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Biochemical and electrophysiological approaches were used to assess possible changes in 5-HT 1A receptors in the rat brain after long-term treatment with an anxiolytic benzodiazepine. Rats were treated with diazepam (2 mg/kg i.p. daily) during 14 days and then untreated for 1 day (protocol A) or 5 days (protocol C) until they were killed for in vitro investigations on 5-HT 1A receptors. In addition, other rats (protocol B) received the same 14-day treatment with diazepam, followed by 1 mg/kg of the drug on days 15 and 16, and 0.5 mg/kg on days 17 and 18, and were killed 24 h after the last injection. In vitro binding and quantitative autoradiographic experiments with [ 3 H]8-hydroxy-2-(di- n -propylamino)tetralin ([ 3 H]8-OH-DPAT) showed that the characteristics of 5-HT 1A receptor binding sites in the hippocampus and the dorsal raphe nucleus were not significantly altered by the administration of diazepam under the treatment protocols A, B and C. Furthermore, in vitro electrophysiological recordings of serotoninergic neurons in the dorsal raphe nucleus of brain stem slices revealed no modification in the sensitivity of somatodendritic 5-HT 1A autoreceptors in rats treated with diazepam according to the protocols A and B. However, under the conditions of protocol C, the potency of 8-OH-DPAT to depress the firing rate of serotoninergic neurons was significantly enhanced, as expected of a hypersensitivity of somatodendritic 5-HT 1A autoreceptors. These data support the hypothesis that some functional changes in these receptors could occur during benzodiazepine withdrawal. However, they do not support the idea of a reduced anxiolytic efficacy of 5-HT 1A receptor agonists as a result of prior treatment with a benzodiazepine. © 1997 Elsevier Science B.V. All rights reserved.

Somatodendritic 5-HT1A autoreceptors play a key role in the control of the electrical and metabolic activity of serotoninergic neurons in the dorsal raphe nucleus. These neurons also possess intracellular glucocorticoid receptors which may be involved in the well established modulation of serotonin (5-hydroxytryptamine, 5-HT) metabolism by corticosterone in stressed animals. The possible mediation by somatodendritic 5-HT1A autoreceptors of such corticosterone-dependent changes in serotoninergic neuron activity was investigated using an in vitro electrophysiological approach. 5-HT1A autoreceptor-mediated inhibition of the firing of serotoninergic neurons was examined in brain stem slices from rats whose serum corticosterone concentrations had been markedly increased (+100-200%) by two different stressful conditions. Immobilization for 30 or 90 min (restraint stress) did not modify the concentration-dependent inhibition of the firing of serotoninergic neurons by the 5-HT1A receptor agonist ipsapirone. In contrast, placing the rats in novel uncontrolled environmental conditions for 16 h significantly reduced the cell response to ipsapirone, indicating a decreased sensitivity of somatodendritic 5-HT1A autoreceptors. Such a change was not observed in adrenalectomized rats subjected to the same stressful conditions. These data show that some forms of stress can reduce the 5-HT1A autoreceptor-dependent inhibitory control of the electrophysiological activity of serotoninergic neurons in the dorsal raphe nucleus. Both the nature and duration of stress seem to be critical factors for triggering the (corticosterone-dependent) mechanism(s) responsible for the functional desensitization of 5-HT1A autoreceptors in stressed rats.

Electrophysiological and autoradiographic approaches were used to assess possible changes in 5-hydroxytryptamine (serotonin) 5-HT1A receptors in the rat dorsal raphe nucleus after a subchronic treatment with fluoxetine or paroxetine, two specific serotonin reuptake inhibitors with antidepressant properties. Fluoxetine or paroxetine were injected daily (5 mg/kg, i.p.) for various time periods up to 21 days. Electrophysiological recordings performed 24 h after the last injection showed that the potency of the 5-HT1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus within brain stem slices was significantly reduced as early as after a 3-day treatment with either drug. The proportion of recorded neurons showing desensitization of somatodendritic 5-HT1A autoreceptors increased along the treatment from approximately 40% on the 3rd day to 60-80% on the 21st day. At no time during the treatment, was the specific binding of [3H]8-OH-DPAT (agonist radioligand) or [3H]WAY-100 635 (antagonist radioligand) to 5-HT1A receptors modified in the dorsal raphe nucleus or in other brain areas, suggesting that neither the density nor the coupling of these receptors to G-proteins were probably altered in rats injected with fluoxetine or paroxetine for up to 21 days. These results show that adaptive desensitization of somatodendritic 5-HT1A autoreceptors within the dorsal raphe nucleus can already be detected after a 3-day treatment with selective serotonin reuptake inhibitors. Rather than the desensitization per se, it may be the progressive increase in the number of serotoninergic neurons with desensitized 5-HT1A autoreceptors which plays a critical role in the (slowly developing) antidepressant action of these drugs.

In vitro biochemical and electrophysiological methods were used to assess the potential antagonist properties of the novel compounds (+)-WAY 100 135 [N-tert-butyl-3,4-(2-methoxyphenyl)piperazin-1-yl-2-phenylpropanamide dihydrochloride] and SDZ 216–525 [methyl 4-(4-(4-(1,1,3-trioxo-2H-1,2-benziosothiazol-2-yl)butyl)-1-piperazinyl)1H-indole-2-carboxylate] at pre- (i.e. somatodendritic autoreceptors) and postsynaptic 5-HT 1A receptors in the rat brain. Both (+)-WAY 100 135 and SDZ 216–525 were pure antagonists at postsynaptic 5-HT 1A receptors negatively coupled to adenylate cyclase in rat hippocampal membranes. Competitive prevention of the inhibition by the 5-HT 1A receptor agonists 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin], 5-HT (5-hydroxytryptamine), S-20499 [(+)-4-(N-(5-methoxychroman-3-yl)-N-propylamino)butyl-8-azaspiro(4,5)decane-7,9-dione] and lesopitron occurred with a pA 2 of 8.7 for (+)-WAY 100 135 and 9.9 for SDZ 216–525. The higher potency of the latter compound was also noted at the level of presynaptic 5-HT 1A receptors where both (+)-WAY 100 135 and SDZ 216–525 prevented the negative influence of 5-HT 1A receptor agonists (8-OH-DPAT, flesinoxan or lesopitron) on the nerve impulse flow within dorsal raphe nucleus 5-HT neurones in brain stem slices. At high concentrations, both (+)-WAY 100 135 (> 1 μ M) and SDZ 216–525 (≥ 0.1 μ M) inhibited the spontaneous cell discharge through different mechanisms. The blockade of α 1 -adrenoceptors by (+)-WAY 100 135 apparently accounted for its inhibitory influence on the firing of 5-HT neurones, whereas 5-HT 1A receptor agonist properties were responsible for the effect of SDZ 216–525. Although ∼ 10 times less potent than SDZ 216–525, (+)-WAY 100 135 is therefore a pure antagonist at both pre- and postsynaptic 5-HT 1A receptors in the rat brain.

Objectives5-HT2C receptors are well known to be involved in anxiety, but their implication in stress-induced changes of 5-HT transmission remained to be investigated. We thus assess the behavioral and neurochemical effects of 5-HT2C receptor activation in naïve and stressed mice, and after chronic paroxetine known to exert anxiolytic effects in humans.Methods and resultsThe effects of the preferential 5-HT2C agonists m-chlorophenylpiperazine (mCPP) and RO60-0175, the selective 5-HT2C receptor antagonist SB242,084 and restraint-stress on anxiety-like behavior in mice were assessed using the social interaction test, while the neurochemical effects of these treatments on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT were determined using HPLC and microdialysis. Both mCPP and restraint-stress increased anxiety-like behavior in the social interaction test, and these effects were blocked by pretreatment with SB242,084. Restraint-stress increased 5-HT turnover in various brain areas, and this effect could be prevented by the 5-HT2C receptor agonist RO60-0175. Acute administration of SB242,084 potentiated the stress-induced increase in 5-HT turnover and blocked the inhibitory effect of RO60-0175. Microdialysis studies in frontal cortex revealed that RO60-0175 has an inhibitory effect on the stress-induced increase in extracellular 5-HT levels, but not on basal 5-HT levels. Chronic paroxetine prevented the anxiogenic effect of mCPP and prevented the inhibitory effect of RO60-0175 on restraint-stress-induced increase in 5-HT turnover.ConclusionsThese data strongly suggest that 5-HT2C receptor activation mediates the anxiogenic effect of stress. In addition, the anxiolytic action of long term treatment with SSRIs might be causally related to a clear-cut 5-HT2C receptor desensitization.

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.

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. 1. (2) Section of the forebrain, caudal to the frontal cortex (rich in 5-HT2 receptors), did not modify the effects on 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.

Quantitative autoradiographic studies indicated that the potency of 10 different 5-HT receptor ligands to displace [ 3 H]8-OH-DPAT specifically bound to 5-HT 1A receptors was not significantly different in the dorsal raphe nucleus and the hippocampus within brain sections. In addition specific anti-rat 5-HT 1A receptor antibodies recognized 5-HT 1A receptors in all brain areas where they have been identified with radioligands. These data strongly support that the presynaptic 5-HT 1A autoreceptors in the dorsal raphe nucleus are pharmacologically and immunologically identical with the postsynaptic 5-HT 1A receptors in limbic regions. However functional differences exist between pre-and postsynaptic 5-HT 1A receptors as only the presynaptic 5-HT 1A autoreceptors appeared to be responsible for the negative effect of 5-HT 1A agonists on the metabolic activity of central serotoninergic neurones.

The stimulation of somatodendritic 5-HT 1A autoreceptors by in vitro superfusion with ipsapirone induced a marked inhibition of the spontaneous firing of serotonergic cells within the dorsal raphe nucleus in a slice preparation. By contrast, ipsapirone exerted no effect on the in vitro 5-HT neuronal discharge after the in vivo local injection of pertussis toxin into the dorsal raphe nucleus. In addition, tissue superfusion with the potassium channel blocker 4-aminopyridine also prevented the inhibitory influence of ipsapirone on the 5-HT neuronal discharge. These data indicate that the somatodendritic 5-HT 1A autoreceptor controls a 4-aminopyridine-sensitive K + conductance via an interaction of the 5-HT 1A binding subunit with a pertussis toxin-sensitive Gi/Go protein. Adaptive regulatory mechanisms control the functional 5-HT 1A autoreceptor complex because long term in vivo treatment with ipsapirone markedly reduced the efficiency of 5-HT 1A agonists to inhibit the in vitro 5-HT neuronal discharge.

Extracellular recordings were made from serotonergic neurons of the rat dorsal raphe nucleus in a slice preparation. In the presence of phenylephrine (3 microM) to restore the pacemaker activity of otherwise silent serotonergic neurons, superfusion with the 5-hydroxytryptamine1A agonist ipsapirone depressed the firing of these neurons with an IC50 of approximately 50 nM. Complete inhibition was achieved with 100-300 nM of the drug. Concomitant superfusion with the 5-hydroxytryptamine1A antagonists spiperone (100 nM) or propranolol (10 microM) markedly reduced the inhibitory effect of ipsapirone (100 nM). Superfusion with K+ channel blockers such as apamin (50-100 nM), charybdotoxin (100 nM) or Ba2+ (1 mM) did not induce any changes in the electrical activity of serotonergic neurons. However, 4-aminopyridine (0.1-1 mM) disrupted the regularity of their discharge without affecting the mean firing rate. The ipsapirone-induced inhibition was unchanged by apamin and charybdotoxin, but was markedly reduced by Ba2+ and 4-aminopyridine. Thus the IC50 of ipsapirone was shifted to approximately 150 nM in the presence of 1 mM of 4-aminopyridine. These results indicate that, in serotonergic neurons within the dorsal raphe nucleus, the K+ channel opened through the stimulation of 5-hydroxytryptamine1A autoreceptors is 4-aminopyridine-sensitive.

Because cannabinoid (CB) and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether CB1 and 5-HT1A receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6J and DBA/2J, with marked differences in native alcohol preference. When offered progressively (3% to 10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (~70%) in DBA/2J than in C57BL/6J mice. In DBA/2J mice, chronic treatment with the CB receptor agonist WIN 55,212-2 increased alcohol intake. This effect was prevented by both chronic CB1 receptor blockade by rimonabant or chronic 5-HT1A receptor stimulation by 8-OH-DPAT, which, on their own, did not affect alcohol intake. In C57BL/6J mice, WIN 55,212-2 had no effect but CB1 receptor blockade or 5-HT1A receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN55,212-2 significantly decreased 5-HT1A-mediated [35S]GTP-γ-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6J mice.These results show that CB neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT1A interactions. However, the differences between C57BL/6J and DBA/2J mice indicate that such modulations of alcohol intake are under genetic control.

Unit activity of raphe dorsalis neurons was recorded in rats between 3 and 24 days of age. Both a regular discharge pattern and a slow firing rate were observed as early as day 3. The frequency of discharge was not statistically different at any age from the mean discharge rate in the adult rat. It was concluded that the electrophysiological activity of 5-HT neurons develops early in the process of maturation, prior to that of the biochemistry and anatomy of these neurons.

1. Intracellular recordings were made from rat striatal neurones in vitro. The cells had resting membrane potentials greater than -60 mV and action potentials greater than 70 mV with spike overshoot of 10-30 mV. 2. In the presence of bicuculline intrastriatal stimulation evoked an excitatory postsynaptic potential (EPSP). The relationship between EPSP amplitude and membrane potential was not linear. The EPSP decreased in amplitude and duration for values of membrane potential more negative than -80 mV and increased in amplitude and duration for values of membrane potential more positive than -50 mV. 3. The mean reversal potential for the EPSP recorded with electrodes filled with potassium methyl-sulphate was -9.2 +/- 1.7 mV (mean +/- S.E.M.) in presence of bicuculline (30 microM). A similar reversal potential was obtained with CsCl-filled electrodes. 4. The endogenous broad-spectrum excitatory amino acid antagonist, kynurenic acid (100-500 microM), reduced the EPSP in a dose-dependent way, maximally by 80% at 500 microM, but a residual depolarization remained even at high antagonist concentrations. This effect was associated sometimes with a membrane depolarization and an increase in input resistance. 5. In normal artificial cerebro-spinal fluid solution and at resting membrane potential the specific N-methyl-D-aspartate (NMDA) antagonist, (D,L)-2-amino-7-phosphonoheptanoic acid (([D,L)-AP7), did not affect the EPSP amplitude. However, this antagonist partially reduced the EPSP amplitude when the membrane was depolarized beyond -50 mV by intracellular current injection. 6. The nicotinic cholinergic antagonist mecamylamine (10 microM) caused a partial (24 +/- 3%) reduction of EPSP amplitude at resting potential in normal medium. However, in the cells where a reduction of EPSP amplitude was observed it was always accompanied by membrane depolarization (7.1 +/- 2.1 mV). (+)-Tubocurarine and hexamethonium were without effect at 10 microM. 7. When Mg2+ was removed from the bathing solution, the EPSP increased in amplitude (89 +/- 9.5%) and duration. In Mg2+-free medium at resting membrane potential (D,L)-AP7 (30 microM) partially reduced EPSP amplitudes (59 +/- 2.5%). 8. It is proposed that a major component of the EPSP evoked by intrastriatal stimulation is mediated by excitatory amino acids. At resting membrane potential and in normal medium only non-NMDA receptors seem to contribute to the synaptic depolarization, but at depolarized potentials and in Mg2+-free medium an NMDA receptor-mediated component of the EPSP can be demonstrated.

Three-day-old rats received an infusion of a small dose of 6-hydroxydopamine (6-OHDA) (12 μg) directly into the locus ceruleus nuclei, in order to induce a subtotal lesion of the noradrenergic system. Significant modifications in cerebellum maturation were observed, mainly a regression failure in the perisomatic Purkinje cell processes in the depth of the fissura prima of 10-day-old rats, a reduction in the whole vermis surface area and in the total number of internal granule cells at 15 days of age, the number of external granule cells was slightly enhanced. To discriminate between the effects of the noradrenergic deafferentation and the nonspecific toxic effects of 6-OHDA, these results were compared to those obtained after an infusion of 6-OHDA + bovine serum albumin (BSA) into the locus ceruleus (which produced a selective deafferentation) and after an injection of 6-OHDA directly into the IVth ventricle (which increased the nonspecific toxic effects). Whatever the procedure used, the same reduction in the size of the vermis was observed. However, the fissuration index was not modified when BSA was added to the 6-OHDA solution. We conclude that noradrenergic deafferentation by itself was sufficient to reduce the growth of the cerebellum at certain periods of postnatal life.

The relationship between the spontaneous unit activity in the raphe dorsalis (RD), and the sleep-wakefulness cycles, was analyzed in the cat from birth to 40 days of age. Electrodes for polygraphic sleep monitoring were implanted under anesthesia, and unit recordings were obtained from bundles of microwires positioned in the RD area in kittens of different ages. Attention was paid only to units with slow firing in wakefulness (W) (1–6 spikes/s), and two types of discharge patterns during this state were obtained: a ‘regular’ type, whose discharge in W had the same characterisitcs of regularity as those described for the adult under the same conditions, was always found inside the RD. An ‘irregular’ type was always found in sites outside the RD. Injections of different doses of 5-methoxy-N, N-dimethyltryptamine (5-MeODMT i.m.) induced a transient decrease in the firing rate of the regular type of cells, and no change for the units of the irregular type, suggesting that the regular neurons were of serotoninergic nature. Whereas the cells of the irregular type exhibited an increase of discharge frequency in active or paradoxical sleep (AS-PS), those which fired in a clock-like manner during W exhibited a rate of discharge which progressively decreased in quiet or slow wave sleep (QS-SWS) and even more in AS-PS. Such a pattern was qualitatively close to the adult one at all ages, but the discharge rate in AS was significantly higher during the first and second weeks of life than later on. The observation that these serotoninergic neurons exhibited at birth an adult-like pattern of discharge during W, indicates that during ontogenesis there was no direct relationship between the RD activity and the behavioral output. It is proposed that the RD neuronal discharge would be largely under genetic influences, and that the maturation of sleep regulations at the brainstem and mesencephalic levels is achieved only after the second week of postnatal age.

In the present report we analyzed the long-term effects of neonatal noradrenergic denervation on the regulation of paradoxical sleep in the rat. The locus coeruleus was destroyed bilaterally at 4 days of age by direct infusion of 6-hydroxydopamine into the nuclei. After they reached adulthood, the rats received either i.p. injections of a β-blocker, propranolol, or the same treatment combined with intraventricular infusion of a β-agonist, isoproterenol. Several doses of each drug were tested. The effects of propranolol, alone and together with isoproterenol, on paradoxical sleep (a decrease for the former drug and a restoration for the latter combination) were significantly more pronounced in the lesioned group than in age-paired controls. These data illustrate a functional aspect of the denervation supersensitivity phenomenon in β-receptors.

Intracerebral injections of 6-hydroxydopamine directly in the nucleus locus coeruleus were performed in 4-day-old rats. The effects of such treatment on the subsequent development of this nucleus were analyzed and compared to those obtained in vehicle-injected age-paired controls. The 3 histological techniques used (Nissl, Glenner reaction, and histochemical fluorescence) indicated in the experimental group a progressive disappearance of the coerulean perikarya within 7 days postinjection. This lesion was permanent and did not induce any noradrenergic sprouting.

The influences of beta adrenergic transmission on the regulation of paradoxical sleep (PS) were analysed in the rat. Pharmacological experiments have used beta 1,2 as well as preferential beta 1 or beta 2 compounds. The blockade of the beta 1 adrenoceptors induced a dose-related PS decrease, and stimulation of these preblocked receptors prevented this PS insomnia. SWS was generally not affected. It is concluded that beta 1 neurotransmission is directly involved in the regulation of PS.

Spontaneous activity of 5-hydroxytryptamine (5-HT) neurons in the dorsal raphe nucleus (DRN) was recorded in adult rats that had undergone a bilateral locus coeruleus (LC) lesion during the neonatal period. The susceptibility of this neuronal firing to beta-adrenergic manipulation was tested. Microiontophoretic application of the beta-blockers d,l-propranolol and acebutolol inhibited the firing of DRN cells in lesioned rats but not in control animals. This effect was specific to beta-receptors since the effects of pharmacological manipulation of other receptors--5-HT, gamma-aminobutyric acid (GABA), alpha-adrenoceptors--were identical in lesioned and control animals. The present data demonstrate that a neonatal noradrenergic lesion allowed the persistence of a beta-regulation of DRN neuronal firing, which in young rats is normally only transient.

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 μM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 μM) 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 μM). The calcium potentials were enhanced by the calcium ‘agonist’ BAY K 8644 (1–5 μM). 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. The current was still inward at the end of the command step and often was followed by an inward tail current. The current/voltage curve was N-shaped with a region of negative slope conductance between −25 and −10 mV. The inward current was larger in the presence of barium (1–3 mM) and tetraethylammonium (5 mM). It was abolished by the calcium antagonists cobalt (2 mM) and cadmium (500 μM) and enhanced by the calcium ‘agonist’ BAY K 8644 (5 μM). In conclusion, like many other central neurons, striatal neurons bear calcium conductances that underlie the calcium potentials observed in current clamp experiments.

Extracellular recordings from raphe dorsalis neurons in rats during development were performed, and the response to iontophoretically applied beta-antagonists was analysed. In pups younger than 2 weeks, raphe dorsalis firing was totally or partially inhibited by application of beta-antagonists, while after 2 weeks of age the neurons were insensitive to the same application. The beta-receptors appeared to be involved in the control of raphe firing during a transient period of ontogenesis and thus the disappearance of beta-regulation at older ages may reflect the regression of this population of neurotransmitter receptors.

The localization of adrenergic receptors in the central nervous system was studied in two physiological conditions of noradrenergic denervation, a 6-hydroxydopamine-induced lesion of the locus coeruleus in newborn rat, and a pathological related degeneration of the locus coeruleus in man, Parkinson's disease. The localization of these receptors in the synapse has been studied with the technique of subcellular fractionation by differential centrifugation. In lesioned rats, an increase in the density of alpha 1 and beta 1 receptors was observed in several brain regions, in contrast to alpha 2 receptors which were not modified. Subcellular fractionation in lesioned rats showed an increase in alpha 1 and beta 1 receptors in synaptosomal fractions. Similar results were found in parkinsonian patients: alpha 1 receptors increased in the synaptosomal fraction; beta receptors increased in synaptosomal and microsomal fractions. These results suggest that alpha 1 and beta 1 receptors may be located on non-noradrenergic nerve terminals in mammalian brain. alpha 2 and beta 2 receptors may be situated on glial cells or neuronal elements unrelated to noradrenergic input.

The potential 5-HT1A antagonist properties of the beta-antagonist tertatolol were assessed using biochemical and electrophysiological assays in the rat. (+/-) Tertatolol bound with high affinity (Ki = 38 nM) to 5-HT1A sites labelled by [3H]8-OH-DPAT in hippocampal membranes. The (-)stereoisomer (Ki = 18 nM) was about 50-fold more potent than the (+)stereoisomer (Ki = 864 nM) to inhibit the specific binding of [3H]-8-OH-DPAT. As expected of a 5-HT1A antagonist, (-)tertatolol prevented in a concentration-dependent manner (Ki = 24 nM) the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal homogenates. Furthermore in vivo pretreatment with (-)tertatolol (5 mg/kg s.c.) significantly reduced the inhibitory influence of 8-OH-DPAT (0.3 mg/kg s.c.) on the accumulation of 5-hydroxytryptophan in various brain areas after the blockade of aromatic L-amino acid decarboxylase by NSD-1015 (100 mg/kg i.p.). In vitro (in brainstem slices; Ki approximately 50 nM) and in vivo (in chloral hydrate anaesthetized rats; ID50 approximately 0.40 mg/kg i.v.), (-)tertatolol prevented the inhibitory effects of the 5-HT1A receptor agonists 8-OH-DPAT, ipsapirone and lesopitron on the firing rate of serotoninergic neurones within the dorsal raphe nucleus. In about 25% of these neurones, the basal firing rate was significantly increased by (-)tertatolol (up to +47% in vitro, and +30% in vivo). These data indicate that (-)tertatolol is a potent competitive antagonist at both pre (in the dorsal raphe nucleus)-and post (in the hippocampus)-synaptic 5-HT1A receptors in the rat brain.

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB1 cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB1 knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB1 knockout mice. The lack of CB1 receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB1 receptor induced a 5-HT1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT2C receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB1 receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice., This study has been supported by grants from the UE Sixth Framework Programme (NEWMOOD, LSHM-CT-2004-503474 to RM and LL), Spanish Ministry of Education & Science (SAF 2007/60249 to OV, BES- 2005-8265 fellowship to EA) and Spanish Ministry of Health (ISCIII-RD/06/0001/1001/ and PNSD Conv-2006 to OV).

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.

Chronic and excessive ethanol consumption triggers neurobiological adaptations within the central nervous system, which are responsible for the development of an addiction. Ethanol induces adaptive mechanisms linked to epigenetic regulations leading to functional and structural changes, and also provokes a neurodegeneration responsible for the cognitive deficits observed in alcohol abusers. Among the different animal models available for studying the effects of chronic ethanol consumption, C57BL/6J mice are among the most relevant. These mice display high ethanol preference, making them a good model for studying the consequences of chronic and free-choice ethanol consumption. The purpose of this work was to study the links between epigenetic mechanisms and hippocampal neuroplasticity and to evaluate the behavioural consequences induced by chronic ethanol consumption in C57BL/6J mice. We showed that, in the hippocampus, chronic ethanol consumption induced global epigenetic modulations that were correlated with chromatin remodelling at the BDNF gene level. These effects involved post-translational histone modifications and DNA methylation. Epigenetic changes at the BDNF gene level probably allowed the increase in BDNF protein expression observed within the hippocampal dentate gyrus in mice having consumed ethanol for 3 weeks. Upregulation of BDNF expression was linked to both the stimulation of intracellular cascades downstream BDNF/TrkB receptor activation, and the increase in neurogenesis within the dentate gyrus. Using a specific TrkB receptor antagonist, ANA-12, we demonstrated that the hippocampal neurogenesis induced by chronic ethanol intake was under the control of BDNF. Behavioural analysis evidenced learning and memory impairments after ethanol consumption without synaptic plasticity alteration within the hippocampus, suggesting the involvement of other mechanisms in the cognitive deficits. Altogether, these data bring new elements for understanding the hippocampal neurogenesis stimulation observed under chronic and voluntary ethanol consumption in C57BL/6J mice. Moreover, this apparent increase in plasticity might probably be considered as an adaptive and compensatory mechanism in response to the cognitive deficits induced by ethanol consumption., La consommation chronique et excessive d’éthanol provoque des modifications neurobiologiques adaptatives. Les mécanismes qui les contrôlent sont multiples et certains ont été reliés à des régulations épigénétiques conduisant à des modifications structurelles et fonctionnelles. L’éthanol induit également une neurodégénérescence de l’hippocampe responsable de déficits cognitifs. Parmi l’ensemble des modèles animaux qui sont utilisés pour étudier les effets d’une consommation chronique d’alcool, figurent les souris de la lignée C57BL/6J. Ces souris possèdent une appétence naturelle pour l’éthanol faisant d’elles un modèle de choix pour étudier les conséquences de la consommation chronique d’éthanol. Le but de ce travail de thèse a été d’étudier les relations entre les mécanismes épigénétiques et la modulation de la neuroplasticité de l’hippocampe à la suite d’une consommation chronique d’éthanol chez les souris C57BL/6J, et d’en évaluer les conséquences comportementales. Nous avons montré que la consommation chronique d’éthanol induit, au niveau de l’hippocampe, des modulations épigénétiques globales corrélées à un remodelage chromatinien au sein du gène du BDNF, impliquant à la fois les modifications post-traductionnelles des histones et la méthylation de l’ADN. Ces modifications épigénétiques sont certainement responsables de l’augmentation d’expression protéique du BDNF observée dans l’hippocampe, et plus particulièrement dans le gyrus denté, après 3 semaines de consommation chronique d’éthanol en libre choix. L’accroissement de l’expression du BDNF induit une stimulation des voies de la signalisation intracellulaire dépendantes de l’activation du récepteur TrkB du BDNF, et une augmentation de la neurogenèse du gyrus denté. Les effets de l’antagoniste spécifique du récepteur TrkB, ANA 12, démontrent que l’augmentation de la neurogenèse observée chez les souris C57BL/6J après la prise chronique d’éthanol, est sous le contrôle unique du complexe BDNF/TrkB. L’analyse comportementale des souris C57BL/6J ayant consommé de l’éthanol, montre une détérioration des capacités d’apprentissage et de mémoire sans modification de la plasticité synaptique dans l’hippocampe, suggérant ainsi que d’autres mécanismes sont impliqués dans ces déficits cognitifs. L’ensemble de ces données apporte de nouveaux éléments de compréhension concernant la stimulation de la neurogenèse hippocampique chez les souris C57BL/6J lors d’une consommation chronique en libre choix d’éthanol. Il est probable que cette apparente augmentation de plasticité soit un mécanisme adaptatif et compensatoire à la détérioration des fonctions cognitives induite par une consommation chronique d’alcool.

Chronic and excessive ethanol consumption triggers neurobiological adaptations within the central nervous system, which are responsible for the development of an addiction. Ethanol induces adaptive mechanisms linked to epigenetic regulations leading to functional and structural changes, and also provokes a neurodegeneration responsible for the cognitive deficits observed in alcohol abusers. Among the different animal models available for studying the effects of chronic ethanol consumption, C57BL/6J mice are among the most relevant. These mice display high ethanol preference, making them a good model for studying the consequences of chronic and free-choice ethanol consumption. The purpose of this work was to study the links between epigenetic mechanisms and hippocampal neuroplasticity and to evaluate the behavioural consequences induced by chronic ethanol consumption in C57BL/6J mice. We showed that, in the hippocampus, chronic ethanol consumption induced global epigenetic modulations that were correlated with chromatin remodelling at the BDNF gene level. These effects involved post-translational histone modifications and DNA methylation. Epigenetic changes at the BDNF gene level probably allowed the increase in BDNF protein expression observed within the hippocampal dentate gyrus in mice having consumed ethanol for 3 weeks. Upregulation of BDNF expression was linked to both the stimulation of intracellular cascades downstream BDNF/TrkB receptor activation, and the increase in neurogenesis within the dentate gyrus. Using a specific TrkB receptor antagonist, ANA-12, we demonstrated that the hippocampal neurogenesis induced by chronic ethanol intake was under the control of BDNF. Behavioural analysis evidenced learning and memory impairments after ethanol consumption without synaptic plasticity alteration within the hippocampus, suggesting the involvement of other mechanisms in the cognitive deficits. Altogether, these data bring new elements for understanding the hippocampal neurogenesis stimulation observed under chronic and voluntary ethanol consumption in C57BL/6J mice. Moreover, this apparent increase in plasticity might probably be considered as an adaptive and compensatory mechanism in response to the cognitive deficits induced by ethanol consumption.; La consommation chronique et excessive d’éthanol provoque des modifications neurobiologiques adaptatives. Les mécanismes qui les contrôlent sont multiples et certains ont été reliés à des régulations épigénétiques conduisant à des modifications structurelles et fonctionnelles. L’éthanol induit également une neurodégénérescence de l’hippocampe responsable de déficits cognitifs. Parmi l’ensemble des modèles animaux qui sont utilisés pour étudier les effets d’une consommation chronique d’alcool, figurent les souris de la lignée C57BL/6J. Ces souris possèdent une appétence naturelle pour l’éthanol faisant d’elles un modèle de choix pour étudier les conséquences de la consommation chronique d’éthanol. Le but de ce travail de thèse a été d’étudier les relations entre les mécanismes épigénétiques et la modulation de la neuroplasticité de l’hippocampe à la suite d’une consommation chronique d’éthanol chez les souris C57BL/6J, et d’en évaluer les conséquences comportementales. Nous avons montré que la consommation chronique d’éthanol induit, au niveau de l’hippocampe, des modulations épigénétiques globales corrélées à un remodelage chromatinien au sein du gène du BDNF, impliquant à la fois les modifications post-traductionnelles des histones et la méthylation de l’ADN. Ces modifications épigénétiques sont certainement responsables de l’augmentation d’expression protéique du BDNF observée dans l’hippocampe, et plus particulièrement dans le gyrus denté, après 3 semaines de consommation chronique d’éthanol en libre choix. L’accroissement de l’expression du BDNF induit une stimulation des voies de la signalisation intracellulaire dépendantes de l’activation du récepteur TrkB du BDNF, et une augmentation de la neurogenèse du gyrus denté. Les effets de l’antagoniste spécifique du récepteur TrkB, ANA 12, démontrent que l’augmentation de la neurogenèse observée chez les souris C57BL/6J après la prise chronique d’éthanol, est sous le contrôle unique du complexe BDNF/TrkB. L’analyse comportementale des souris C57BL/6J ayant consommé de l’éthanol, montre une détérioration des capacités d’apprentissage et de mémoire sans modification de la plasticité synaptique dans l’hippocampe, suggérant ainsi que d’autres mécanismes sont impliqués dans ces déficits cognitifs. L’ensemble de ces données apporte de nouveaux éléments de compréhension concernant la stimulation de la neurogenèse hippocampique chez les souris C57BL/6J lors d’une consommation chronique en libre choix d’éthanol. Il est probable que cette apparente augmentation de plasticité soit un mécanisme adaptatif et compensatoire à la détérioration des fonctions cognitives induite par une consommation chronique d’alcool.