Ethanol-induced epigenetic regulations at the Bdnf gene in C57BL/6J mice.

High ethanol intake is well known to induce both anxiolytic and anxiogenic effects, in correlation with chromatin remodeling in the amygdaloid brain region and deficits in cell proliferation and survival in the hippocampus of rodents. Whether only moderate but chronic ethanol intake in C57BL/6J mice could also have an impact on chromatin remodeling and neuroplasticity was addressed here. Chronic ethanol consumption in a free choice paradigm was found to induce marked changes in the expression of genes implicated in neural development and histone post-translational modifications in the mouse hippocampus. Transcripts encoding neural bHLH activators and those from Bdnf exons II, III and VI were upregulated, whereas those from Bdnf exon VIII and Hdacs were downregulated by ethanol compared with water consumption. These ethanol-induced changes were associated with enrichment in both acetylated H3 at Bdnf promoter PVI and trimethylated H3 at PII and PIII. Conversely, acetylated H3 at PIII and PVIII and trimethylated H3 at PVIII were decreased in ethanol-exposed mice. In parallel, hippocampal brain-derived neurotrophic factor (BDNF) levels and TrkB-mediated neurogenesis in the dentate gyrus were significantly enhanced by ethanol consumption. These results suggest that, in C57BL/6J mice, chronic and moderate ethanol intake produces marked epigenetic changes underlying BDNF overexpression and downstream hippocampal neurogenesis.

Repeated exposure to MDMA triggers long-term plasticity of noradrenergic and serotonergic neurons.

3,4-Methylenedioxymethamphetamine (MDMA or 'ecstasy') is a psychostimulant drug, widely used recreationally among young people in Europe and North America. Although its neurotoxicity has been extensively described, little is known about its ability to strengthen neural circuits when administered in a manner that reproduces human abuse (i.e. repeated exposure to a low dose). C57BL/6J mice were repeatedly injected with MDMA (10 mg kg(-1), intraperitoneally) and studied after a 4-day or a 1-month withdrawal. We show, using in vivo microdialysis and locomotor activity monitoring, that repeated injections of MDMA induce a long-term sensitization of noradrenergic and serotonergic neurons, which correlates with behavioral sensitization. The development of this phenomenon, which lasts for at least 1 month after withdrawal, requires repeated stimulation of α(1B)-adrenergic and 5-hydroxytryptamine (5-HT)(2A) receptors. Moreover, behavioral and neuroendocrine assays indicate that hyper-reactivity of noradrenergic and serotonergic networks is associated with a persistent desensitization of somatodendritic α(2A)-adrenergic and 5-HT1A autoreceptor function. Finally, molecular analysis including radiolabeling, western blot and quantitative reverse transcription-polymerase chain reaction reveals that mice repeatedly treated with MDMA exhibit normal α(2A)-adrenergic and 5-HT(1A) receptor binding, but a long-lasting downregulation of Gαi proteins expression in both locus coeruleus and dorsal raphe nucleus. Altogether, our results show that repeated MDMA exposure causes strong neural and behavioral adaptations and that inhibitory feedback mediated by α(2A)-adrenergic and 5-HT(1A) autoreceptors has an important role in the physiopathology of addictive behaviors.

RNA splicing and editing modulation of 5-HT(2C) receptor function: relevance to anxiety and aggression in VGV mice.

Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

Epigenetic regulation of the BDNF gene: implications for psychiatric disorders.

Abnormal brain-derived neurotrophic factor (BDNF) signaling seems to have a central role in the course and development of various neurological and psychiatric disorders. In addition, positive effects of psychotropic drugs are known to activate BDNF-mediated signaling. Although the BDNF gene has been associated with several diseases, molecular mechanisms other than functional genetic variations can impact on the regulation of BDNF gene expression and lead to disturbed BDNF signaling and associated pathology. Thus, epigenetic modifications, representing key mechanisms by which environmental factors induce enduring changes in gene expression, are suspected to participate in the onset of various psychiatric disorders. More specifically, various environmental factors, particularly when occurring during development, have been claimed to produce long-lasting epigenetic changes at the BDNF gene, thereby affecting availability and function of the BDNF protein. Such stabile imprints on the BDNF gene might explain, at least in part, the delayed efficacy of treatments as well as the high degree of relapses observed in psychiatric disorders. Moreover, BDNF gene has a complex structure displaying differential exon regulation and usage, suggesting a subcellular- and brain region-specific distribution. As such, developing drugs that modify epigenetic regulation at specific BDNF exons represents a promising strategy for the treatment of psychiatric disorders. Here, we present an overview of the current literature on epigenetic modifications at the BDNF locus in psychiatric disorders and related animal models.

Deficiency of the 5-hydroxytryptamine transporter gene leads to cardiac fibrosis and valvulopathy in mice.

BACKGROUND: Serotonin (5-hydroxytryptamine; 5-HT) overproduction is responsible for cardiac valvular disease in patients with carcinoid tumors. Reduced 5-HT inactivation is one proposed mechanism of the valvulopathy observed in individuals treated with the appetite suppressants fenfluramine and phentermine. One key protein limiting systemic availability of 5-HT is the 5-HT transporter (5-HTT) expressed by platelets and pulmonary vascular cells; 5-HTT is responsible for 5-HT uptake and subsequent inactivation of the amine passing through the lung. Here we investigated whether 5-HTT-deficient (5-HTT-KO) mice developed structural and/or functional cardiac abnormalities and valvulopathy. METHODS AND RESULTS: Cardiac endothelial cells expressed large amounts of 5-HTT in wild-type mice. 5-HTT deficiency appeared to be associated with marked interstitial, perivascular, and valvular fibrosis as evidenced by staining of cardiac collagen in 5-HTT-KO mice. Histological analysis provided evidence for valvulopathy characterized by valvular hyperplasia and prominent fibrosis at the attachment site and base of the leaflets. Echocardiography revealed an increase in left ventricular lumen diameter and a decrease in left ventricular diameter fractional shortening. Although 5-HT1B receptors mediated the 5-HT-induced collagen secretion by human cardiac myofibroblasts, the contribution of this receptor type to valvulopathy was ruled out because double-KO mice deficient in both 5-HTT and 5-HT1B receptors showed the same cardiac alterations as 5-HTT-KO mice. CONCLUSIONS: The present results establish a link between 5-HTT and the development of cardiac fibrosis and valvulopathy in vivo. 5-HTT-KO mice represent an especially relevant model for studying the mechanisms by which 5-HT induces valvulopathy.

Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene.

Hypoxia is a well-recognized stimulus for pulmonary blood vessel remodeling and pulmonary hypertension development. One mechanism that may account for these effects is the direct action of hypoxia on the expression of specific genes involved in vascular smooth muscle cell (SMC) proliferation. Previous studies demonstrated that the serotonin (5-hydroxytryptamine; 5-HT) transporter (5-HTT) mediates the mitogenic activity of 5-HT in pulmonary vascular SMCs and is overexpressed during hypoxia. Thus, 5-HT-related mitogenic activity is increased during hypoxia. Here, we report that mice deficient for 5-HTT (5-HTT(-/-)) developed less hypoxic pulmonary hypertension and vascular remodeling than paired 5-HTT(+/+) controls. When maintained under normoxia, 5-HTT(-/-)-mutant mice had normal hemodynamic parameters, low blood 5-HT levels, deficient platelet 5-HT uptake, and unchanged blood levels of 5-hydroxyindoleacetic acid, a metabolite of 5-HT. After exposure to 10% O(2) for 2 or 5 weeks, the number and medial wall thickness of muscular pulmonary vessels were reduced in hypoxic 5-HTT(-/-) mice as compared with wild-type paired controls. Concomitantly, right ventricular systolic pressure was lower and right ventricle hypertrophy less marked in the mutant mice. This occurred despite potentiation of acute hypoxic pulmonary vasoconstriction in the 5-HTT(-/-) mice. These data further support a key role of 5-HTT in hypoxia-induced pulmonary vascular SMC proliferation and pulmonary hypertension.

Adult neurogenesis in serotonin transporter deficient mice.

Serotonin (5-HT) is a regulator of morphogenetic activities during early brain development and neurogenesis, including cell proliferation, migration, differentiation, and synaptogenesis. The 5-HT transporter (5-HTT, SLC6A4) mediates high-affinity reuptake of 5-HT into presynaptic terminals and thereby fine-tunes serotonergic neurotransmission. Inactivation of the 5-HTT gene in mice reduces 5-HT clearance resulting in persistently increased concentrations of synaptic 5-HT. In the present study, we investigated the effects of elevated 5-HT levels on adult neurogenesis in the hippocampus of 5-HTT deficient mice, including stem cell proliferation, survival, and differentiation. Using an in vivo approach, we showed an increase in proliferative capacity of hippocampal adult neural stem cells in aged 5-HTT knockout mice (approximately 14.5 months) compared to wildtype controls. In contrast, in vivo and additional in vitro analyses of younger adult 5-HTT knockout mice (approximately 7 weeks and approximately 3.0 months) did not reveal significant changes in proliferation of neural stem cells or survival of newborn cells. We showed that the cellular fate of newly generated cells in 5-HTT knockout mice is not different with respect to the total number and percentage of neurons or glial cells from wildtype controls. Our findings indicate that elevated synaptic 5-HT concentration throughout early development and later life of 5-HTT deficient mice does not induce adult neurogenesis in adult mice, but that elevated 5-HT levels in aged mice influence stem cell proliferation.

Behavioral and neurochemical characterization of TrkB-dependent mechanisms of agomelatine in glucocorticoid receptor-impaired mice.

Growing evidence indicates that impairment of the stress response, in particular the negative feedback regulation mechanism exerted by the hypothalamo-pituitary-adrenal (HPA) axis, might be responsible for the hippocampal atrophy observed in depressed patients. Antidepressants, possibly through the activation of BDNF signaling, may enhance neuroplasticity and restore normal hippocampal functions. In this context, glucocorticoid receptor-impaired (GR-i) mice-a transgenic mouse model of reduced GR-induced negative feedback regulation of the HPA axis-were used to investigate the role of BDNF/TrkB signaling in the behavioral and neurochemical effects of the new generation antidepressant drug, agomelatine. GR-i mice exhibited marked alterations in depressive-like and anxiety-like behaviors, together with a decreased cell proliferation and altered levels of neuroplastic and epigenetic markers in the hippocampus. GR-i mice and their wild-type littermates were treated for 21 days with vehicle, agomelatine (50mg/kg/day; i.p) or the TrkB inhibitor Ana-12 (0.5mg/kg/day, i.p) alone, or in combination with agomelatine. Chronic treatment with agomelatine resulted in antidepressant-like effects in GR-i mice and reversed the deficit in hippocampal cell proliferation and some of the alterations of mRNA plasticity markers in GR-i mice. Ana-12 blocked the effect of agomelatine on motor activity as well as its ability to restore a normal hippocampal cell proliferation and expression of neurotrophic factors. Altogether, our findings indicate that agomelatine requires TrkB signaling to reverse some of the molecular and behavioral alterations caused by HPA axis impairment.

Functional consequences of 5-HT transporter gene disruption on 5-HT(1a) receptor-mediated regulation of dorsal raphe and hippocampal cell activity.

The consequences of the absence of 5-HT reuptake on the functional properties of 5-HT(1A) receptors were examined in the dorsal raphe nucleus and the hippocampus of knock-out mice lacking the serotonin transporter (5-HTT). Extracellular recordings showed that application of selective 5-HT reuptake inhibitors such as paroxetine and citalopram onto brainstem slices resulted in a concentration-dependent inhibition of 5-HT neuron firing in the dorsal raphe nucleus of wild-type 5-HTT+/+ mice, but not 5-HTT-/- mutants. By contrast, the 5-HT(1A) receptor agonists ipsapirone and 5-carboxamidotryptamine inhibited the discharge in both groups. However, the potency of these agonists was markedly decreased (by approximately 55- and approximately 6-fold, respectively) in 5-HTT-/- compared with 5-HTT+/+ animals. Similarly, intracellular recordings showed that the potency of 5-carboxamidotryptamine to hyperpolarize 5-HT neurons in the dorsal raphe nucleus was significantly lower in 5-HTT-/- than in 5-HTT+/+ animals. These data contrasted with those obtained with hippocampal slices in which 5-carboxamidotryptamine was equipotent to hyperpolarize CA1 pyramidal neurons in both mutant and wild-type mice. As expected from their mediation through 5-HT(1A) receptors, the effects of ipsapirone and 5-carboxamidotryptamine were competitively inhibited by the selective 5-HT(1A) antagonist WAY 100635 in both groups. These data showed that 5-HTT gene knock-out induced a marked desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus without altering postsynaptic 5-HT(1A) receptor functioning in the hippocampus. Similarities between these changes and those evoked by chronic treatment with 5-HT reuptake inhibitors emphasize the existence of regional differences in 5-HT(1A) receptor regulatory mechanisms.

Homeostatic regulation of serotonergic function by the serotonin transporter as revealed by nonviral gene transfer.

With the aim of exploring the relationship between the serotonin transporter (5-HTT or SERT) and the activity level of serotonin (5-HT) neurotransmission, in vivo expression of this protein was specifically altered using a nonviral DNA transfer method. Plasmids containing the entire coding sequence or a partial antisense sequence of the 5-HTT gene were complexed with the cationic polymer polyethylenimine and injected into the dorsal raphe nucleus of adult male rats. Significant increase or decrease in both [(3)H]citalopram binding and [(3)H]5-HT synaptosomal uptake were observed in various brain areas up to 2 weeks after a single administration of the sense plasmid or 7 d after injection of the short antisense plasmid, respectively. Such changes in 5-HTT expression were associated with functional alterations in 5-HT neurotransmission, as shown by the increased capacity of 5-HT(1A) receptor stimulation to enhance [(35)S]GTP-gamma-S binding onto the dorsal raphe nucleus in sections from rats injected with the sense plasmid. Conversely, both a decrease in 5-HT(1A)-mediated [(35)S]GTP-gamma-S binding and a reduced potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit neuronal firing were observed in the dorsal raphe nucleus of antisense plasmid-injected rats. Furthermore, changes in brain 5-HT and/or 5-HIAA levels, and sleep wakefulness circadian rhythm in the latter animals demonstrated that altered expression of 5-HTT by recombinant plasmids has important functional consequences on central 5-HT neurotransmission in adult rats.

5-hydroxytryptamine (5-HT)1A autoreceptor adaptive changes in substance P (neurokinin 1) receptor knock-out mice mimic antidepressant-induced desensitization.

Antagonists at substance P receptors of the neurokinin 1 (NK1) type have been shown to represent a novel class of antidepressant drugs, with comparable clinical efficacy to the selective serotonin (5-HT) reuptake inhibitors (SSRIs). Because 5-HT(1A) receptors may be critically involved in the mechanisms of action of SSRIs, we examined whether these receptors could also be affected in a model of whole-life blockade of NK1 receptors, i.e. knock-out mice lacking the latter receptors (NK1-/-). 5-HT(1A) receptor labeling by the selective antagonist radioligand [(3)H]N-[2-[4-(2-methoxyphenyl)1-piperazinyl]-ethyl]-N-(2-pyridinyl)-cyclohexanecarboxamide (WAY 100635) and 5-HT(1A)-dependent [(35)S]GTP-gamma-S binding at the level of the dorsal raphe nucleus (DRN) in brain sections, as well as the concentration of 5-HT(1A) mRNA in the anterior raphe area were significantly reduced (-19 to -46%) in NK1-/- compared with NK1+/+ mice. Furthermore, a approximately 10-fold decrease in the potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit the discharge of serotoninergic neurons in the dorsal raphe nucleus within brainstem slices, and reduced hypothermic response to 8-OH-DPAT, were noted in NK1-/- versus NK1+/+ mice. On the other hand, cortical 5-HT overflow caused by systemic injection of the SSRI paroxetine was four- to sixfold higher in freely moving NK1-/- mutants than in wild-type NK1+/+ mice. Accordingly, the constitutive lack of NK1 receptors appears to be associated with a downregulation/functional desensitization of 5-HT(1A) autoreceptors resembling that induced by chronic treatment with SSRI antidepressants. Double immunocytochemical labeling experiments suggest that such a heteroregulation of 5-HT(1A) autoreceptors in NK1-/- mutants does not reflect the existence of direct NK1-5-HT(1A) receptor interactions in normal mice.

Brain plasticity and cognitive functions after ethanol consumption in C57BL/6J mice.

Acute or chronic administrations of high doses of ethanol in mice are known to produce severe cognitive deficits linked to hippocampal damage. However, we recently reported that chronic and moderate ethanol intake in C57BL/6J mice induced chromatin remodeling within the Bdnf promoters, leading to both enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal neurogenesis under free-choice protocol. We performed here a series of cellular and behavioral studies to analyze the consequences of these modifications. We showed that a 3-week chronic free-choice ethanol consumption in C57BL/6J mice led to a decrease in DNA methylation of the Bdnf gene within the CA1 and CA3 subfields of the hippocampus, and upregulated hippocampal BDNF signaling pathways mediated by ERK, AKT and CREB. However, this activation did not affect long-term potentiation in the CA1. Conversely, ethanol intake impaired learning and memory capacities analyzed in the contextual fear conditioning test and the novel object recognition task. In addition, ethanol increased behavioral perseveration in the Barnes maze test but did not alter the mouse overall spatial capacities. These data suggested that in conditions of chronic and moderate ethanol intake, the chromatin remodeling leading to BDNF signaling upregulation is probably an adaptive process, engaged via epigenetic regulations, to counteract the cognitive deficits induced by ethanol.

Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors.

Among the recently cloned serotonin (5-hydroxytryptamine, 5-HT) receptors, the 5-HT6 subtype is of special interest for at least two reasons: 1) it is abundant in limbic areas which participate in the control of mood and emotion; and 2) some antidepressants and antipsychotics are potent 5-HT6 receptor antagonists. Studies using polyclonal anti-5-HT6 receptor antibodies and an antisense oligonucleotide were performed in order to investigate further the function(s) of 5-HT6 receptors in the rat brain. Immunocytochemistry at the light and electron microscope levels showed that 5-HT6 receptors are mainly confined to the dendritic compartment, suggesting that they could mediate 5-HT actions on neuronal firing. In some limbic areas, 5-HT6 receptor-like immunoreactivity is also associated with neuronal cilia with yet unknown functions. Continuous i.c.v. infusion with an antisense oligonucleotide for 3-4 days resulted in decreased 5-HT6 receptor-like immunostaining of the nucleus accumbens and anxiogenic behaviours in the social interaction and elevated plus maze tests. Selective 5-HT6 receptor ligands are eagerly expected to investigate further the potential implication of these receptors in limbic-dependent behaviours.

The main features of central 5-HT1 receptors.

The 5-HT1 receptor family comprises five different pharmacologic subtypes, designated 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT1E, whose common property is to bind 5-HT with nanomolar affinity. Recent investigations with molecular biology approaches led to the cloning and sequencing of 5-HT1A receptors in the rat and in the human, and of the 5-HT1C receptor in the rat. Although the 5-HT1A and 5-HT1C protein binding subunits exhibit the same structure with seven hydrophobic transmembrane domains, an extracellular N terminal and an intracellular C tail, their respective amino-acid sequences are markedly different. Indeed, a higher degree of sequence homology is found between the 5-HT1C and 5-HT2 receptors than between the former and 5-HT1A receptors, suggesting that the 5-HT1C subtype in fact belongs to the 5-HT2 class of central 5-HT receptors. All other 5-HT1 receptor subtypes are negatively coupled to adenylyl cyclase, whereas the 5-HT1C subtype, like 5-HT2 receptors, is positively coupled to phospholipase C. The respective regional distributions and regulatory properties, as well as pending questions regarding the ultrastructural localization, synthesis, mutual interactions, and axonal flow of 5-HT1 receptor subtypes, are also discussed.

Glucocorticoid receptor-mediated inhibition by corticosterone of 5-HT1A autoreceptor functioning in the rat dorsal raphe nucleus.

In the rat brain, the dorsal raphe nucleus contains a large proportion of serotoninergic neurons, which are mostly regulated by somato-dendritic 5-HT1A autoreceptors. This nucleus also possesses intracellular glucocorticoid receptors (GR), which may be involved in the well established modulation of serotonin (5-hydroxytryptamine, 5-HT) metabolism by glucocorticoids. Control by corticosteroids of 5-HT1A receptor-mediated inhibitory control of the firing of serotoninergic neurons in the dorsal raphe nucleus was investigated using an in vitro electrophysiological approach. The spontaneous firing rate of serotoninergic neurons recorded in brain stem slices and its inhibition due to 5-HT1A autoreceptor stimulation by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) were similar in adrenalectomized rats and sham-operated animals. In vitro pretreatment with corticosterone (30-100 nM) significantly reduced 8-OH-DPAT-induced inhibition of the 5-HT cell discharge in slices from adrenalectomized rats. This effect could be prevented by the GR antagonist, 11 beta-(4-dimethyl-amino-phenyl)- 17 beta-hydroxy-17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one (RU) 38486, 30 nM), and mimicked by the GR agonist, 11 beta, 17 beta-dihydroxy-6-methyl-17 alpha (prop-1-ynyl) androsta-1,4,6-trien-3-one (RU 28362, 500 nM). In contrast, the mineralocorticoid receptor (MR) agonist, aldosterone (10 nM), did not alter 8-OH-DPAT-induced inhibition in tissues from adrenalectomized animals. Complementary autoradiographic experiments showed that [3H]8-OH-DPAT specific binding to 5-HT1A sites in the dorsal raphe nucleus (and the hippocampus) was not significantly altered following adrenalectomy and exposure of brain stem slices to corticosterone. These data suggest that GR are involved in the suppressive effects of high levels of corticosterone on the 5-HT1A receptor-dependent regulation of 5-HT neuronal activity in the rat dorsal raphe nucleus.

Regulation of noradrenergic coerulean neuronal firing mediated by 5-HT2 receptors: involvement of the prepositus hypoglossal nucleus.

Previous studies have indicated a 5-HT2-mediated inhibitory influence on unit activity in the locus coeruleus. In the present work, attempts were made to determine which area(s) of the brain is (are) involved in this effect: (1) Microiontophoretic application of serotoninergic compounds (quipazine, ketanserin, RU 24969 (Roussel Uclaf), 8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT), metergoline, serotonin) in the locus coeruleus, did not alter the coerulean discharge. Local microinjection of quipazine or ketanserin in the area of the locus coeruleus, as well as in one of its major afferents, the prepositus hypoglossi, had no effect on the unit activity in the locus coeruleus. (2) Section of the forebrain, caudal to the frontal cortex (rich in 5-HT2 receptors), did not modify the effects of coerulean activity of quipazine-ketanserin injected systemically: quipazine induced an inhibition which was reversed by ketanserin. In contrast, these effects were significantly reduced after the bilateral or contralateral lesion of the prepositus hypoglossi. It is concluded that the prepositus hypoglossal nucleus is part of the network responsible for the 5-HT2-mediated control of unit activity in the locus coeruleus.

Central action of 5-HT3 receptor ligands in the regulation of sleep-wakefulness and raphe neuronal activity in the rat.

Anxiolytic drugs, such as the benzodiazepines and the azapirones (ipsapirone, gepirone, buspirone), are well known to affect states of vigilance and to decrease the firing rate of serotoninergic neurones within the dorsal raphe nucleus in rats. In order to examine whether the newly developed 5-HT3 antagonists with potential anxiolytic properties act through similar mechanisms, the effects of several of such antagonists: MDL 72222, ICS 205-930, ondansetron and/or zacopride on both sleep-wakefulness and the discharge of serotoninergic neurones within the dorsal raphe nucleus were investigated in rats. When tested in a wide range of doses (0.05-10 mg/kg, i.p.), none of these drugs significantly affected the states of vigilance, except ondansetron, at 0.1 mg/kg, which increased paradoxical sleep for the first 2 hr after administration and MDL 72222, at 10 mg/kg, which reduced both paradoxical and slow wave sleep and increased wakefulness for the same initial period after treatment. In vivo, in chloral hydrate anaesthetized rats, as well as in vitro, in slices of brain stem, none of the 5-HT3 antagonists tested affected the firing rate of serotoninergic neurones. Similarly, no change in the electrical activity of serotoninergic neurones could be evoked in vitro by superfusion of the tissue with the 5-HT3 agonists, phenylbiguanide (10 microM) and 2-methyl-5-HT (1 microM). At a larger concentration (10 microM), the latter compound reduced the neuronal discharge probably through the stimulation of somatodendritic 5-HT1A autoreceptors since this effect, as that of ipsapirone, could be prevented by 10 microM l-propranolol. Comparison of these data with those obtained with benzodiazepines and 5-HT1A agonists of the azapirone series, supports the concept that different mechanisms are responsible for the anxiolytic-like properties of 5-HT3 agonists, compared to those of other anxiolytic drugs.

Chronic treatment with imipramine reverses immobility behaviour, hippocampal corticosteroid receptors and cortical 5-HT(1A) receptor mRNA in prenatally stressed rats.

Prenatal stress in the rat induces enhanced reactivity of the hypothalamus-pituitary-adrenal (HPA) axis, disturbances in a variety of circadian rhythms and increased anxiety-like behaviour. Such abnormalities parallel those found in human depressed patients. Prenatally stressed (PS) rats could represent, therefore, an interesting animal model for the evaluation of the efficacy of pharmacotherapeutic intervention in psychiatric disorders that has often been addressed using control animals. In the present study, PS and non-stressed rats were chronically treated with the tricyclic antidepressant imipramine (10 mg/kg i.p. for 21 days) and assessed in the forced swim test. Glucocorticoid receptor binding sites in the hippocampus were measured and 5-HT(1A) receptor mRNA levels in the frontal cortex were also assessed. PS rats were characterised by increased immobility in the forced swim test, reduced hippocampal corticosteroid receptor binding and increased levels of cortical 5-HT(1A) mRNA. All these parameters were significantly reversed by chronic imipramine treatment. Conversely, no significant effects were observed for non-stressed rats. All these effects are consistent with the expected pharmacotherapy of depression-like abnormalities in PS rats. These results further indicate that PS rats are a relevant animal model of depression.

Differential adaptation of brain 5-HT1A and 5-HT1B receptors and 5-HT transporter in rats treated chronically with fluoxetine.

Quantification of receptor binding sites and their encoding mRNAs, and electrophysiological recordings, were used to assess central serotonin (5-HT) neurotransmission in rats 24 h after a 2-3 week treatment with the selective 5-HT reuptake inhibitor fluoxetine (8 mg/kg i.p., daily). Binding studies showed that this treatment affected neither 5-HT1A nor 5-HT1B binding sites in all brain areas examined. However, a significant decrease (-38%) in 5-HT1A mRNA levels in the anterior raphe area (but not forebrain regions) and increases in 5-HT1B mRNA levels in the striatum (+127%) and the cerebral cortex (+34%) were noted in fluoxetine-treated rats. Electrophysiological recordings in brain slices showed that chronic fluoxetine treatment reduced the potency of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin to inhibit neuronal activity in the dorsal raphe nucleus, but did not affect 5-HT1A-evoked responses of CA1 pyramidal cells in the hippocampus. These data further demonstrate that fluoxetine-induced adaptive changes in 5-HT neurotransmission exhibit marked regional differences. The decrease in 5-HT1A mRNA levels in the anterior raphe suggests that fluoxetine-induced desensitization of 5-HT1A autoreceptors involves changes at the transcription level.

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644.

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)