Mice lacking the serotonin Htr2B receptor gene present an antipsychotic-sensitive schizophrenic-like phenotype

Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. Recently, a population-specific serotonin 2B (5-HT2B) receptor stop codon (ie, HTR2B Q20*) was reported to segregate with severely impulsive individuals, whereas 5-HT2B mutant (Htr2B−/−) mice also showed high impulsivity. Interestingly, in the same cohort, early-onset schizophrenia was more prevalent in HTR2B Q*20 carriers. However, the putative role of 5-HT2B receptor in the neurobiology of schizophrenia has never been investigated. We assessed the effects of the genetic and the pharmacological ablation of 5-HT2B receptors in mice subjected to a comprehensive series of behavioral test screenings for schizophrenic-like symptoms and investigated relevant dopaminergic and glutamatergic neurochemical alterations in the cortex and the striatum. Domains related to the positive, negative, and cognitive symptom clusters of schizophrenia were affected in Htr2B−/− mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr2B−/− mice presented with enhanced locomotor response to the psychostimulants dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment. We report herein that 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provide first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders

Glutamatergic Modulation of Cue-Induced Drug-Seeking Behavior in the Rat

The characteristics of drug addiction include compulsive drug use despite negative consequences and re-occurring relapses, returns to drug use after a period of abstinence. Therefore, relapse prevention is one of the major challenges for the treatment of drug addiction. There are three main factors capable of inducing craving for drugs and triggering relapse long after cessation of drug use and dissipation of physical withdrawal signs: stress, re-exposure to the drug, and environmental stimuli (cues) that have been previously associated with drug use. The neurotransmitters dopamine and glutamate have been implicated in the modulation of drug-seeking behavior. The aim of this project was to examine the role of glutamatergic neurotransmission in relapse triggered by conditioned drug-associated stimuli. The focus was on clarifying whether relapse to drug seeking can be attenuated by blockade of glutamate receptors. In addition, as the nucleus accumbens has been proposed to participate in the modulation of drug-seeking behavior, the effects of glutamate receptor blockade in this brain structure on cue-induced relapse were investigated. The studies employed animals models in which rats were trained to press a lever in a test cage to obtain alcohol or intravenous cocaine. Drug availability was paired with distinct olfactory, auditory, or visual stimuli. This phase was followed by extinction training, during which lever presses did not result in the presentation of the drug or the drug-associated stimuli. Extinction training led to a gradual decrease in the number of lever presses during test sessions. Relapse was triggered by presenting the rats with the drug-associated stimuli in the absence of alcohol or cocaine. The drug-associated stimuli were alone capable of inducing resumption of lever pressing and maintaining this behavior during repeated testing. The number of lever presses during a session represented the intensity of drug-seeking and relapse behavior. The results suggest that glutamatergic neurotransmission is involved in the modulation of drug-seeking behavior. Both alcohol and cocaine relapse were attenuated by systemic pretreatment with glutamate receptor antagonists. However, differences were found in the ability of ionotropic AMPA/kainate and NMDA receptor antagonists to regulate drug-seeking behavior. The AMPA/kainate antagonists CNQX and NBQX, and L-701,324, an antagonist with affinity for the glycine site of the NMDA receptor, attenuated cue-induced drug seeking, whereas the competitive NMDA antagonist CGP39551 and the NMDA channel blocker MK-801 were without effect. MPEP, an antagonist at metabotropic mGlu5 glutamate receptors, also decreased drug seeking, but its administration was found to lead to conditioned suppression of behavior during subsequent treatment sessions, suggesting that MPEP may have undesirable side effects. The mGluR2/3 agonist LY379268 and the mGluR8 agonist (S)-3,4-DCPG decreased both cue-induced relapse to alcohol drinking and alcohol consumption. Control experiments showed however that administration of the agonists was accompanied by motor suppression limiting their usefulness. Administration of the AMPA/kainate antagonist CNQX, the NMDA antagonist D-AP5, and the mGluR5 antagonist MPEP into the nucleus accumbens resulted also in a decrease in drug-seeking behavior, suggesting that the nucleus accumbens is at least one of the anatomical sites regulating drug seeking and mediating the effects of glutamate receptor antagonists on this behavior.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4-10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+-permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+-permeable AMPAR modulation). Co-inhibiting PKA, GSK-3 beta and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

Inibição do receptor de glutamato do tipo NMDA em um modelo de hipóxia-isquemia prenatal: avaliação morfofuncional do cerebelo

Lesões sistêmicas peri e pré-natais alteram o desenvolvimento do SNC, levando a problemas cognitivos e motores em crianças que podem perdurar por toda a vida. Um tipo particular de lesão é a hipóxia-isquemia (HI), caracterizada pela interrupção momentânea ou permanente do fluxo sanguíneo. Um dos mecanismos propostos para as lesões decorrentes da HI é a excitotoxicidade glutamatérgica. O uso de inibidores da neurotransmissão glutamatérgica tem sido estudados em diversos modelos de HI. Neste trabalho, avaliamos os efeitos morfofuncionais da administração de um antagonista não-competitivo do receptor de glutamato NMDA sobre o desenvolvimento do cerebelo. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo H). Animais controle tiveram os úteros expostos, sem a obstrução (Grupo S). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um dia após o nascimento, metade de cada ninhada foi designada para receber MK801, 0,3mg/kg/dia, (grupos SM e HM) e a outra metade recebeu solução salina (grupos SS e HS), por 5 dias. Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23, 30 e 60 dias pós-natais, cortes parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para calbindina, GFAP, GLAST, PDGFRα e MBP. A partir de 45 dias de vida, os animais foram testados em vários de testes comportamentais: labirinto em cruz elevado (LCE), campo vazado (CV), ROTAROD, teste de caminhada sobre barras (ladder test) e teste do comprimento da passada (stride length). Aos 9 dias, a espessura da árvore dendrítica era menor nos animais SM, HS/HM, demonstrando efeitos deletérios tanto do MK801 quanto da HI. Menor número de células PDGFRα+ foi observado nos animais HS/HM, sem efeitos da administração de MK801. Aos 23 dias, maior número de células PDGFRα+ foi observado nos animais HM comparado aos outros 3 grupos, indicando efeito neuroprotetor do MK801. Nessa idade, menor número de fibras mielinizadas (MBP+) foi observada nos animais HS, e a administração de MK801 parece reverter estes efeitos. Aos 9 dias a distribuição de GLAST estava alterada nos animais HS, com os efeitos da HI parcialmente revertidos pelo MK801. Não foram observados efeitos da HI ou do MK801 sobre comportamentos relacionados a ansiedade pelo LCE, assim como na latência de queda no ROTAROD. HI piora a performance motora no ladder test. No teste do CV, não observamos efeitos da HI sobre a busca por novidade assim como sobre a atividade locomotora espontânea. No entanto, MK801 diminui comportamentos de autolimpeza e a atividade locomotora espontânea. Menor variação das passadas foi observada em decorrência da administração de MK801 no stride length, com nenhum efeito da HI. Nossos resultados demonstram que a inibição do receptor NMDA tem um efeito neuroprotetor sobre os progenitores de oligodendrócitos e mielinização, provavelmente pela manutenção da capacidade proliferativa por um período maior. A atividade do receptor NMDA exerce importante papel na diferenciação das células de Purkinje, assim como na distribuição do transportador GLAST, corroborando a importância deste receptor na gênese das lesões causadas pela HI.

GABAA receptor chloride channels are involved in the neuroprotective role of GABA following oxygen and glucose deprivation in the rat cerebral cortex but not in the hippocampus.

Assays on "ex vivo" sections of rat hippocampus and rat cerebral cortex, subjected to oxygen and glucose deprivation (OGD) and a three-hour reperfusion-like (RL) recovery, were performed in the presence of either GABA or the GABA(A) receptor binding site antagonist, bicuculline. Lactate dehydrogenase (LDH) and propidium iodide were used to quantify cell mortality. We also measured, using real-time quantitative polymerase chain reaction (qPCR), the early transcriptional response of a number of genes of the glutamatergic and GABAergic systems. Specifically, glial pre- and post-synaptic glutamatergic transporters (namely GLAST1a, EAAC-1, GLT-1 and VGLUT1), three GABAA receptor subunits (α1, β2 and γ2), and the GABAergic presynaptic marker, glutamic acid decarboxylase (GAD65), were studied. Mortality assays revealed that GABAA receptor chloride channels play an important role in the neuroprotective effect of GABA in the cerebral cortex, but have a much smaller effect in the hippocampus. We also found that GABA reverses the OGD-dependent decrease in GABA(A) receptor transcript levels, as well as mRNA levels of the membrane and vesicular glutamate transporter genes. Based on the markers used, we conclude that OGD results in differential responses in the GABAergic presynaptic and postsynaptic systems.

An investigation into mechanisms of visceral pain in rodents

Visceral pain is a debilitating symptom of irritable bowel syndrome (IBS), a disorder affecting up to 30% of adults. A better understanding of the mechanisms underlying visceral hypersensitivity may facilitate development of more targeted therapies, improving the quality of life of these individuals. The studies performed in this thesis were designed to investigate important factors of visceral pain, including early-life manipulations, genetic predisposition and sex hormones. Maternal separation (MS) consistently reproduces visceral hypersensitivity and altered anxiety-like behaviours in rats, symptoms associated with IBS. It has been found that 5-HT2B receptor antagonism blocks visceral pain but no difference in relative 5-HT2B receptor mRNA expression was found in hippocampus, amygdala and colon. The neuronal activation patterns of prefrontal cortex and amygdala of MS rats were then investigated. MS animals are characterised by differential activation of the prefrontal cortex (anterior cingulate cortex (ACC), infralibic cortex, prelimbic cortex) as well as the central nucleus of the amygdala (CeA). Genetic factors also contribute to pain syndromes such as IBS. We utilised the Wistar Kyoto (WKY) rat, a stress-sensitive strain, as an animal model of brain-gut axis dysfunction. WKY rats have a lower expression of the glutamate transporter EAAT2 and mGlu4 receptor in the ACC. Another early-life factor that can increase susceptibility to functional gastrointestinal symptoms later life is disruption of the gut microbiota, thus early-life antibiotic treatment was used to assess this effect. Antibiotic treatment induced visceral hypersensitivity in adulthood and may be related to observed reductions in spinal cord alpha-2A adrenoreceptor (adra2A) mRNA. Lastly, we investigated sex differences in visceral sensitivity. EAAT1 & 2 mRNA levels are lower in females, potentially increasing glutamatergic concentration at the symaptic level. Moreover, NR1 and NR2B subunits mRNA of NMDA receptor were increased in caudal ACC of females. These findings may account for sex differences in visceral sensitivity.

Changes in midbrain pain receptor expression, gait and behavioral sensitivity in a rat model of radiculopathy.

Intervertebral disc herniation may contribute to inflammatory processes that associate with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait and the expression of key pain receptors in the midbrain in a rodent model of radiculopathy. Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in rats (NP-treated, n=12). Tail NP was discarded in sham-operated animals (n=12). Mechanical allodynia, weight-bearing, and gait were evaluated in all animals over time. At 1 and 4 weeks after surgery, astrocyte and microglial activation was tested in DRG sections. Midbrain sections were similarly evaluated for immunoreactivity to serotonin (5HT(2B)), mu-opioid (µ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the duration of the study. Astroctye activation was observed at DRGs only at 4 weeks after surgery. Findings for pain receptors in the midbrain of NP-treated rats included an increased expression of 5HT(2B) at 1, but not 4 weeks; increased expression of µ-OR and mGluR5 at 1 and 4 weeks (periaqueductal gray region only); and no changes in expression of mGluR4 at any point in this study. These observations provide support for the hypothesis that the midbrain responds to DRG injury with a transient change in receptors regulating pain responses.

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Y1 receptor knockout increases nociception and prevents the anti-allodynic actions of NPY.

OBJECTIVE: Recent pharmacologic studies in our laboratory have suggested that the spinal neuropeptide Y (NPY) Y1 receptor contributes to pain inhibition and to the analgesic effects of NPY. To rule out off-target effects, the present study used Y1-receptor-deficient (-/-) mice to further explore the contribution of Y1 receptors to pain modulation. METHODS AND RESULTS: Y1(-/-) mice exhibited reduced latency in the hotplate test of acute pain and a longer-lasting heat allodynia in the complete Freund's adjuvant (CFA) model of inflammatory pain. Y1 deletion did not change CFA-induced inflammation. Upon targeting the spinal NPY systems with intrathecal drug delivery, NPY reduced tactile and heat allodynia in the CFA model and the partial sciatic nerve ligation model of neuropathic pain. Importantly, we show for the first time that NPY does not exert these anti-allodynic effects in Y1(-/-) mice. Furthermore, in nerve-injured CD1 mice, concomitant injection of the potent Y1 antagonist BIBO3304 prevented the anti-allodynic actions of NPY. Neither NPY nor BIBO3304 altered performance on the Rotorod test, arguing against an indirect effect of motor function. CONCLUSION: The Y1 receptor contributes to pain inhibition and to the analgesic effects of NPY.

A possible role for chemokine signalling through CXCR4 as a downstream effector of retinoic acid signalling in the regenerating tail and spinal cord of Notophthalmus viridescens

The newt, Notopthalmus viridescens is one of the few tet rapod vertebrates capable of extensive regeneration of the central nervous system, however, the factors involved in this process are still unknown. Chemokine signalling through the receptor CXCR4, has been found to be involved in the development of the central nervous system of mammals and more recently in epimorphic fin regeneration in zebrafish. We have hypothesized that the CXCR4 signalling pathway is involved in spinal cord and tail regeneration in the adul t newt , possibly as a downstream target of retinoic acid signalling. We found that CXCR4 mRNA expression was observed in the brain, spinal cord, heart, gut, liver and regenerating tail blastemas. CXCR4 expression increased over the f i rst 12 days of tail regeneration and returned to basal expression levels at day 21 of regeneration. Inhibition of CXCR4 wi th AMD3100, a specific receptor antagonist, led to a decrease in CXCR4 mRNA in the regenerating tail 14 days post amputation. Histological analysis suggests a delay in the early stages of tail and spinal cord regeneration. Spinal cord explants t reated wi th CXCL12, the ligand to CXCR4, displayed enhanced neurite outgrowth in vitro. Explants t reated wi th AMD3100 abolished any retinoic acid enhanced neurite outgrowth effects suggesting a link between these signalling pathways.

Liquid Chromatography-Electrospray Linear Ion Trap Mass Spectrometry Analysis of Targeted Neuropeptides in Tac1-/- Mouse Spinal Cords Reveal Significant Lower Concentration of Opioid Peptides.

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop a HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1-/- mouse endogenous opioid system is hampered and therefore affect significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1-/- spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p < 0.0001). No significant concentration differences were observed in mouse Tac1-/- spinal cords for Met-Enk and CGRP. The analysis of Tac1-/- mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Neurotensinergic modulation of glutamatergic neurotransmission in VTA neurons.

L’aire tegmentaire ventrale (VTA) contient une forte densité de terminaisons neurotensinergiques ainsi que des récepteurs à la surface des neurones dopaminergiques et non-dopaminergiques. Le VTA a été impliqué dans des maladies comme la schizophrénie, les psychoses et l’abus de substance. Les drogues d’abus sont connues pour induire le phénomène de sensibilisation - un processus de facilitation par lequel l’exposition à un stimulus produit une réponse augmentée lors de l’exposition subséquente au même stimulus. La sensibilisation se développe dans le VTA et implique mécanismes dopaminergiques et glutamatergiques. Il a été montré que les antagonistes neurotensinergiques bloquaient le développement de la sensibilisation et certains mécanismes de récompense et ces effets pourraient être médiés indirectement par une modulation de la neurotransmission glutamatergique. Cependant, on connaît peu les mécanismes de modulation de la transmission glutamatergique par la neurotensine (NT) dans le VTA. Le but de la présente thèse était d’étudier la modulation neurotensinergique de la neurotransmission glutamatergique dans les neurones dopaminergiques et non-dopaminergiques du VTA. Pour ce faire, nous avons utilisé la technique du patch clamp dans la cellule entière dans des tranches horizontales du VTA pour étudier les effets de différents agonistes et antagonistes neurotensinergiques. Les neurones ont été identifié comme Ih+ (présumés dopaminergiques) ou Ih- (présumés non-dopaminergiques) selon qu’ils exprimaient ou non un courant cationique activé par l’hyperpolarisation (Ih). Des techniques d’immunocytochimie ont été utilisées pour marquer les neurones et vérifier leur localisation dans le VTA. Dans une première étude nous avons trouvé que la neurotensine indigène (NT1-13) ou son fragment C-terminal, NT8-13, induisait une augmentation comparable des courants postsynaptiques excitateurs glutamatergiques (CPSEs) dans les neurones Ih+ ou Ih- du VTA. L'augmentation induite dans les neurones Ih+ par la NT8-13 a été bloquée par le SR48692, un antagoniste des récepteurs NTS1, et par le SR142948A, un antagoniste des récepteurs NTS1 et NTS2, suggérant que l'augmentation était médiée par l’activation des récepteurs NTS1. Dans les neurones Ih- l'augmentation n’a été bloquée que par le SR142948A indiquant une implication des récepteurs NTS2. Dans une deuxième étude, nous avons testé les effets de la D-Tyr[11]NT (un analogue neurotensinergique ayant différentes affinités de liaison pour les sous-types de récepteurs neurotensinergiques) sur les CPSEs glutamatergiques dans les neurones Ih+ et Ih- en parallèle avec une série d’expériences comportementales utilisant un paradigme de préférence de place conditionnée (PPC) menée dans le laboratoire de Pierre-Paul Rompré. Nous avons constaté que la D-Tyr[11]NT induisaient une inhibition dépendante de la dose dans les neurones Ih+ médiée par l'activation de récepteurs NTS2. En revanche, la D-Tyr[11]NT a produit une augmentation des CPSEs glutamatergiques médiée par des récepteurs NTS1 dans les neurones Ih-. Les résultats des expériences comportementales ont montré que des microinjections bilatérales de D-Tyr[11]NT dans le VTA induisait une PPC bloquée uniquement par la co-injection de SR142948A et SR48692, indiquant un rôle pour les deux types de récepteurs, NTS1 et NTS2. Cette étude nous a permis de conclure que i) la D-Tyr[11]NT agit dans le VTA via des récepteurs NTS1 et NTS2 pour induire un effet de récompense et ii) que cet effet est dû, au moins en partie, à une augmentation de la neurotransmission glutamatergique dans les neurones non-dopaminergiques (Ih-). Dans une troisième étude nous nous sommes intéressés aux effets de la D-Tyr[11]NT sur les réponses isolées médiées par les récepteurs N-méthyl-D-aspartate (NMDA) et acide α-amino-3- hydroxy-5-méthyl-4-isoxazolepropionique (AMPA) dans les neurones du VTA. Nous avons constaté que dans les neurones Ih+ l’amplitude des CPSEs NMDA et AMPA étaient atténuées de la même manière par la D-Tyr[11] NT. Cette modulation des réponses était médiée par les récepteurs NTS1 et NTS2. Au contraire, dans les neurones Ih-, l’amplitude des réponses NMDA et AMPA étaient augmentées en présence de D-Tyr[11]NT et ces effets dépendaient de l’activation des récepteurs NTS1 localisés sur les terminaisons glutamatergiques. Ces résultats fournissent une preuve supplémentaire que le NT exerce une modulation bidirectionnelle sur la neurotransmission glutamatergique dans les neurones du VTA et met en évidence un nouveau type de modulation peptidergique des neurones non-dopaminergiques qui pourrait être impliqué dans la sensibilisation. En conclusion, la modulation neurotensinergique de la neurotransmission glutamatergique dans les neurones dopaminergiques et non-dopaminergiques du VTA se fait en sens opposé soit, respectivement, par une inhibition ou par une excitation. De plus, ces effets sont médiés par différents types de récepteurs neurotensinergiques. En outre, nos études mettent en évidence une modulation peptidergique de la neurotransmission glutamatergique dans le VTA qui pourrait jouer un rôle important dans les mécanismes de lutte contre la toxicomanie.

Glutamatergic Nmda And Ampa Receptors Functional Regulation In Streptozotocin Induced Diabetic Rats: Effect Of Vitamin D3 And Curcumin Supplementation

The present study was designed to investigate the protective effect of curcumin and vitamin D3 in the functional regulation of glutamatergic NMDA and AMPA receptors in streptozotocin (STZ) induced diabetic rats. Alterations in glutamatergic neurotransmission in the brain were evaluated by analyzing the glutamate content, glutamate receptors - NMDA and AMPA receptors binding parameters and gene expression, GAD and GLAST gene expression. Immunohistochemistry studies using confocal microscope were carried out to confirm receptor density and gene expression results of NMDA and AMPA receptors. The role of glutamatergic receptors in pancreas was studied using the following parameters; glutamate content, GLAST expression, glutamate receptors - NMDA and AMPA receptor binding and gene expression. Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of diabetes. In the present study SOD assay and GPx gene expression were done to evaluate the activity of antioxidant enzymes in the brain regions and pancreas. NeuroD1 and Pdx1 gene expression were performed in pancreas of experimental rats to evaluate pancreatic islet survival. Gene expression profiles of caspase 8, Bax, and Akt in brain regions and pancreas were studied to understand the possible mechanism behind curcumin and vitamin D3 mediated neuroprotection and islet survival. Gene expression studies of vitamin D3 receptor localisation in the pancreas was done to understand the mechanism of vitamin D3 in insulin secretion. Curcumin and vitamin D3 mediated insulin secretion via Ca2+ release were studied using confocal microscope.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous alpha,beta-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Protease-activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease-activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2-mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene-related peptide (CGRP), mediators of pain transmission. In PAR2-expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4alpha-phorbol 12,13-didecanoate (4alphaPDD) and hypotonic solutions. PAR2-agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cbeta and protein kinases A, C and D inhibited PAR2-induced sensitization of TRPV4 Ca2+ signals and currents. 4alphaPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4-dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4alphaPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist-induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4-dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.