties in vitro and in vivo. Immuncytochemical studies using specific polyclonal anti-peptide antibodies have shown that this peptide is distributed heterogeneously in mouse brain and located in areas which also contain serotonin-1B receptors. Moreover, the content of these cerebral tissues in 5-hydroxytryptamine-moduline is affected by stress. In the present work, polyclonal anti-5-hydroxytryptamine-moduline antibodies were administered to mice via intracerebroventricular injections to study the in vivo effects of a lowering (or suppression) of this neuropeptide in the central nervous system. The inactivation of the peptide by the specific antibodies significantly modified the behaviour of the animals in two behavioural tests, the open-field and elevated plus-maze, known to be animal models related to anxiety behaviour. Treated mice displayed behaviour consistent with an anxiolytic effect of the antibody, suggesting a potential role of 5-hydroxytryptamine-moduline in the control of anxiety.

Endurance training effects on 5-HT1B receptors mRNA expression in cerebellum, striatum, frontal cortex and hippocampus of rats

The 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. The present study investigated the effects of a 7 week period of physical training on the expression of cerebral 5-HT(1B) receptors by measuring corresponding mRNA levels in rat. Using RNase protection assay technique, we have observed no change in 5-HT(1B) receptor mRNA levels in the striatum and in the hippocampus after moderate as well as after intensive training. In contrast, a significant decrease in 5-HT(1B) receptor mRNA levels was observed in cerebellum of intensively trained rats. Moreover, in frontal cortex, a significant decrease in 5-HT(1B) receptors mRNA level occurred in both groups of trained rats. These data suggest the existence of regional differences in the effect of physical exercise on the expression of 5-HT(1B) receptors.

Acute stress induces a differential increase of 5-HT-moduline (LSAL) tissue content in various rat brain areas.

5-HT-moduline is an endogenous cerebral tetrapeptide (LSAL) which specifically interacts as an allosteric modulator with 5-HT1B receptors controlling serotonergic activity [O. Massot, J.C. Rousselle, M.P. Fillion, B. Grimaldi, I. Cloez-Tayarani, A. Fugelli, N. Prudhomme, L. Seguin, B. Rousseau, M. Plantefol, R. Hen, G. Fillion, 5-Hydroxytryptamine-moduline, a new endogenous cerebral peptide, controls the serotonergic activity via its specific interaction with 5-hydroxytryptamine1B/1D receptors, Mol. Pharmacol. 50 (1996) 752-762; J.C. Rousselle, O. Massot, M. Delepierre, E. Zifa, G. Fillion, Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic1B receptor subtypes, J. Biol. Chem. 271 (1996) 726-735; J.C. Rousselle, M. Plantefol, M.P. Fillion, O. Massot, P.J. Pauwels, G. Fillion, Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells, Naunyn-Schmiedebergs Arch. Pharmacol. 358 (1998) 279-286]. Cerebral tissue contents of 5-HT-moduline were determined in various rat brain areas after an acute restraint stress, and after repetition of this stress, to examine whether or not mechanisms involving this peptide could be affected by stress situations. The measurement of the peptide was carried out using specific polyclonal antibodies [B. Grimaldi, M.P. Fillion, A. Bonnin, J.C. Rousselle, O. Massot, G. Fillion, Immunocytochemical localization of neurons expressing 5-HT-moduline in the mouse brain, Neuropharmacology 36 (1997) 1079-1087] in a dot-ELISA (enzyme-linked-immunosorbent assay) assay in cortex, hippocampus, hypothalamus, substantia nigra, striatum and in adrenal glands. Tissue contents of 5-HT-moduline progressively and transiently increased in most studied brain regions and reached a maximal value 20 min after the beginning of the restraint stress. The increase in 5-HT-moduline tissue contents represented 323% of the value observed in unstressed control animals in the cortex, 207% in the hippocampus, 149% in the hypothalamus and 156% in the substantia nigra. Thereafter, the peptide content of the latter tissues diminished during the last 20 min of restraint and returned to control values within 1 h after the end of the stress period. The striatum did not show any significant variation of 5-HT-moduline content during restraint stress. In adrenal glands, the 5-HT-moduline content rapidly decreased (60% of controls) after the beginning of the restraint stress, the effect of this stress being progressively less pronounced, still representing 80% of controls after 40 min. Repetition of the restraint stress daily for 3 weeks totally abolished the effect of the stress on variations of 5-HT-moduline tissue content in all the studied brain regions. These results show that an acute restraint stress induces a rapid and significant increase in the amount of 5-HT-moduline contained in various brain areas. This phenomenon is likely to be related to the stress-induced 5-HT1B receptor desensitization which was previously demonstrated.

Production and characterization of an antibody directed against the mouse 5HT1B receptor.

We have developed a polyclonal antibody directed against a peptide located in the third intracellular loop of the 5HT1B receptor. Its characterization was carried out using NIH cells stably transfected with a eukaryotic expression vector containing the mouse 5HT1B receptor cDNA. The synthetic antigenic peptide had a unique sequence to the mouse 5HT1B receptor corresponding to amino acids 273-287 localized in the third intracellular loop. In dot blot analysis, antisera detected 2 ng to 2 micrograms of synthetic peptide at dilutions of 1/200-1/20,000 and bound antibody was visualized using an immunoperoxidase procedure. Preimmune serum showed no immunoreactivity to the synthetic peptide. NIH cells stably transfected expressing mouse 5HT1B receptor displayed an intense immunoreactivity with the antiserum. In contrast no immunoreactivity was seen in any of the control experiments. In the present study, we have produced a specific antibody which is an essential tool suitable for immunocytochemical applications such as regional distribution, anatomical localization and phenotypical characterization of the cells expressing the 5HT1B receptors in brain by double immunolabelling procedure.

Application of the polymerase chain reaction to the RNase protection assay for 5-HT1B receptor mRNA levels measurement in rat brain tissues

The RNase protection assay (RPA) is an extremely sensitive procedure for detection of messenger RNA (mRNA) in complex sample mixture of total RNA. However, its usefulness has been limited by the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction (PCR) to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for the 5-hydroxytryptamine(1B) (5-HT(1B)) receptor. Radiolabeled riboprobe was then synthesized using the PCR product as a template and used in RPA to detect mRNA for 5-HT(1B) receptor in rat brain. The internal control was the beta-Actin mRNA. Due to the simplicity of its design and the lack of need for subcloning, the DNA template synthesis by PCR facilitates the implementation of the RPA. Since the 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters and the protocol described here permits the determination of 5-HT(1B) receptor mRNA levels in the rat cerebellum, striatum, hippocampus and frontal cortex, this protocol is helpful in understanding the involvement of 5-HT(1B) receptors in various physiological phenomena.

Interaction of the serotonergic and the immune systems: 5HT-Moduline

The serotonergic system is known as a neuromodulatory system present in the brain and participating to the homeostasis of the brain. Its major morphological and anatomical features are favoring this role. Accordingly it is involved in many physiological functions and a number of psychiatric dysfunctions. Amongst the numerous receptors involved in its functions, 5HT1B receptors constitute a particular subtype located on neuron terminals and regulating the release of the corresponding neurotransmitter. These receptors are specifically the target of an endogenous tetrapeptide (5HT-Moduline) characterized in mammalian brain and regulating the functional activity of the receptor as an allosteric modulator.

5-HT-Moduline

The serotoninergic system (serotonin = 5-hydroxytryptamine = 5-HT) has been implicated in a vast number of physiological and pathological events in vertebrates (12). Surprisingly, serotoninergic activity does not appear essential for any of the physiological processes in which it has been implicated. This characteristic is consistent with the hypothesis that the main role of the 5-HT system is to exert a modulatory control on such physiological functions.