Jean-Guy Chabot

Neuroprotective effects of resveratrol against β-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C

Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. The present study evaluated the neuroprotective effects of resveratrol against amyloid β(Aβ)‐induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. Pre‐, co‐ and post‐treatment with resveratrol significantly attenuated Aβ‐induced cell death in a concentration‐dependent manner, with a concentration of 25 μM being maximally effective. Pretreatment (1 h) of hippocampal cells with phorbol‐12‐myristate‐13‐acetate, a PKC activator, at increasing concentrations (1–100 ng ml−1), resulted in a dose‐dependent reduction in Aβ‐induced toxicity, whereas the inactive 4α‐phorbol had no effect. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 μM), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Aβ‐induced cell death. Treatment of hippocampal cells with resveratrol (20 μM) also induced the phosphorylation of various isoforms of PKC leading to activation. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Aβ‐induced toxicity.

Insulin-like growth factor-1 (somatomedin-C) receptors in the rat brain: distribution and interaction with the hippocampal cholinergic system

The present work characterizes the autoradiographic distribution of insulin-like growth factor-1 (IGF-1)/somatomedin-C binding sites in neonatal and adult rat brain, and attempts to correlate the distribution of IGF-1 sites, in certain regions of the rat brain, with functional IGF-1 receptors. In neonatal brain, [125I]IGF-1 binding sites are especially concentrated in superficial cortical layers, nucleus accumbens and hippocampus. In the adult rat brain, the distribution of IGF-1 sites is broader, with a high density of sites observed in superficial and deep cortical layers, olfactory bulb, endopiriform nucleus, basomedial nucleus of the amygdala, thalamic nuclei and hippocampus. Specific binding of [125I]IGF-1 to its sites in these brain regions was almost completely inhibited by 100 nM nonradioactive IGF-1. In contrast, similar concentrations of either IGF-2 or insulin did not significantly alter [125I]IGF-1 binding to its sites. Therefore, under our incubation conditions, [125I]IGF-1 appears to label specifically the type-I IGF receptor. In the hippocampus, which is highly enriched with specific [125I]IGF-1 binding sites in both neonatal and adult rat brain, IGF-1 significantly altered the potassium-evoked (25 mM) release of acetylcholine (ACh) from slices of adult, but not immature (6- and 18-day-old), rat brain. This IGF-1-induced decrease in ACh release from adult rat brain slices was concentration-dependent and appeared to be specific to hippocampus; ACh release from frontal cortical slices was not affected by this GF. The spontaneous release of ACh in the presence of IGF-1 in either tissue was not significantly different from control.(ABSTRACT TRUNCATED AT 250 WORDS)

Knockdown of spinal metabotropic glutamate receptor 1 (mGluR1) alleviates pain and restores opioid efficacy after nerve injury in rats

Nerve injury often produces long‐lasting spontaneous pain, hyperalgesia and allodynia that are refractory to treatment, being only partially relieved by clinical analgesics, and often insensitive to morphine. With the aim of assessing its therapeutic potential, we examined the effect of antisense oligonucleotide knockdown of spinal metabotropic glutamate receptor 1 (mGluR1) in neuropathic rats. We chronically infused rats intrathecally with either vehicle, or 50 μg day−1 antisense or missense oligonucleotides beginning either 3 days prior to or 5 days after nerve injury. Cold, heat and mechanical sensitivity was assessed prior to any treatment and again every few days after nerve injury. Here we show that knockdown of mGluR1 significantly reduces cold hyperalgesia, heat hyperalgesia and mechanical allodynia in the ipsilateral (injured) hindpaw of neuropathic rats. Moreover, we show that morphine analgesia is reduced in neuropathic rats, but not in sham‐operated rats, and that knockdown of mGluR1 restores the analgesic efficacy of morphine. We also show that neuropathic rats are more sensitive to the excitatory effects of intrathecally injected N‐methyl‐D‐aspartate (NMDA), and have elevated protein kinase C (PKC) activity in the spinal cord dorsal horn, two effects that are reversed by knockdown of mGluR1. These results suggest that activity at mGluR1 contributes to neuropathic pain through interactions with spinal NMDA receptors and PKC, and that knockdown of mGluR1 may be a useful therapy for neuropathic pain in humans, both to alleviate pain directly, and as an adjunct to opioid analgesic treatment.

LOCALIZATION AND MODULATION OF CALCITONIN GENE-RELATED PEPTIDE-RECEPTOR COMPONENT PROTEIN-IMMUNOREACTIVE CELLS IN THE RAT CENTRAL AND PERIPHERAL NERVOUS SYSTEMS

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats.

&NA; Coincident with nociception, both noxious chemical stimulation of the hind paw and chronic constriction injury (CCI) of the sciatic nerve produce an increase in protein kinase C (PKC) translocation in the spinal cord of rats. Noxious stimulus‐induced PKC translocation likely depends on glutamate activity at either N‐methyl‐D‐aspartate (NMDA) receptors or group I metabotropic glutamate receptors (mGluR1/5) in the spinal cord dorsal horn. This study compares nociceptive responses to, and the alterations in membrane‐associated PKC, induced by noxious chemical stimulation of the hindpaw and CCI of the sciatic nerve, as well as their modulation by both NMDA and mGluR1/5 receptor antagonists. Three groups of rats were given a single intrathecal (i.t.) injection of either vehicle, dizocilpine maleate (MK‐801, 60 nmol), an NMDA receptor antagonist, or (S)‐4‐carboxyphenylglycine (S)‐4CPG, (150 nmol), an mGluR1/5 antagonist, 10 min prior to a 50 &mgr;l of 2.5% formalin injection into the ventral surface of one hind paw. Another three groups of rats were given twice daily injections of either vehicle, MK‐801 (30 nmol) or (S)‐4CPG (90 nmol) i.t. for 5 days starting 30 min before CCI or sham injury of the sciatic nerve. Nociceptive responses were assessed for a 60 min period after the formalin injection in the first three groups, and tests of mechanical and cold allodynia were performed on days 4, 8, 12 and 16 after CCI for the latter three groups. Furthermore, changes in the levels of membrane‐associated PKC, as assayed by quantitative autoradiography of the specific binding of [3H]‐phorbol 12,13‐dibutyrate ([3H]‐PDBu) in the dorsal horn of the lumbar spinal cord sections, were assessed in formalin‐injected rats (at 5, 25 and 60 min) and in neuropathic rats 5 days after CCI, treated (as above) with vehicle, MK‐801 or (S)‐4CPG. The results indicate that i.t. treatment with MK‐801 significantly reduced nociceptive scores in the formalin test and also produced a significant suppression of formalin‐induced increases in [3H]‐PDBu binding in laminae I–II, III–VI and X of the lumbar spinal cord. In contrast, i.t. treatment with (S)‐4CPG failed to significantly affect either nociceptive behaviours in the formalin test or formalin‐induced increases in [3H]‐PDBu binding in laminae I–II and III–VI of the lumbar spinal cord. On the other hand, i.t. treatment with either MK‐801 or (S)‐4CPG produced a significant reduction in mechanical and cold hypersensitivity, as well as [3H]‐PDBu binding in laminae I–II and III–VI of the lumbar spinal cord, after CCI. These results suggest that while NMDA, but not mGluR1/5, receptors are involved in translocation of PKC and nociception in a model of persistent acute pain, both types of receptors influence the translocation of PKC in dorsal horn and mechanical and cold allodynia in a model of chronic neuropathic pain.

Expression of the purported sigma1 (σ1) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of sigma receptor binding sites (sigma(1) and sigma(2)) is now well established. The recent cloning of the mouse, guinea pig and human sigma(1) receptors has allowed the study of the discrete distribution of the sigma(1) receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific sigma(1) receptor mRNA signals were in accordance to the anatomical distribution of sigma(1) receptor protein first established by quantitative receptor autoradiography. Specific sigma(1) receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I-II) and deeper (IV-VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of sigma(1) receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the sigma(1) receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of sigma(1) receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV-VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the sigma(1) receptor mRNA. Antisense oligodeoxynucleotides against the purported sigma(1) receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific sigma(1) receptor antisense into the third ventricle (0.4 nmol/h for 5 days), sigma(1)/[3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the sigma(1) receptor antisense treatments (5 nmol/injection, every 12 hx3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the sigma(1) receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported sigma(1) receptor gene reduced specific sigma(1)/[3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Cell-type specific activation of p38 and ERK mediates calcitonin gene-related peptide involvement in tolerance to morphine-induced analgesia

Tolerance to morphine‐induced analgesia is a well‐established phenomenon, often limiting its usefulness in the long‐term treatment of pain. The mechanisms underlying tolerance are not well understood. We previously suggested a possible role for spinal calcitonin gene‐related peptide (CGRP) in the development of tolerance to morphine‐induced analgesia. In the present study, we demonstrate that CGRP is involved in morphine tolerance by differentially regulating the ERK‐dependent up‐regulation of IL‐iβ, TNF‐α, and microsomal prostaglandin E synthase‐1 (mPGES‐1) in astrocytes and p38‐dependent up‐regulation of IL‐6 in microglia in the rat spinal cord. A 7‐d treatment with morphine induced tolerance to the antinociceptive effect and increased phosphorylated ERK localized in astrocytes and phosphorylated p38 enriched in microglia, both effects being inhibited by blocking CGRP receptors. Interestingly, the inhibition of the ERK pathway suppressed the development of tolerance and morphine‐induced up‐regulation of IL‐ iβ, TNF‐α, and mPGES‐1. Blockade of p38 activity also inhibited the development of tolerance and morphine‐ induced IL‐6 up‐regulation. Taken together, these data suggest that chronic morphine induces the synthesis of CGRP, which in turn acts on CGRP receptors located on astrocytes and microglia to stimulate ERK and p38, respectively, leading to increased synthesis and release of proinflammatory mediators resulting in tolerance to morphine‐induced analgesia.— Wang, Z.,Ma, W., Chabot, J.‐G., Quirion, R. Cell‐type specific activation of p38 and ERK mediates calcitonin gene‐related peptide involvement in tolerance to morphine‐induced analgesia. FASEBJ. 23, 2576–2586 (2009)

Expression of calcitonin gene-related peptide, substance P and protein kinase C in cultured dorsal root ganglion neurons following chronic exposure to mu, delta and kappa opiates.

The mechanisms involved in morphine tolerance are poorly understood. It was reported by our group that calcitonin gene-related peptide (CGRP)-like immunoreactivity (IR) was increased in the spinal dorsal horn during morphine tolerance [Ménard et al. (1996) J. Neurosci. 16, 2342-2351]. More recently, we observed that it was possible to mimic these results in cultured dorsal root ganglion (DRG) neurons allowing for more detailed mechanistic studies [Ma et al. (2000) Neuroscience 99, 529-539]. The aim of the present series of experiments was to further validate the DRG cell culture model by establishing which subtypes of opioid receptors are involved in the induction of CGRP in cultured rat DRG neurons, and to examine the signaling pathway possibly involved in the induction of CGRP-like IR following repeated opiate treatments. Other neuropeptides known to be expressed in DRG neurons, such as substance P (SP), neuropeptide Y (NPY) and galanin, were investigated to assess specificity. Following treatment with any of the three opioid agonists (mu, DAMGO; delta, DPDPE; kappa, U50488H), the number of CGRP- and SP-IR cultured DRG neurons increased significantly, and in a concentration-dependent manner, with the effects of kappa agonist being less pronounced. NPY and galanin were not affected.Double-immunofluorescence staining showed that the three opioid receptors were co-localized with both CGRP- and SP-like IR.Protein kinase C (PKC)-like IR was found to be significantly increased following a repetitive treatment with DAMGO. Double-immunofluorescence staining showed the co-localization of PKCalpha with CGRP- and SP-IR in cultured DRG neurons. Moreover, a combined treatment with DAMGO and a PKC inhibitor (chelerythrine chloride or Gö 6976) was able to block the effects of the opioid on increased CGRP-like IR. These data suggest that the three opioid receptors may be involved in the induction of CGRP and SP observed following chronic exposure to opiates, and that PKC probably plays a role in the signaling pathway leading to the up-regulation of these neuropeptides. These findings further validate the DRG cell culture as a suitable model to study intracellular pathways that govern changes seen following repeated opioid treatments possibly leading to opioid tolerance.

Agonist‐ and antagonist‐induced sequestration/internalization of neuropeptide Y Y1 receptors in HEK293 cells

Neuropeptide Y Y1 receptors are known to internalize following the binding of agonists. In the present study, a pseudopeptide Y1 receptor antagonist, homodimeric Ile‐Glu‐Pro‐Dpr‐Tyr‐Arg‐Leu‐Arg‐Tyr‐CONH2 (GR231118), also induced Y1 receptor internalization in human embryonic kidney (HEK293) cells. We demonstrated first that both specifically bound radiolabeled antagonist ([125I]GR231118) and agonist ([125I][Leu31, Pro34]PYY) underwent receptor‐mediated sequestration/internalization in transfected HEK293 cells. Agonist‐induced Y1 receptor internalization was dependent on clathrin‐coated pits and was regulated in part by Gi/o‐protein activation as revealed by pertussin toxin sensitivity. In contrast, antagonist‐induced sequestration of Y1 receptors was partly dependent on clathrin‐coated pits, but independent from Gi/o‐protein activation. Exposure to high concentrations of agonist or antagonist caused a 50 and 75% loss of cell surface binding, respectively. The loss caused by the agonist rapidly recovered. This phenomenon was blocked by monensin, an inhibitor of endosome acidification, suggesting that cell surface receptor recovery is due to recycling. In contrast to the agonist, GR231118 induced a long‐lasting sequestration of Y1 receptors in HEK293 cells. Immunofluorescence labeling indicated that following 40 min of incubation with either the agonist or the antagonist, Y1 receptors followed markedly different intercellular trafficking pathways. Taken together, these findings provided evidence that a pseudopeptide Y1 receptor antagonist can induce long‐lasting disappearance of cell surface receptors through a pathway distinct from the classical endocytic/recycling pathway followed by stimulation with an agonist.