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Chronic treatment with serotonin selective reuptake inhibitors or tryciclic antidepressant drugs in rodents has been shown to increase the expression of GluA1 and/or GluA2 AMPA receptor (AMPAR) subunits in several brain areas, including the hippocampus. These changes in AMPAR composition have been suggested to result in increased glutamatergic neurotransmission and possibly underlie enhanced hippocampal synaptic plasticity through the increased availability of calcium-permeable AMPARs, specifically at CA3/CA1 synapses. However, the possibility that chronic treatment with antidepressants actually results in strengthened glutamatergic neurotransmission in CA1 has poorly been investigated. Here, we studied whether chronic treatment with the multimodal antidepressant drug trazodone mimicked the effect of paroxetine on the expression of AMPAR subunits in male wistar rat hippocampus and whether these drugs produced a parallel facilitation of field excitatory postsynaptic potentials (fEPSP) responses evoked by activation of CA3/CA1 synapses in dorsal hippocampal slices. In addition, we investigated whether the quality of glutamatergic AMPARs involved in basal neurotransmission was changed by altered subunit expression, e.g. leading to appearance of calcium-permeable AMPARs. We found a significant increase in GluA2 subunit expression following treatment with trazodone or paroxetine for twenty-one days, but not after seven-days treatment. In contrast, we did not find any significant changes in fEPSP responses supporting either a facilitation of glutamatergic neurotransmission in basal conditions or the appearance of functional calcium-permeable AMPARs at CA3/CA1 pyramidal neuron synapses. Thus, neurochemically-detected increases in the expression of AMPAR subunits cannot directly be extrapolated in increased number of functioning receptors and/or facilitated basal neurotransmission.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced IK and reduced INa currents by stimulating Gi -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.

Recent years, particularly the COVID-19 pandemic, can be considered a turning point for pharmacovigilance and pharmacoepidemiology in terms of their role in drug safety and drug utilisation monitoring in clinical practice [...]

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced I

The hippocampus is functionally differentiated along its longitudinal axis, with the dorsal and ventral segments preferentially involved in cognitive and emotional processing, respectively. Serotonergic modulation of hippocampal function has been extensively studied, but its relation to the dorsoventral axis has remained largely unknown. To examine the modulation of hippocampal output along the dorsoventral axis by endogenous serotonin (5-HT) we compared the effect of the 5-HT/noradrenaline (NA)-releaser, 3,4-methylenedioxymethamphetamine (MDMA), in transversal slices encompassing the entire rat hippocampus. Co-release of 5-HT and NA by MDMA resulted in a gradient of effects on evoked population spikes in the CA1 area along the dorsoventral axis of the hippocampus. Selective 5-HT release decreased population spike amplitude in slices from dorsal hippocampus, whereas an increase was produced in the ventral hippocampus, indicating differential modulation of CA1 impulse flow along the dorsoventral axis by endogenous 5-HT. Selective NA release increased population spike amplitude with no gradient indicating facilitatory effect of endogenous NA along the entire dorsoventral axis. Blockade of 5-HT1A receptors prevented the inhibitory component of MDMA action and the emergence of the gradient, indicating that activation of 5-HT1A receptors is required for differential modulation of CA1 impulse flow by endogenous 5-HT. These findings suggest that a dorsoventral shift in CA1 output level may represent an integral component of 5-HT action on hippocampal information processing. Given the preferential role of ventral hippocampus in emotional and anxiety-related behavior, it can be proposed that serotonin tone encodes the emotional salience of the signal processed by hippocampus.

Firing activity of serotonergic neurons is under regulatory control by somatodendritic 5-HT1A autoreceptors (5-HT(1A)ARs). Enhanced 5-HT(1A)AR functioning may cause decreased serotonergic signaling in brain and has thereby been implicated in the etiology of mood and anxiety disorders. Tryptophan hydroxylase-2 knockout (Tph2(-/-)) mice exhibit sensitization of 5-HT1A agonist-induced inhibition of serotonergic neuron firing and thus represents a unique animal model of enhanced 5-HT(1A)AR functioning. To elucidate the mechanisms underlying 5-HT(1A)AR supersensitivity in Tph2(-/-) mice, we characterized the activation of G protein-coupled inwardly rectifying potassium (GIRK) conductance by the 5-HT1A receptor agonist 5-carboxamidotryptamine using whole-cell recordings from serotonergic neurons in dorsal raphe nucleus. Tph2(-/-) mice exhibited a mean twofold leftward shift of the agonist concentration-response curve (p

Trazodone is an antidepressant drug with considerable affinity for 5-HT1A receptors and α1-adrenoceptors for which the drug is competitive agonist and antagonist, respectively. In this study, we used cell-attached or whole-cell patch-clamp recordings to characterize the effects of trazodone at somatodendritic 5-HT1A receptors (5-HT1AARs) and α1-adrenoceptors of serotonergic neurons in rodent dorsal raphe slices. To reveal the effects of trazodone at α1-adrenoceptors, the baseline firing of 5-HT neurons was facilitated by applying the selective α1-adrenoceptor agonist phenylephrine at various concentrations. In the absence of phenylephrine, trazodone (1-10 μM) concentration-dependently silenced neurons through activation of 5-HT1AARs. The effect was fully antagonized by the selective 5-HT1A receptor antagonist Way-100635. With 5-HT1A receptors blocked by Way-100635, trazodone (1-10 μM) concentration-dependently inhibited neuron firing facilitated by 1 μM phenylephrine. Parallel rightward shift of dose-response curves for trazodone recorded in higher phenylephrine concentrations (10-100 μM) indicated competitive antagonism at α1-adrenoceptors. Both effects of trazodone were also observed in slices from Tph2-/- mice that lack synthesis of brain serotonin, showing that the activation of 5-HT1AARs was not mediated by endogenous serotonin. In whole-cell recordings, trazodone activated 5-HT1AAR-coupled G protein-activated inwardly-rectifying (GIRK) channel conductance with weak partial agonist efficacy (~35%) compared to that of the full agonist 5-CT. Collectively our data show that trazodone, at concentrations relevant to its clinical effects, exerts weak partial agonism at 5-HT1AARs and disfacilitation of firing through α1-adrenoceptor antagonism. These two actions converge in inhibiting dorsal raphe serotonergic neuron activity, albeit with varying contribution depending on the intensity of α1-adrenoceptor stimulation.

The serotonin mediating autoinhibition of neurons in the raphe nucleus is released from a nonvesicular pool., The firing activity of serotonergic neurons in raphe nuclei is regulated by negative feedback exerted by extracellular serotonin (5-HT)o acting through somatodendritic 5-HT1A autoreceptors. The steady-state [5-HT]o, sensed by 5-HT1A autoreceptors, is determined by the balance between the rates of 5-HT release and reuptake. Although it is well established that reuptake of 5-HTo is mediated by 5-HT transporters (SERT), the release mechanism has remained unclear. It is also unclear how selective 5-HT reuptake inhibitor (SSRI) antidepressants increase the [5-HT]o in raphe nuclei and suppress serotonergic neuron activity, thereby potentially diminishing their own therapeutic effect. Using an electrophysiological approach in a slice preparation, we show that, in the dorsal raphe nucleus (DRN), continuous nonexocytotic 5-HT release is responsible for suppression of phenylephrine-facilitated serotonergic neuron firing under basal conditions as well as for autoinhibition induced by SSRI application. By using 5-HT1A autoreceptor-activated G protein–gated inwardly rectifying potassium channels of patched serotonergic neurons as 5-HTo sensors, we show substantial nonexocytotic 5-HT release under conditions of abolished firing activity, Ca2+ influx, vesicular monoamine transporter 2–mediated vesicular accumulation of 5-HT, and SERT-mediated 5-HT transport. Our results reveal a cytosolic origin of 5-HTo in the DRN and suggest that 5-HTo may be supplied by simple diffusion across the plasma membrane, primarily from the dense network of neurites of serotonergic neurons surrounding the cell bodies. These findings indicate that the serotonergic system does not function as a sum of independently acting neurons but as a highly interdependent neuronal network, characterized by a shared neurotransmitter pool and the regulation of firing activity by an interneuronal, yet activity-independent, nonexocytotic mechanism.

Firing activity of serotonergic neurons is under regulatory control by somatodendritic 5-HT

Tonic spiking of serotonergic neurons establishes serotonin levels in the brain. Since the first observations, slow regular spiking has been considered as a defining feature of serotonergic neurons. Recent studies, however, have revealed the heterogeneity of serotonergic neurons at multiple levels, comprising their electrophysiological properties, suggesting the existence of functionally distinct cellular subpopulations. In order to examine in an unbiased manner whether serotonergic neurons of the dorsal raphe nucleus are heterogeneous, we used a non-invasive loose-seal cell-attached method to record α1 adrenergic receptor-stimulated spiking of a large sample of neurons in brain slices obtained from transgenic mice lines that express fluorescent marker proteins under the control of serotonergic system-specific Tph2 and Pet-1 promoters. We found wide homogeneous distribution of firing rates, well fitted by a single Gaussian function (r2 = 0.93) and independent of anatomical location (P = 0.45), suggesting that in terms of intrinsic firing properties, serotonergic neurons in the dorsal raphe nucleus represent a single cellular population. Characterization of the population in terms of spiking regularity was hindered by its dependence on the firing rate. For instance, the coefficient of variation of the interspike intervals, a common measure of spiking irregularity, is of limited usefulness since it correlates negatively with the firing rate (r = -0.33, P < 0.0001). Nevertheless, the majority of neurons exhibited regular, pacemaker-like activity, with coefficient of variance of the interspike intervals lower than 0.5 in ~97% of cases. Unexpectedly, a small percentage of neurons (~1%) exhibited a particular spiking pattern, characterized by low frequency (~0.02 - 0.1 Hz) oscillations in the firing rate. Transitions between regular and oscillatory firing were observed, suggesting that the oscillatory firing is an alternative firing pattern of serotonergic neurons.

Psychiatric and neurological diseases combined represent a considerable social and economic burden in Europe. A recent study conducted by the European Brain Council (EBC) quantified the 'cost and burden' of major brain diseases in Europe, amounting to €386bn per year. Considering that these costs will increase exponentially in the years to come due to ageing of the European population, it is necessary to act now in order to curb this increase and possibly reverse the trend. Thus, establishing a strong European platform supporting basic and clinical research in neuroscience is needed to confront the economic and social challenge posed by management of brain diseases in European countries. To setup a platform for discussion, EBC published in 2006 a Consensus Document on European Brain Research, describing needs and achievements of research in Europe and presenting proposals for future research programs. Since 2006, European research in neuroscience has advanced tremendously. The present document represents an update elaborated to reflect changes in research priorities and advances in brain research that have taken place since 2006. The same approach and format have been used here as in the previous version. Multinational and multidisciplinary teams have once again come together to express their views, not only on the current strengths in European research, but also on what needs to be done in priority, hoping that this update will inspire policy makers and stakeholders in directing funding for research in Europe.

Sudden infant death syndrome is the leading cause of death in the postneonatal period in developed countries. Postmortem studies show alterations in serotonin neurons in the brainstem of such infants. However, the mechanism by which altered serotonin homeostasis might cause sudden death is unknown. We investigated the consequences of altering the autoinhibitory capacity of serotonin neurons with the reversible overexpression of serotonin 1A autoreceptors in transgenic mice. Overexpressing mice exhibited sporadic bradycardia and hypothermia that occurred during a limited developmental period and frequently progressed to death. Moreover, overexpressing mice failed to activate autonomic target organs in response to environmental challenges. These findings show that excessive serotonin autoinhibition is a risk factor for catastrophic autonomic dysregulation and provide a mechanism for a role of altered serotonin homeostasis in sudden infant death syndrome.

It is well documented that N-methyl-3,4-methylenedioxyamphetamine (MDMA, ecstasy) releases brain serotonin (5-HT; 5-hydroxytryptamine), noradrenaline (NE; norepinephrine), and dopamine, but the consequent effect on brain functioning remains elusive. In this study, we characterized the effects of MDMA on electrically evoked responses in the ventral CA1 region of a rat hippocampal slice preparation. Superfusion with MDMA (10 microM, 30 min) increased the population spike amplitude (PSA) by 48.9+/-31.2% and decreased population spike latency (PSL) by 103+/-139 mus (both: mean+/-SD, n=123; p

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT1A autoreceptor-activated inwardly rectifying K+ conductance in rat dorsal raphe 5-HT neurons. 5-HT1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba2+ (EC50 = 9.4 μM, full block with 100 μM) and by SCH23390 (EC50 = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT1A autoreceptor-activated GIRK conductance with high potency (EC50 = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC50s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K+ channels coupled to 5-HT1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.

Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia a primary damage due to the early massive increase of extracellular glutamate is followed by activation of resident immune cells, i.e microglia, and production or activation of inflammation mediators. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. Extracellular concentrations of ATP and adenosine in the brain increase dramatically during ischemia in concentrations able to stimulate their respective specific P2 and P1 receptors. Both ATP P2 and adenosine P1 receptor subtypes exert important roles in ischemia. Although adenosine exerts a clear neuroprotective effect through A1 receptors during ischemia, the use of selective A1 agonists is hampered by undesirable peripheral effects. Evidence up to now in literature indicate that A2A receptor antagonists provide protection centrally by reducing excitotoxicity, while agonists at A2A (and possibly also A2B) and A3 receptors provide protection by controlling massive infiltration and neuroinflammation in the hours and days after brain ischemia. Among P2X receptors most evidence indicate that P2X7 receptor contribute to the damage induced by the ischemic insult due to intracellular Ca(2+) loading in central cells and facilitation of glutamate release. Antagonism of P2X7 receptors might represent a new treatment to attenuate brain damage and to promote proliferation and maturation of brain immature resident cells that can promote tissue repair following cerebral ischemia. Among P2Y receptors, antagonists of P2Y12 receptors are of value because of their antiplatelet activity and possibly because of additional anti-inflammatory effects. Moreover strategies that modify adenosine or ATP concentrations at injury sites might be of value to limit damage after ischemia. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Considerable evidence links dysfunction of serotonin (5-hydroxytryptamine, 5-HT) transmission to neurodevelopmental and psychiatric disorders characterized by compromised "social" cognition and emotion regulation. It is well established that the brain 5-HT system is under autoregulatory control by its principal transmitter 5-HT via its effects on activity and expression of 5-HT system-related proteins. To examine whether 5-HT itself also has a crucial role in the acquisition and maintenance of characteristic rhythmic firing of 5-HT neurons, we compared their intrinsic electrophysiological properties in mice lacking brain 5-HT, i.e. tryptophan hydroxylase-2 null mice (Tph2(-/-)) and their littermates, Tph2(+/-) and Tph2(+/+), by using whole-cell patch-clamp recordings in a brainstem slice preparation and single unit recording in anesthetized animals. We report that the active properties of dorsal raphe nucleus (DRN) 5-HT neurons in vivo (firing rate magnitude and variability; the presence of spike doublets) and in vitro (firing in response to depolarizing current pulses; action potential shape) as well as the resting membrane potential remained essentially unchanged across Tph2 genotypes. However, there were subtle differences in subthreshold properties, most notably, an approximately 25% higher input conductance in Tph2(-/-) mice compared with Tph2(+/-) and Tph2(+/+) littermates (p

Backgound: The neurobiological changes underlying depression resistant to treatments remain poorly understood, and failure to respond to selective serotonin reuptake inhibitors may result from abnormalities of neurotransmitter systems that excite serotonergic neurons, such as histamine. Methods: Using behavioral (tail suspension test) and neurochemical (in vivo microdialysis, Western-blot analysis) approaches, here we report that antidepressant responses to selective serotonin reuptake inhibitors (citalopram or paroxetine) are abolished in mice unable to synthesize histamine due to either targeted disruption of histidine decarboxylase gene (HDC-/-) or injection of alpha-fluoromethylhistidine, a suicide inhibitor of this enzyme. Results: In the tail suspension test, all classes of antidepressants tested reduced the immobility time of controls. Systemic reboxetine or imipramine reduced the immobility time of histamine-deprived mice as well, whereas selective serotonin reuptake inhibitors did not even though their serotonergic system is functional. In in vivo microdialysis experiments, citalopram significantly increased histamine extraneuronal levels in the cortex of freely moving mice, and methysergide, a serotonin 5-HT1/5-HT2 receptor antagonist, abolished this effect, thus suggesting the involvement of endogenous serotonin. CREB phosphorylation, which is implicated in the molecular mechanisms of antidepressant treatment, was abolished in histamine-deficient mice treated with citalopram. The CREB pathway is not impaired in HDC-/- mice, as administration of 8-bromoadenosine 3’, 5’-cyclic monophosphate increased CREB phosphorylation, and in the tail suspension test it significantly reduced the time spent immobile by mice of both genotypes. Conclusions: Our results demonstrate that selective serotonin reuptake inhibitors selectively require the integrity of the brain histamine system to exert their preclinical responses.

The role of adenosine A3 receptor activation during ischaemia-like conditions produced by oxygen and glucose deprivation (OGD) was evaluated with extracellular recordings from the CA1 region of rat hippocampal slices. In all, 7 min of OGD evoked tissue anoxic depolarisation (AD, peak at approximately 7 min from OGD start, n=20) and were invariably followed by irreversible loss of electrically evoked field epsps (fepsps, n=42).The selective adenosine A3 antagonists 3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS 1523, 1-100 nM, n=31), N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-yl]benzeneacetamide (MRS 1220, 100 nM, n=7), N-(2-methoxyphenyl)-N'-[2-(3-pyrindinyl)-4-quinazolinyl]-urea, (VUF 5574, 100 nM, n=3) and 5-[[(4-pyridyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)-pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine hydrochloride (1 nM, n=4), prevented the irreversible failure of neurotransmission induced by 7 min OGD (n=45) and the development of AD in 20 out of 22 monitored slices. When tested on OGD episodes of longer duration (8-10 min, n=18), 100 nM MRS 1523 prevented or delayed the appearance of AD and exerted a protective effect on neurotransmission for episodes of up to 9 min duration. In the absence of AD, the fepsp recovery was almost total, regardless of OGD episode duration. These findings support the notion that A3 receptor stimulation is deleterious during ischaemia and suggest that selective A3 receptor block may substantially increase the resistance of the CA1 hippocampal region to ischaemic damage.

1. Ischemic preconditioning in the brain consists of reducing the sensitivity of neuronal tissue to further, more severe, ischemic insults. We recorded field epsps (fepsps) extracellularly from hippocampal slices to develop a model of in vitro ischemic preconditioning and to evaluate the role of A1, A2A and A3 adenosine receptors in this phenomenon. 2. The application of an ischemic insult, obtained by glucose and oxygen deprivation for 7 min, produced an irreversible depression of synaptic transmission. Ischemic preconditioning was induced by four ischemic insults (2 min each) separated by 13 min of normoxic conditions. After 30 min, an ischemic insult of 7 min was applied. This protocol substantially protected the tissue from the irreversible depression of synaptic activity. 3. The selective adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nm), completely prevented the protective effect of preconditioning. The selective adenosine A2A receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 100 nm) did not modify the magnitude of fepsp recovery compared to control slices. The selective A3 adenosine receptor antagonists, 3-propyl-6-ethyl-5[ethyl(thio)carbonyl]-2-phenyl-4-propyl-3-pyridinecarboxylate (MRS 1523, 100 nm) significantly improved the recovery of fepsps after 7 min of ischemia. 4. Our results show that in vitro ischemic preconditioning allows CA1 hippocampal neurons to become resistant to prolonged exposure to ischemia. Adenosine, by stimulating A1 receptors, plays a crucial role in eliciting the cell mechanisms underlying preconditioning; A2A receptors are not involved in this phenomenon, whereas A3 receptor activation is harmful to ischemic preconditioning.

1 In the hippocampus, axon collaterals of CA1 pyramidal cells project locally onto neighbouring CA1 pyramidal cells and interneurones, forming a local excitatory network which, in disinhibited conditions, feeds polysynaptic epscs (poly-epscs). 5-hydroxytryptamine (5-HT) has been shown to inhibit poly-epscs through activation of a presynaptic receptor. The aim of the present work was the pharmacological characterization of the 5-HT receptor involved in this 5-HT action. 2 Poly-epscs, evoked by electrical stimulation of the stratum radiatum and recorded in whole-cell voltage-clamp from CA1 pyramidal neurones, were studied in mini-slices of the CA1 region under pharmacological block of GABA(A), GABA(B), and 5-HT(1A) receptors. 3 The 5-HT(1B) receptor selective agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129) inhibited poly-epscs (EC(50)=55 nM), an effect mimicked by the 5-HT(1B) ligands 5-carboxamidotryptamine (5-CT; EC(50)=14 nM) and methylergometrine (EC(50)=78 nM), but not by 1-(3-chlorophenyl)piperazine dihydrochloride (mCPP; 10 micro M) or 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline dimaleate (CGS 12066B; 10 micro M). 4 The effects of CP 93129 and 5-CT were blocked by the selective 5-HT(1B) receptor antagonist 3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR 55562; K(B) approximately 100 nM) and by cyanopindolol (K(B)=6 nM); methiothepin (10 micro M) and dihydroergotamine (1 micro M). For both GR 55562 and methiothepin, application times of at least two hours were required in order to achieve their full antagonistic effects. 5 Our results demonstrate that 5-HT(1B) receptors are responsible for the presynaptic inhibition of neurotransmission at CA1/CA1 local excitatory synapses exerted by 5-HT.

Encoding of episodic memory requires long-term potentiation (LTP) of neurotransmission at excitatory synapses of the hippocampal circuitry. Previous data obtained with the application of exogenous 5-hydroxytryptamine (5-HT) in hippocampal slices indicate that 5-HT blocks LTP, which contrasts with the facilitatory effect of selective serotonin reuptake inhibitors (SSRIs) on learning and memory observed in vivo. Here, we investigated the effects of endogenous 5-HT, released from terminals by the monoamine releaser 3,4-methylenedioxymethamphetamine (MDMA), on LTP of field EPSPs induced by theta-burst stimulation and recorded at CA3/CA1 synapses of rat hippocampal slices. LTP was greater in the presence of MDMA (10 µM; 45.76 ± 15.75%; n = 28) than in controls (31.26 ± 11.03; n = 21; p < 0.01). This facilitatory effect on LTP persisted when the entry of MDMA in noradrenergic terminals was prevented by the selective noradrenaline reuptake inhibitor nisoxetine (44.90 ± 14.07%; n = 27 vs. 34.49 ± 12.94%; n = 20 in controls; p < 0.05). In both conditions, the facilitation of LTP was abolished by the SSRI citalopram that prevented the entry of MDMA in 5-HT terminals and the subsequent 5-HT release. These data show that, unlike exogenous 5-HT application, release of endogenous 5-HT does not impair cellular mechanisms responsible for induction of LTP, indicating that 5-HT is not detrimental to learning and memory. Moreover, facilitation of LTP by endogenous 5-HT may underlie the in vivo positive effects of augmented 5-HT tone on cognitive performance.

1. In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia-like conditions. 2. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoade nos ine ), and of selective A2A receptor antagonists, ZM 241385 (4-(2-[7-amino-2-(2-furyl)-?1,2,4?-triazolo?2,3-a??1,3, 5?triazin-5-ylamino]ethyl)phenol) and SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. 3. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1, 3-dipropyl-8-cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. 4. A prolonged application of either adenosine (100 micronM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). 5. SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia-induced depression. 6. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1-mediated depression of synaptic transmission induced by in vitro ischaemia.

The pharmacological profile of (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) and of other group 1 metabotropic glutamate (mGlu) receptor agents were studied in BHK cells transfected with mGlu receptor subtypes or in native receptors in brain slices by measuring second messenger responses. The mGlu receptor-mediated changes in the electrophysiological properties of CA1 pyramidal cells of the hippocampus were also evaluated. In mGlu5a receptor transfected cells, CBPG behaved as a partial agonist, while in mGlu1alpha receptor transfected cells, it behaved as a glutamate antagonist. No effect was found on cAMP formation in cells transfected with mGlu2 receptors or mGlu4 receptors. In brain slices, CBPG neither affected phospholipase D-coupled glutamate receptors nor did it modify the responses to ionotropic receptor stimulation (at concentrations up to 1 mM). When tested in CA1 pyramidal cells of the hippocampus, CBPG (50-100 microM) caused depolarization, increased cell input resistance, and decreased action potential frequency adaptation and afterhyperpolarization. DHPG (3-100 microM), an agonist of both mGlu1 and mGlu5 receptors, and CHPG (1000 microM), a low affinity mGlu5 agonist, produced qualitatively similar effects. The actions of CBPG or CHPG were not modified by AIDA (300 microM), a selective mGlu1 receptor antagonist. Our results suggest that CBPG could be a useful tool for discriminating between mGlu1 receptor and mGlu5 receptor effects and that mGlu5 receptors are the receptors which are mainly responsible for the direct excitatory effects of mGlu receptor agonists on CA1 pyramidal cells.

1. The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. 2. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. 3. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50-500 nM). 8-Phenyltheophylline (10 microM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12 +/- 4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. 4. In the time-interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 microM. 5. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode.

1 The aim of the present work was to investigate the electrophysiological eAects of oxindole, a tryptophan metabolite present in rat blood and brain, and recently proposed as a contributing factor in the pathogenesis of hepatic encephalopathy. 2 Using rat hippocampal slices in vitro and extra- or intracellular recordings, we evaluated oxindole eAects on the neurotransmission of the CA1 region following orthodromic stimulation of the SchaAer collaterals. 3 Oxindole (0.3‐3 mM) decreased the amplitude of population spikes extracellularly recorded at the somatic level and of the fEPSPs recorded at the dendritic level. In intracellular recordings, oxindole (0.1‐3 mM) did not aAect the resting membrane potential or the neuronal input resistance, but reduced the probability of firing action potentials upon either synaptic or direct activation of the pyramidal cells. 4 Oxindole (0.3‐3 mM) increased the threshold and the latency of firing action potentials elicited by depolarizing steps without changing the duration or the peak amplitude of the spikes. It also significantly increased the spike frequency adaptation induced by long lasting (400 ms) depolarizing stimuli. 5 In separate experiments, performed by measuring AMPA or NMDA-induced responses in cortical slices, oxindole (1‐3 mM) did not modify glutamate receptor agonist responses. 6 Our results show that concentrations of oxindole which may be reached in pathological conditions, significantly decrease neuronal excitability by modifying the threshold of action potential generation.

Felbamate is a broad spectrum antiepileptic drug recently introduced into clinical practice for controlling seizures in patients affected by Lennox-Gastaut epilepsy, complex partial seizures or otherwise intractable epilepsies. However, the cellular mechanisms by which the drug exerts its anticonvulsant actions are not fully understood. The aim of the present article is to outline the possible mechanisms of action of felbamate as suggested by findings obtained with electrophysiological approaches.

1. The aim of the present work was to characterize the 5-hydroxytryptamine1A (5-HT1A) antagonistic actions of (-)-pindolol and WAY 100635 (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide). Studies were performed on 5-HT1A receptors located on 5-hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices. 2. Intracellular electrophysiological recording of CA1 pyramidal cells and 5-hydroxytryptaminergic DRN neurones showed that the 5-HT1A receptor agonist 5-carboxamidotryptamine (5-CT) evoked in both cell types a concentration-dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (-)-pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 microM, but it antagonized the 5-CT effects in a concentration-dependent manner. Similar antagonism of 5-CT effects was observed in the CA3 hippocampal region. (-)-Pindolol also prevented the 5-HT1A receptor-mediated hyperpolarization of CA1 pyramidal cells due to 5-HT (15 microM). In contrast, the 5-HT-induced depolarization mediated by presumed 5-HT4 receptors persisted in the presence of 3 microM (-)-pindolol. 3. In the hippocampus, (-)-pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5-CT (IC50=92 nM; apparent KB=20.1 nM), and of CA3 neurones by 300 nM 5-CT (IC50=522 nM; apparent KB= 115.1 nM). The block by (-)-pindolol was surmounted by increasing the concentration of 5-CT, indicating a reversible and competitive antagonistic action. 4. Extracellular recording of the firing rate of 5-hydroxytryptaminergic neurones in the DRN showed that (-)-pindolol blocked, in a concentration-dependent manner, the decrease in firing elicited by 100 nM 5-CT (IC50=598 nM; apparent KB= 131.7 nM) or 100 nM ipsapirone (IC50= 132.5 nM; apparent KB= 124.9 nM). The effect of (-)-pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 microM (-)-pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5-CT. 5. In vivo labelling of brain 5-HT1A receptors by i.v. administration of [3H]-WAY 100635 ([O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1 -piperazinyl)ethyl-N-(2-pyridyl)cyclo-hexane-carboxamide) was used to assess their occupancy following in vivo treatment with (-)-pindolol. (-)-Pindolol (15 mg kg[-1]) injected i.p. either subchronically (2 day-treatment before i.v. injection of [3H]-WAY 100635) or acutely (20 min before i.v. injection of [3H]-WAY 100635) markedly reduced [3H]-WAY 100635 accumulation in all 5-HT1A receptor-containing brain areas. In particular, no differences were observed in the capacity of (-)-pindolol to prevent [3H]-WAY 100635 accumulation in the DRN and the CAI and CA3 hippocampal areas. 6. Intracellular electrophysiological recording of 5-hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5-CT in a concentration-dependent manner (IC50=4.9 nM, apparent KB=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5-CT was also antagonized by WAY 100635 (IC50 = 0.80 nM, apparent KB= 0.28 nM).

In the CA1 region of rat hippocampal slices, the antiepileptic drug 2-phenyl-1,3-propanediol dicarbamate (felbamate; 100-1300 microM) concentration-dependently decreased extracellularly recorded synaptic potentials. The effect was significant at 200 microM, and became maximal at 700 microM felbamate, with a 70% decrease in population spike amplitude and 25% reduction of dendritic field excitatory postsynaptic potential (fEPSP) slope. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor-mediated components of the fEPSP were decreased by 700 microM felbamate. Up to 300 microM felbamate did not affect long-term potentiation (LTP), whereas 500 microM decreased the magnitude of LTP. Higher concentrations of felbamate (700-1300 microM) blocked induction of somatic and dendritic LTP completely, but reversibly. It appears that the concentrations of felbamate which affect LTP are higher than those needed for its antiepileptic action.

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT\(_{1A}\) and 5-HT\(_{1B}\) receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis.

1. The aim of the present study was to test the effects of DAU 6215 (endo-N-(8-methyl-8-azabicyclo-[3.2.1]-octo-3-yl)-2,3-dihydro-2-ox o-1H- benzimidazole-1-carboxamide carboxamide hydrochloride), a newly synthesized, selective 5-hydroxytryptamine3 (5-HT3) antagonist, on the cell membrane properties and on characterized 5-HT-mediated responses of pyramidal neurones in the hippocampal CA1 region. 2. Administration of DAU 6215, even at concentrations several hundred fold its Ki, did not affect the cell membrane properties of pyramidal neurones, nor modify extracellularly recorded synaptic potentials, evoked by stimulating the Schaffer's collaterals. 3. Micromolar concentrations (15-30 microM) of 5-HT elicited several responses in pyramidal neurones that are mediated by distinct 5-HT receptor subtypes. DAU 6215 did not antagonize the 5-HT1A-induced membrane hyperpolarization and conductance increase, a response that was blocked by the selective 5-HT1A antagonist NAN-190 (1-(2-methoxyphenyl)-4-[4-(2-phtalamido)butyl- piperazine). Similarly, DAU 6215 did not affect the membrane depolarization and decrease in amplitude of the afterhyperpolarization, elicited by the activation of putative 5-HT4 receptors. 4. 5-HT increased the frequency of spontaneous postsynaptic potentials (s.p.s.ps) recorded in pyramidal neurones loaded with chloride. In agreement with previous observations, most of the s.p.s.ps were reversed GABAergic events, produced by the activation of 5-HT3 receptors on interneurones, because they persisted in the presence of the glutamate NMDA and non NMDA antagonists, D-aminophosphonovaleric acid (APV; 50 microM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX; 25 microM), and were elicited by the selective 5-HT3 agonist, 2-methyl-5-HT (2-Me-5-HT, 50 microM). 5. The increase in frequency of s.p.s.ps induced by 5-HT was significantly antagonized by DAU 6215 in 70% of the cases, whereas the 5-HT3 antagonist always suppressed the effect of 2-Me-5-HT, at concentrations as low as 60 nM.6. The antagonistic effect of DAU 6215 was also tested on the 5-HT3-mediated block of induction of long-term potentiation (LTP), elicited by a primed burst (PB) stimulation. Extracellular recordings showed that low concentrations (60 nM) of DAU 6215 suppressed the inhibitory action of 5-HT onPB-induced LTP, without affecting the 5-HTlA-induced reduction in the amplitude of the population spike.7. These results provide evidence that DAU 6215 is an effective antagonist of the 5-HT3-mediated responses in the central nervous system and may offer a cellular correlate for the pharmacological effects of DAU 6215 as an anxiolytic and cognition enhancer.

In rats and primates, (±)3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) produces both long-lasting damage to serotonergic axons and memory impairment. Our objective was to determine effects of neurotoxic dose of MDMA on long-term potentiation (LTP) in hippocampal area CA1 in Dark-Agouti (DA) rats. One week after neurotoxic MDMA treatment in vivo (12.5mg/kg i.p., once a week, per three weeks), serotonergic deficit was evident in hippocampal slices as 56.3% reduction in 5-HT content (p=0.04) and as 68.4% reduction in the effect of endogenous 5-HT release on synaptic neurotransmission (p0.01). In hippocampal slices from the same animals, LTP was on average 46% greater than that observed in sham-treated controls (42.9 ± 3.5%; n=12 vs. 29.2 ± 3.2%; n=12; p0.01). Non-neurotoxic dose of MDMA (12.5 mg/kg, i.p., one time) did not change LTP one week after the treatment, suggesting correlation between serotonergic deficit and enhanced synaptic plasticity. We conclude that MDMA-induced impairment of learning and memory is not a consequence of hippocampal LTP inhibition.

The role of adenosine A 3 receptors in synaptic transmission under severe (7 min) and shorter (2–5 min) ischemic conditions, obtained by oxygen and glucose deprivation (OGD), was investigated in rat hippocampal slices. The effects of selective A 3 agonists or antagonists were examined on field excitatory postsynaptic potentials (fEPSPs) extracellularly recorded at the dendritic level of the CA1 pyramidal region. The novel, selective A 3 antagonist LJ1251 ((2 R ,3 R ,4 S )-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol, 0.1–10 nM) protected hippocampal slices from irreversible fEPSP depression induced by severe OGD and prevented or delayed the appearance of anoxic depolarization. Similar results were obtained when severe OGD was carried out with a long, receptor-desensitizing exposure to various selective A 3 agonists: 5′- N -methylcarboxamidoadenosine derivatives Cl-IB-MECA ( N 6 -(3-iodobenzyl)-2-chloro), VT72 ( N 6 -methoxy-2-phenylethynyl), VT158 ( N 6 -methoxy-2-phenylethynyl), VT160 ( N 6 -methoxy-2-(2-pyridinyl)-ethynyl), and VT163 ( N 6 -methoxy-2- p -acetylphenylethynyl) and AR132 ( N 6 -methyl-2-phenylethynyladenosine). The selective A 3 antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate, 100 nM) reduced fEPSP depression evoked by 2-min OGD and induced a faster recovery of fEPSP amplitude after 5-min OGD. Similar results were obtained for 2- or 5-min OGD applied in the presence of each of the A 3 agonists tested. Shorter exposure to A 3 agonists significantly delayed the recovery of fEPSP amplitude after 5-min OGD. This indicates that A 3 receptors, stimulated by selective A 3 agonists, undergo desensitization during OGD. It is inferred that CA1 hippocampal A 3 receptors stimulated by adenosine released during brief ischemia (2 and 5 min) might exert A 1 -like protective effects on neurotransmission. Severe ischemia would transform the A 3 receptor-mediated effects from protective to injurious.

Recent studies implicated involvement of the 5-hydroxytryptamine 4 (5-HT4) receptor in cognitive and emotional processes. The highest 5-HT4 receptor densities in the brain are found in the limbic system including the hippocampus. Here we used the selective 5-HT4 receptor full agonist, N-pentyl-N'-aminoguanidine carbazimidamide (SDZ-216454) to characterize effects of 5-HT4 receptor activation in whole-cell and field recordings in the area CA1 in hippocampal slices prepared from 3 to 4- and 6 to 9-week-old rats, respectively. Extracellular recordings showed that transient 5-HT4 receptor activation by 10-20 min application of SDZ-216454 induces field excitatory postsynaptic potential (fEPSP)-population spike potentiation (ESP 5-HT4 ), which persisted for as long as we held the recordings (> 2 h). ESP 5-HT4 displayed characteristics different from EPSP-spike potentiation that accompanies long-term potentiation; it developed without an associated increase in synaptic transmission, was independent on afferent input, activity of postsynaptic neurons and N-methyl-D-aspartate receptor activation; and was expressed in the presence of GABA receptor antagonists. ESP 5-HT4 was also induced by transient application of the natural neurotransmitter, 5-HT. The increase in the evoked population spike (PS) induced by SDZ-216454 was not prevented by blockers of hyperpolarization-activated cation current (I h ), Cs + and ZD-7288, but was mimicked and occluded by 150 μM Ba 2+ . Whole-cell voltage-clamp recordings from pyramidal neurons demonstrated that SDZ-216454 application increases membrane resistance with a concomitant decrease in a Ba 2+ -sensitive inwardly rectifying K + current and the Ba 2+ -insensitive K + current underlying slow afterhyperpolarization (I sAHP ). We conclude that 5-HT4 receptor activation may cause a long-lasting excitability increase in CA1 pyramidal neurons by inhibition of a Ba 2+ -sensitive inwardly rectifying K + current.

5-Hydroxytryptamine neurons in the dorsal raphe nucleus are under autoinhibitory control by endogenous 5-hydroxytryptamine. Tonic activation of 5-hydroxytryptamine1A autoreceptors was demonstrated in awake animals, but was inconsistently observed in anaesthetized animals and slice preparations, leading to questioning of its physiological signi¢cance. We re-evaluated autoinhibition in single-unit recordings from deeply seated 5-hydroxytryptamine neurons in slices in which endogenous 5-hydroxytryptamine bioavailability was restored by supplementing its precursor L-tryptophan. In these conditions, the application of the neutral 5-hydroxytryptamine 1A receptor antagonist WAY-100635 markedly increased 5-hydroxytryptamine neuron ¢ring. Responses to WAY-100635 in single experiments ranged from a lack of eiect to a several-fold increase in ¢ring rate, suggesting that 5-hydroxytryptamine neurons in the dorsal raphe nucleus represent a heterogeneous population regarding their susceptibility to autoinhibition by endogenous 5-hydroxytryptamine. NeuroReport 16:1351^1355 � c 2005 Lippincott Williams & Wilkins.

The pharmacological properties of cyclohexanecarboxylic acid, {2-[4-(2-bromo-5-methoxybenzyl)piperazin-1-yl]ethyl}-(2-trifluoromethoxyphenyl)amide (Rec 27/0224), and cyclohexanecarboxylic acid, (2-methoxy-phenyl)-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethyl}amide (Rec 27/0074), were characterized using radioligand displacement and guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding assays, as well as electrophysiological experiments, in rat hippocampal and dorsal raphe nucleus (DRN) slices. Both compounds showed a high affinity (Ki, approximately 1 nM) and selectivity (70-fold) at human 5-hydroxytryptamine (5-HT)1A receptors versus other 5-HT receptors. In [35S]GTPgammaS binding assays on HeLa cells stably expressing human 5-HT1A receptors, Rec 27/0224 and Rec 27/0074 inhibited basal [35S]GTPgammaS binding by 44.8 +/- 1.7% (pEC50 = 8.58) and 25 +/- 2.5% (pEC50 = 8.86), respectively. In intracellularly recorded CA1 pyramidal cells, 5-HT1A (hetero)receptor-mediated hyperpolarization, elicited by 100 nM 5-carboxamidoytryptamine (5-CT), was partially antagonized by Rec 27/0224 (approximately 50%; IC50 = 18.0 nM) and Rec 27/0074 (74%; IC50 = 0.8 nM). In extracellularly recorded DRN serotonergic neurons, Rec 27/0224 and Rec 27/0074 fully antagonized the inhibition of firing caused by the activation of 5-HT1A (auto)receptors by 30 nM 5-CT with an IC50 of 34.9 nM and 16.5 nM, respectively. The antagonism had a slow time course, reaching a steady state within 60 min. Both compounds also antagonized the citalopram-elicited, endogenous 5-HT-mediated inhibition of cell firing. In conclusion, Rec 27/0224 and Rec 27/0074 exhibited inverse agonism in [35S]GTPgammaS binding assays and differential antagonistic properties on 5-HT1A receptor-mediated responses in the hippocampus but not in the DRN. Whether this differential effect is causally related to inverse agonist activity is unclear. The qualitatively different nature of the antagonism in the hippocampus versus the DRN clearly distinguishes the compounds from neutral antagonists, such as N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclo-hexanecarboxamide (WAY-100635).

A multitude of different serotonin (5-HT) receptor types are expressed in the hippocampus, but the identity of receptors actually mediating the physiological response to endogenous 5-HT has not been determined. We combined pharmacologically induced release of 5-HTwith patch-clamp recordings on disinhibited rat CA1 minislices to determine effects of endogenous 5-HT on the excitability of pyramidal neurons and synaptic transmission among them. We found that application of 5-HT releasers, 3,4-methylenedioxy-methamphetamine (MDMA) or p-methylthioamphetamine, at concentrations ranging from 2 to 50 μM, reduced the excitatory synaptic transmission between CA1 pyramidal neurons without altering their basal electrical properties. This effect of MDMA was blocked by the selective 5-HT 1 B antagonist GR 55562, was dependent on endogenous 5-HT content and was mediated by presynaptically located, pertussis-toxin sensitive mechanisms. We found no other MDMA effects in our preparation, which indicates that the release of endogenous 5-HT preferentially stimulates 5-HT 1 B receptors on CA1 pyramidal neurons. Therefore, 5-HT 1 B receptor activation may represent a predominant component of the physiological response to endogenous 5-HT in the CA1. The high sensitivity of the 5-HT 1 B receptor-mediated reduction of polysynaptic excitatory responses to the extracellular 5-HT level enabled us to study mechanisms of the 5-HT releasing action of MDMA. Block of the serotonin transporter (SERT) with citalopram slowed the time course and reduced overall 5-HT release by MDMA. Depletion of vesicular 5-HT, by inhibition of vesicular monoamine transporter type 2 with tetrabenazine prevented the release. Thus although the SERT reversal contributes, a direct vesicle-depleting action is essential for MDMA release of 5-HT.