Experimental neuropsychopharmacology, yesterday, today and tomorrow. A conversation with Michel Hamon.

For this issue, Luc Zimmer, professor of pharmacology and chair of the Neuropsychopharmacology Committee of the French Society of Pharmacology and Therapeutics, talked with Michel Hamon, honorary director of research at the French National Institute of Health and Medical Research (INSERM) and honorary professor of neuropharmacology at Paris-Sorbonne (Pierre et Marie Curie) University. Some of the leading names in neuropsychopharmacology research are mentioned, pointing to significant conceptual advances that founded this discipline. The links between psychopharmacology and neuropharmacology are also discussed in the light of past collaborations. Finally, priorities are proposed for the emergence of the psychopharmacology of the future.

Pharmaco-fUS for Characterizing Drugs for Alzheimer's Disease - The Case of THN201, a Drug Combination of Donepezil Plus Mefloquine.

Donepezil is a potent acetylcholinesterase inhibitor, largely used worldwide to alleviate cognitive symptoms in Alzheimer's disease (AD). Beyond the widely described neuronal impact of donepezil, it was recently shown that targeting connexins, the proteins involved in astrocyte network organization, potentiates donepezil efficacy profile using behavioral tests in AD rodent models. We herein present data demonstrating the potential of functional ultrasound imaging to monitor cerebral activity changes after pharmacological challenge in mice. As an example, we showed that although administration of donepezil or mefloquine alone at low dose had only very limited effects on the signal compared to the baseline, their combination produced marked hemodynamic effects in the hippocampus, in line with previously published behavioral data demonstrating a synergic interaction between both drugs. Thus, the present study provides new perspectives, (i) through the use of pharmaco-fUS, a new non-clinical imaging modality, to move forward drug discovery in AD and (ii) by the profiling of two drug treatments on brain dynamics, one used in AD: donepezil, and the other in development: donepezil combined with mefloquine (THN201) as a modulator of astrocyte network.

Efficacy of THN201, a Combination of Donepezil and Mefloquine, to Reverse Neurocognitive Deficits in Alzheimer's Disease.

Donepezil (DPZ) is an acetylcholinesterase inhibitor used in Alzheimer's disease to restore cognitive functions but is endowed with limited efficacy. Recent studies pointed out the implication of astroglial networks in cognitive processes, notably via astrocyte connexins (Cxs), proteins involved in gap junction intercellular communications. Hence, we investigated the impact on cognition of pharmacological or genetic modulations of those astrocyte Cxs during DPZ challenge in two rodent models of Alzheimer's disease-like memory deficits. We demonstrated that the Cx modulator mefloquine (MEF) significantly enhanced the procognitive effect of DPZ in both models. In parallel, we determined that MEF potentiated DPZ-induced release of acetylcholine in hippocampus. Finally, local genetic silencing of astrocyte Cxs in the hippocampus was also found to enhance the procognitive effect of DPZ, pointing out the importance of Cx-dependent astrocyte networks in memory processes.

Key role of the 5-HT1A receptor addressing protein Yif1B in serotonin neurotransmission and SSRI treatment.

Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.

A New Tool for In Vivo Study of Astrocyte Connexin 43 in Brain.

Astrocytes are glial cells organized in dynamic and structured networks in the brain. These plastic networks, involving key proteins such as connexin 43 (Cx43), are engaged in fine neuronal tuning and have recently been considered as emerging therapeutic targets in central nervous system disorders. We developed and validated a new application of the manganese-enhanced magnetic resonance imaging (MEMRI) technique allowing in vivo investigations of astrocyte-neuron interactions through quantification of brain Cx43 functional activity. The proof of concept has been achieved by quantification of MEMRI signals in brain after either local astrocyte-specific Cx43 knockdown with shRNA or systemic administration of Cx43 blockers. Unilateral hippocampal Cx43 genetical silencing was associated with an ipsilateral local increase of MEMRI signal. Furthermore, Cx43 blockers also enhanced MEMRI signal responses in hippocampus. Altogether, these data reveal the MEMRI technique as a tool for quantitative imaging of in vivo Cx43-dependent function in astrocytes under physiological and pathological conditions.

A new painkiller nanomedicine to bypass the blood-brain barrier and the use of morphine.

The clinical use of endogenous neuropeptides has historically been limited due to pharmacokinetic issues, including plasma stability and blood-brain barrier permeability. In this study, we show that the rapidly metabolized Leu-enkephalin (LENK) neuropeptide may become pharmacologically efficient owing to a simple conjugation with the lipid squalene (SQ). The corresponding LENK-SQ bioconjugates were synthesized using different chemical linkers in order to modulate the LENK release after their formulation into nanoparticles. This new SQ-based nanoformulation prevented rapid plasma degradation of LENK and conferred on the released neuropeptide a notable antihyperalgesic effect that lasted longer than after treatment with morphine in a rat model of inflammation (Hargreaves test). The biodistribution study as well as the use of brain-permeant and -impermeant opioid receptor antagonists indicated that LENK-SQ NPs act through peripherally located opioid receptors. This study represents a novel nanomedicine approach, allowing the specific delivery of LENK neuropeptide into inflamed tissues for pain control.

α2- and ß2-Adrenoreceptor-Mediated Efficacy of the Atypical Antidepressant Agomelatine Combined With Gabapentin to Suppress Allodynia in Neuropathic Rats With Ligated Infraorbital or Sciatic Nerve.

Previous data showed that neuropathic pain induced by mechanical lesion of peripheral nerves has specific characteristics and responds differently to alleviating drugs at cephalic versus extracephalic level. This is especially true for tricyclic antidepressants currently used for alleviating neuropathic pain in humans which are less effective against cephalic neuropathic pain. Whether this also applies to the antidepressant agomelatine, with its unique pharmacological properties as MT1/MT2 melatonin receptor agonist and 5-HT2B/5-HT2C serotonin receptor antagonist, has been investigated in two rat models of neuropathic pain. Acute treatments were performed 2 weeks after unilateral chronic constriction (ligation) injury to the sciatic nerve (CCI-SN) or the infraorbital nerve (CCI-ION), when maximal mechanical allodynia had developed in ipsilateral hindpaw or vibrissal pad, respectively, in Sprague-Dawley male rats. Although agomelatine (45 mg/kg i.p.) alone was inactive, co-treatment with gabapentin, at an essentially ineffective dose (50 mg/kg i.p.) on its own, produced marked anti-allodynic effects, especially in CCI-ION rats. In both CCI-SN and CCI-ION models, suppression of mechanical allodynia by 'agomelatine + gabapentin' could be partially mimicked by the combination of 5-HT2C antagonist (SB 242084) + gabapentin, but not by melatonin or 5-HT2B antagonist (RS 127445, LY 266097), alone or combined with gabapentin. In contrast, pretreatment by idazoxan, propranolol or the ß2 antagonist ICI 118551 markedly inhibited the anti-allodynic effect of 'agomelatine + gabapentin' in both CCI-SN and CCI-ION rats, whereas pretreatment by the MT1/MT2 receptor antagonist S22153 was inactive. Altogether these data indicate that 'agomelatine + gabapentin' is a potent anti-allodynic combination at both cephalic and extra-cephalic levels, whose action implicates α2- and ß2-adrenoreceptor-mediated noradrenergic neurotransmission.

The GABAergic Gudden's dorsal tegmental nucleus: A new relay for serotonergic regulation of sleep-wake behavior in the mouse.

Serotonin (5-HT) neurons are involved in wake promotion and exert a strong inhibitory influence on rapid eye movement (REM) sleep. Such effects have been ascribed, at least in part to the action of 5-HT at post-synaptic 5-HT1A receptors (5-HT1AR) in the brainstem, a major wake/REM sleep regulatory center. However, the neuroanatomical substrate through which 5-HT1AR influence sleep remains elusive. We therefore investigated whether a brainstem structure containing a high density of 5-HT1AR mRNA, the GABAergic Gudden's dorsal tegmental nucleus (DTg), may contribute to 5-HT-mediated regulatory mechanisms of sleep-wake stages. We first found that bilateral lesions of the DTg promote wake at the expense of sleep. In addition, using local microinjections into the DTg in freely moving mice, we showed that local activation of 5-HT1AR by the prototypical agonist 8-OH-DPAT enhances wake and reduces deeply REM sleep duration. The specific involvement of 5-HT1AR in the latter effects was further demonstrated by ex vivo extracellular recordings showing that the selective 5-HT1AR antagonist WAY 100635 prevented DTg neuron inhibition by 8-OH-DPAT. We next found that GABAergic neurons of the ventral DTg exclusively targets glutamatergic neurons of the lateral mammillary nucleus (LM) in the posterior hypothalamus by means of anterograde and retrograde tracing techniques using cre driver mouse lines and a modified rabies virus. Altogether, our findings strongly support the idea that 5-HT-driven enhancement of wake results from 5-HT1AR-mediated inhibition of DTg GABAergic neurons that would in turn disinhibit glutamatergic neurons in the mammillary bodies. We therefore propose a Raphe→DTg→LM pathway as a novel regulatory circuit underlying 5-HT modulation of arousal.

Lentiviral vector-driven inhibition of 5-HT synthesis in B3 bulbo-spinal serotonergic projections - Consequences on nociception, inflammatory and neuropathic pain in rats.

Although it is well established that bulbo-spinal serotonergic projections contribute to pain control mechanisms, whether they exert anti- or pro-nociceptive modulations is still a matter of debate. In order to reappraise the role of 5-HT in descending controls, we used RNA interference to selectively inhibit 5-HT synthesis in B3 neurons and assess resulting changes in nociception. Rats were injected into the bulbar B3 group with a recombinant lentiviral vector, LV-shTPH2, encoding RNA interfering with tryptophan hydroxylase 2 expression. Together with the long term disappearance of this enzyme in the whole rostro-caudal extent of B3 group, 5-HT was markedly depleted selectively in the dorsal horn at all levels of the spinal cord. In contrast, immunolabeling of the 5-HT transporter was unaffected by LV-shTPH2 injection, indicating the preservation of serotonergic fibers integrity. Whereas mechanical and thermal nociceptive thresholds were unchanged by 5-HT depletion, marked reductions in intraplantar formalin (but not carrageenin)-evoked nocifensive responses, and, in contrast, significant increases in mechanical and thermal hyperalgesia evoked by sciatic nerve ligation were noted in LV-shTPH2-injected rats versus controls. Parallel changes in c-Fos immunolabeling within the dorsal horn confirmed that bulbo-spinal serotonergic projections modulate pain signaling under these various conditions. These results suggest that serotonergic neurons of the B3 group are only moderately concerned, if any, by acute nociception but exert modulatory influences under pain sensitizing conditions. The opposite changes in formalin injected- versus sciatic nerve ligated rats might be related to the implication of different receptors in 5-HT-mediated modulation of inflammatory versus neuropathic pain.

Potentiation of Amitriptyline Anti-Hyperalgesic-Like Action By Astroglial Connexin 43 Inhibition in Neuropathic Rats.

Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants.

Respective pharmacological features of neuropathic-like pain evoked by intrathecal BDNF versus sciatic nerve ligation in rats.

Numerous reported data support the idea that Brain Derived Neurotrophic Factor (BDNF) is critically involved in both depression and comorbid pain. The possible direct effect of BDNF on pain mechanisms was assessed here and compared with behavioral/neurobiological features of neuropathic pain caused by chronic constriction injury to the sciatic nerve (CCI-SN). Sprague-Dawley male rats were either injected intrathecally with BDNF (3.0 ng i.t.) or subjected to unilateral CCI-SN. Their respective responses to anti-hyperalgesic drugs were assessed using the Randall-Selitto test and both immunohistochemical and RT-qPCR approaches were used to investigate molecular/cellular mechanisms underlying hyperalgesia in both models. Long lasting hyperalgesia and allodynia were induced by i.t. BDNF in intact healthy rats like those found after CCI-SN. Acute treatment with the BDNF-TrkB receptor antagonist cyclotraxin B completely prevented i.t. BDNF-induced hyperalgesia and partially reversed this symptom in both BDNF-pretreated and CCI-SN lesioned rats. Acute administration of the anticonvulsant pregabalin, the NMDA receptor antagonist ketamine, the opioid analgesics morphine and tapentadol or the antidepressant agomelatine also transiently reversed hyperalgesia in both i.t. BDNF injected- and CCI-SN lesioned-rats. Marked induction of microglia activation markers (OX42, Iba1, P-p38), proinflammatory cytokine IL-6, NMDA receptor subunit NR2B and BDNF was found in spinal cord and/or dorsal root ganglia of CCI-SN rats. A long lasting spinal BDNF overexpression was also observed in BDNF i.t. rats, indicating an autocrine self-induction, with downstream long lasting TrkB-mediated neuropathic-like pain. Accordingly, TrkB blockade appeared as a relevant approach to alleviate not only i.t. BDNF- but also nerve lesion-evoked neuropathic pain.

5-HT2C receptor desensitization moderates anxiety in 5-HTT deficient mice: from behavioral to cellular evidence.

BACKGROUND: Desensitization and blockade of 5-HT2C receptors (5-HT2CR) have long been thought to be central in the therapeutic action of antidepressant drugs. However, besides behavioral pharmacology studies, there is little in vivo data documenting antidepressant-induced 5-HT2CR desensitization in specific brain areas. METHODS: Mice lacking the 5-HT reuptake carrier (5-HTT(-/-)) were used to model the consequences of chronic 5-HT reuptake inhibition with antidepressant drugs. The effect of this mutation on 5-HT2CR was evaluated at the behavioral (social interaction, novelty-suppressed feeding, and 5-HT2CR-induced hypolocomotion tests), the neurochemical, and the cellular (RT-qPCR, mRNA editing, and c-fos-induced expression) levels. RESULTS: Although 5-HTT(-/-) mice had an anxiogenic profile in the novelty-suppressed feeding test, they displayed less 5-HT2CR-mediated anxiety in response to the agonist m-chlorophenylpiperazine in the social interaction test. In addition, 5-HT2CR-mediated inhibition of a stress-induced increase in 5-HT turnover, measured in various brain areas, was markedly reduced in 5-HTT(-/-) mutants. These indices of tolerance to 5-HT2CR stimulation were associated neither with altered levels of 5-HT2CR protein and mRNA nor with changes in pre-mRNA editing in the frontal cortex. However, basal c-fos mRNA production in cells expressing 5-HT2CR was higher in 5-HTT(-/-) mutants, suggesting an altered basal activity of these cells following sustained 5-HT reuptake carrier inactivation. Furthermore, the increased c-fos mRNA expression in 5-HT2CR-like immune-positive cortical cells observed in wild-type mice treated acutely with the 5-HT2CR agonist RO-60,0175 was absent in 5-HTT(-/-) mutants. CONCLUSIONS: Such blunted responsiveness of the 5-HT2CR system, observed at the cell signaling level, probably contributes to the moderation of the anxiety phenotype in 5-HTT(-/-) mice.

BDNF-dependent plasticity induced by peripheral inflammation in the primary sensory and the cingulate cortex triggers cold allodynia and reveals a major role for endogenous BDNF as a tuner of the affective aspect of pain.

Painful experiences are multilayered, composed of sensory, affective, cognitive and behavioral facets. Whereas it is well accepted that the development of chronic pain is due to maladaptive neuronal changes, the underlying molecular mechanisms, their relationship to the different pain modalities, and indeed the localization of these changes are still unknown. Brain-derived neurotrophic factor (BDNF) is an activity-dependent neuromodulator in the adult brain, which enhances neuronal excitability. In the spinal cord, BDNF underlies the development and maintenance of inflammatory and neuropathic pain. Here, we hypothesized that BDNF could be a trigger of some of these plastic changes. Our results demonstrate that BDNF is upregulated in the anterior cingulate cortex (ACC) and the primary sensory cortex (S1) in rats with inflammatory pain. Injections of recombinant BDNF (into the ACC) or a viral vector synthesizing BDNF (into the ACC or S1) triggered both neuronal hyperexcitability, as shown by elevated long-term potentiation, and sustained pain hypersensitivity. Finally, pharmacological blockade of BDNF-tropomyosin receptor kinase B (TrkB) signaling in the ACC, through local injection of cyclotraxin-B (a novel, highly potent, and selective TrkB antagonist) prevented neuronal hyperexcitability, the emergence of cold hypersensitivity, and passive avoidance behavior. These findings show that BDNF-dependent neuronal plasticity in the ACC, a structure known to be involved in the affective-emotional aspect of pain, is a key mechanism in the development and maintenance of the emotional aspect of chronic pain.

Effect of genetic and pharmacological blockade of GABA receptors on the 5-HT2C receptor function during stress.

Serotonin (5-HT)2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5-HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5-HT2C receptor-induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5-HT turnover by a 5-HT2C receptor agonist (RO 60-0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA-A or GABA-B receptors in mice. Neither the GABA-B receptor antagonist phaclofen nor the specific genetic ablation of either GABA-B1a or GABA-B1b subunits altered the inhibitory effect of RO 60-0175, although 5-HT turnover was markedly decreased in GABA-B1a knock-out mice in both basal and stress conditions. In contrast, the 5-HT2C receptor-mediated inhibition of 5-HT turnover was reduced by the GABA-A receptor antagonist bicuculline. However, a significant effect of 5-HT2C receptor activation persisted in mutant mice deficient in the α3 subunit of GABA-A receptors. It can be inferred that non-α3 subunit-containing GABA-A receptors, but not GABA-B receptors, mediate the 5-HT2C -induced inhibition of stress-induced increase in hippocampal 5-HT turnover in mice.

Spinal cord transection-induced allodynia in rats--behavioral, physiopathological and pharmacological characterization.

In humans, spinal cord lesions induce not only major motor and neurovegetative deficits but also severe neuropathic pain which is mostly resistant to classical analgesics. Better treatments can be expected from precise characterization of underlying physiopathological mechanisms. This led us to thoroughly investigate (i) mechanical and thermal sensory alterations, (ii) responses to acute treatments with drugs having patent or potential anti-allodynic properties and (iii) the spinal/ganglion expression of transcripts encoding markers of neuronal injury, microglia and astrocyte activation in rats that underwent complete spinal cord transection (SCT). SCT was performed at thoracic T8-T9 level under deep isoflurane anaesthesia, and SCT rats were examined for up to two months post surgery. SCT induced a marked hyper-reflexia at hindpaws and strong mechanical and cold allodynia in a limited (6 cm2) cutaneous territory just rostral to the lesion site. At this level, pressure threshold value to trigger nocifensive reactions to locally applied von Frey filaments was 100-fold lower in SCT- versus sham-operated rats. A marked up-regulation of mRNAs encoding ATF3 (neuronal injury) and glial activation markers (OX-42, GFAP, P2×4, P2×7, TLR4) was observed in spinal cord and/or dorsal root ganglia at T6-T11 levels from day 2 up to day 60 post surgery. Transcripts encoding the proinflammatory cytokines IL-1ß, IL-6 and TNF-α were also markedly but differentially up-regulated at T6-T11 levels in SCT rats. Acute treatment with ketamine (50 mg/kg i.p.), morphine (3-10 mg/kg s.c.) and tapentadol (10-20 mg/kg i.p.) significantly increased pressure threshold to trigger nocifensive reaction in the von Frey filaments test, whereas amitriptyline, pregabalin, gabapentin and clonazepam were ineffective. Because all SCT rats developed long lasting, reproducible and stable allodynia, which could be alleviated by drugs effective in humans, thoracic cord transection might be a reliable model for testing innovative therapies aimed at reducing spinal cord lesion-induced central neuropathic pain.

Controversies on the role of 5-HT(2C) receptors in the mechanisms of action of antidepressant drugs.

Evidence from the various sources indicates alterations in 5-HT2C receptor functions in anxiety, depression and suicide, and other stress-related disorders treated with antidepressant drugs. Although the notion of a 5-HT2C receptor desensitization following antidepressant treatments is rather well anchored in the literature, this concept is mainly based on in vitro assays and/or behavioral assays (hypolocomotion, hyperthermia) that have poor relevance to anxio-depressive disorders. Our objective herein is to provide a comprehensive overview of the studies that have assessed the effects of antidepressant drugs on 5-HT2C receptors. Relevant molecular (second messengers, editing), neurochemical (receptor binding and mRNA levels), physiological (5-HT2C receptor-induced hyperthermia and hormone release), behavioral (5-HT2C receptor-induced changes in feeding, anxiety, defense and motor activity) data are summarized and discussed. Setting the record straight about drug-induced changes in 5-HT2C receptor function in specific brain regions should help to determine which pharmacotherapeutic strategy is best for affective and anxiety disorders.

Neuronal phenotype dependency of agonist-induced internalization of the 5-HT(1A) serotonin receptor.

Selective serotonin reuptake inhibitors (SSRI) are aimed at increasing brain 5-HT tone; however, this expected effect has a slow onset after starting SSRI treatment because of initial activation of 5-HT(1A) autoreceptor-mediated negative feedback of 5-HT release. After chronic SSRI treatment, 5-HT(1A) autoreceptors desensitize, which allows 5-HT tone elevation. Because 5-HT(1A) receptor (5-HT(1A)R) internalization has been proposed as a possible mechanism underlying 5-HT(1A) autoreceptor desensitization, we examined whether this receptor could internalize under well controlled in vitro conditions in the LLC-CPK1 cell line and in raphe or hippocampal neurons from rat embryos. To this goal, cells were transfected with recombinant lentiviral vectors encoding N-terminal tagged 5-HT(1A)R, and exposed to various pharmacological conditions. Constitutive endocytosis and plasma membrane recycling of tagged-5-HT(1A)R was observed in LLC-PK1 cells as well as in neurons. Acute exposure (for 1 h) to the full 5-HT(1A)R agonists, 5-HT and 5-carboxamido-tryptamine, but not the partial agonist 8-OH-DPAT, triggered internalization of tagged 5-HT(1A)R in serotonergic neurons only. In contrast, sustained exposure (for 24 h) to all agonists induced tagged-5-HT(1A)R endocytosis in raphe serotonergic neurons and a portion of hippocampal neurons, but not LLC-PK1 cells and partial agonist displayed an effect only in serotonergic neurons. In all cases, agonist-induced tagged 5-HT(1A)R endocytosis was prevented by the 5-HT(1A)R antagonist, WAY-100635, which was inactive on its own. These data showed that agonist-induced 5-HT(1A)R internalization does exist in neurons and depends on agonist efficacy and neuronal phenotype. Its differential occurrence in serotonergic neurons supports the idea that 5-HT(1A)R internalization might underlie 5-HT(1A) autoreceptor desensitization under SSRI antidepressant therapy.

Differential pharmacological alleviation of oxaliplatin-induced hyperalgesia/allodynia at cephalic versus extra-cephalic level in rodents.

Previous data showed that neuropathic pain induced by mechanical lesion of peripheral nerves responds differently to alleviating drugs at cephalic versus extracephalic level. Because neuropathic pain evoked by anti-cancer drugs differs from that triggered by mechanical nerve lesion, we investigated whether differences between cephalic and extracephalic levels could also be characterized in rodents rendered neuropathic by treatment with the anti-cancer platinum derivative oxaliplatin. C57BL/6J mice received two injections and Sprague-Dawley rats three injections of oxaliplatin (10 mg/kg, i.p.) or its vehicle, with three days intervals. Supersensitivity to mechanical (von Frey filaments), cold (acetone drop) and chemical/inflammatory (formalin) stimulations was assessed in vibrissae and hindpaw territories. Transcripts of neuroinflammatory markers were quantified by real-time RT-qPCR in rat ganglia and central tissues. Oxaliplatin induced mechanical allodynia, cold hyperalgesia and chemical/inflammatory supersensitivity at both hindpaw and vibrissal levels in mice and rats. Acute treatment with gabapentin (30 mg/kg i.p.), morphine (3 mg/kg s.c.) or the 5-HT1A receptor agonist 8-OH-DPAT (0.16 mg/kg s.c.) significantly reduced oxaliplatin-induced supersensitivity in hindpaw but not vibrissal territory. In contrast, the antimigraine drugs naratriptan (0.1 mg/kg s.c.) and olcegepant (0.6 mg/kg i.v.) decreased oxaliplatin-induced supersensitivity in vibrissal territory only. Among the various markers investigated, only TRPA1 transcript was upregulated in ganglia of oxaliplatin-treated rats. These data showed that oxaliplatin induced supersensitivity to various stimuli in both cephalic and extra-cephalic territories in rodents. Regional differences in the efficacy of drugs to alleviate oxaliplatin-induced allodynia/hyperalgesia further support the idea that mechanisms underlying neuropathic pain have peculiarities at cephalic versus extra-cephalic level.

Juvenile ethanol exposure increases rewarding properties of cocaine and morphine in adult DBA/2J mice.

Convergent data showed that ethanol exposure during adolescence can alter durably ethanol-related behaviour at adulthood. However, the consequences of juvenile ethanol exposure on the reinforcing effects of other drugs of abuse remain unclear. In the present work, we evaluated in adult male DBA/2J mice the effects of early ethanol exposure on the sensitivity to the incentive effects of cocaine and morphine, and on extracellular signal-regulated kinase (ERK) activation in response to cocaine. Juvenile male mice received intragastric administration of ethanol (2×2.5g/kg/day) or water for 5 days starting on postnatal day 28. When reaching adult age (10 week-old), animals were subjected to an unbiased procedure to assess conditioned place preference (CPP) to cocaine or morphine. In addition, activation of ERK in response to an acute injection of cocaine was investigated using immunoblotting in the striatum and the nucleus accumbens. Mice that have been subjected to early ethanol exposure developed CPP to doses of cocaine (5mg/kg) or morphine (10mg/kg) below the threshold doses to induce CPP in water pre-exposed mice. In addition, early ethanol administration significantly increased striatal ERK phosphorylation normally induced by acute cocaine (10 and 20mg/kg) in adult mice. These results show that, in DBA/2J mice, early exposure to ethanol enhanced the perception of the incentive effects of cocaine and morphine. Ethanol pre-exposure also induced a positive modulation of striatal ERK signalling, in line with the inference that juvenile ethanol intake may contribute to the development of addictive behaviour at adult age.

Monoamine neurocircuitry in depression and strategies for new treatments.

Extensive studies showed that monoaminergic neurotransmission that involves serotonin (5-HT), norepinephrine (NE) and dopamine (DA) exerts major influence on brain circuits concerned by the regulation of mood, reactivity to psychological stress, self-control, motivation, drive, and cognitive performance. Antidepressants targeting monoamines directly affect the functional tone of these circuits, notably in limbic and frontocortical areas, and evidence has been provided that this action plays a key role in their therapeutic efficacy. Indeed, at least some of functional changes detected by functional magnetic resonance imaging in emotion- and cognitive-related circuits such as the one involving limbic-cortical-striatal-pallidal-thalamic connections in depressed patients can be reversed by monoamine-targeted antidepressants. However, antidepressants acting selectively on only one monoamine, such as selective inhibitors of 5-HT or NE reuptake, alleviate depression symptoms in a limited percentage of patients, and are poorly effective to prevent recurrence. Thorough investigations for the last 30 years allowed the demonstration of the existence of functional interactions between 5-HT, NE and DA systems, and the identification of the specific receptors involved. In particular, 5-HT systems were shown to exert negative influence on NE and DA systems through 5-HT2A and 5-HT2C receptor- mediated mechanisms, respectively. On the other hand, complex positive and negative influences of NE system on 5-HT neurotransmission are mediated through α1- and α2-adrenergic receptors, respectively. These data provided a rationale for the design of new, multimodal, therapeutic strategies involving drugs acting not only at the "historical" targets such as the 5-HT and/or the NE transporter, but also at other molecular targets to improve their efficacy and their tolerability.