Do glial cells control pain?

Management of chronic pain is a real challenge, and current treatments focusing on blocking neurotransmission in the pain pathway have only resulted in limited success. Activation of glia cells has been widely implicated in neuroinflammation in the central nervous system, leading to neruodegeneration in many disease conditions such as Alzheimer's and multiple sclerosis. The inflammatory mediators released by activated glial cells, such as tumor necrosis factor-α and interleukin-1β can not only cause neurodegeneration in these disease conditions, but also cause abnormal pain by acting on spinal cord dorsal horn neurons in injury conditions. Pain can also be potentiated by growth factors such as BDNF and bFGF that are produced by glia to protect neurons. Thus, glia cells can powerfully control pain when they are activated to produce various pain mediators. We will review accumulating evidence supporting an important role of microglia cells in the spinal cord for pain control under injury conditions (e.g. nerve injury). We will also discuss possible signaling mechanisms in particular MAP kinase pathways that are critical for glia control of pain. Investigating signaling mechanisms in microglia may lead to more effective management of devastating chronic pain.

Gene expression mapping in neuropathic pain : molecular plasticity in nociceptors and superficial dorsal horn

RESUME : La douleur neuropathique est le résultat d'une lésion ou d'un dysfonctionnement du système nerveux. Les symptômes qui suivent la douleur neuropathique sont sévères et leur traitement inefficace. Une meilleure approche thérapeutique peut être proposée en se basant sur les mécanismes pathologiques de la douleur neuropathique. Lors d'une lésion périphérique une douleur neuropathique peut se développer et affecter le territoire des nerfs lésés mais aussi les territoires adjacents des nerfs non-lésés. Une hyperexcitabilité des neurones apparaît au niveau des ganglions spinaux (DRG) et de la corne dorsale (DH) de la moelle épinière. Le but de ce travail consiste à mettre en évidence les modifications moléculaires associées aux nocicepteurs lésés et non-lésés au niveau des DRG et des laminae I et II de la corne dorsale, là où l'information nociceptive est intégrée. Pour étudier les changements moléculaires liés à la douleur neuropathique nous utilisons le modèle animal d'épargne du nerf sural (spared nerve injury model, SNI) une semaine après la lésion. Pour la sélection du tissu d'intérêt nous avons employé la technique de la microdissection au laser, afin de sélectionner une sous-population spécifique de cellules (notamment les nocicepteurs lésés ou non-lésés) mais également de prélever le tissu correspondant dans les laminae superficielles. Ce travail est couplé à l'analyse à large spectre du transcriptome par puce ADN (microarray). Par ailleurs, nous avons étudié les courants électriques et les propriétés biophysiques des canaux sodiques (Na,,ls) dans les neurones lésés et non-lésés des DRG. Aussi bien dans le système nerveux périphérique, entre les neurones lésés et non-lésés, qu'au niveau central avec les aires recevant les projections des nocicepteurs lésés ou non-lésés, l'analyse du transcriptome montre des différences de profil d'expression. En effet, nous avons constaté des changements transcriptionnels importants dans les nocicepteurs lésés (1561 gènes, > 1.5x et pairwise comparaison > 77%) ainsi que dans les laminae correspondantes (618 gènes), alors que ces modifications transcriptionelles sont mineures au niveau des nocicepteurs non-lésés (60 gènes), mais important dans leurs laminae de projection (459 gènes). Au niveau des nocicepteurs, en utilisant la classification par groupes fonctionnels (Gene Ontology), nous avons observé que plusieurs processus biologiques sont modifiés. Ainsi des fonctions telles que la traduction des signaux cellulaires, l'organisation du cytosquelette ainsi que les mécanismes de réponse au stress sont affectés. Par contre dans les neurones non-lésés seuls les processus biologiques liés au métabolisme et au développement sont modifiés. Au niveau de la corne dorsale de la moelle, nous avons observé des modifications importantes des processus immuno-inflammatoires dans l'aire affectée par les nerfs lésés et des changements associés à l'organisation et la transmission synaptique au niveau de l'aire des nerfs non-lésés. L'analyse approfondie des canaux sodiques a démontré plusieurs changements d'expression, principalement dans les neurones lésés. Les analyses fonctionnelles n'indiquent aucune différence entre les densités de courant tétrodotoxine-sensible (TTX-S) dans les neurones lésés et non-lésés même si les niveaux d'expression des ARNm des sous-unités TTX-S sont modifiés dans les neurones lésés. L'inactivation basale dépendante du voltage des canaux tétrodotoxine-insensible (TTX-R) est déplacée vers des potentiels positifs dans les cellules lésées et non-lésées. En revanche la vitesse de récupération des courants TTX-S et TTX-R après inactivation est accélérée dans les neurones lésés. Ces changements pourraient être à l'origine de l'altération de l'activité électrique des neurones sensoriels dans le contexte des douleurs neuropathiques. En résumé, ces résultats suggèrent l'existence de mécanismes différenciés affectant les neurones lésés et les neurones adjacents non-lésés lors de la mise en place la douleur neuropathique. De plus, les changements centraux au niveau de la moelle épinière qui surviennent après lésion sont probablement intégrés différemment selon la perception de signaux des neurones périphériques lésés ou non-lésés. En conclusion, ces modulations complexes et distinctes sont probablement des acteurs essentiels impliqués dans la genèse et la persistance des douleurs neuropathiques. ABSTRACT : Neuropathic pain (NP) results from damage or dysfunction of the peripheral or central nervous system. Symptoms associated with NP are severe and difficult to treat. Targeting NP mechanisms and their translation into symptoms may offer a better therapeutic approach.Hyperexcitability of the peripheral and central nervous system occurs in the dorsal root ganglia (DRG) and the dorsal horn (DH) of the spinal cord. We aimed to identify transcriptional variations in injured and in adjacent non-injured nociceptors as well as in corresponding laminae I and II of DH receiving their inputs.We investigated changes one week after the injury induced by the spared nerve injury model of NP. We employed the laser capture microdissection (LCM) for the procurement of specific cell-types (enrichment in nociceptors of injured/non-injured neurons) and laminae in combination with transcriptional analysis by microarray. In addition, we studied functionál properties and currents of sodium channels (Nav1s) in injured and neighboring non-injured DRG neurons.Microarray analysis at the periphery between injured and non-injured DRG neurons and centrally between the area of central projections from injured and non-injured neurons show significant and differential expression patterns. We reported changes in injured nociceptors (1561 genes, > 1.5 fold, >77% pairwise comparison) and in corresponding DH laminae (618 genes), while less modifications occurred in non-injured nociceptors (60 genes) and in corresponding DH laminae (459 genes). At the periphery, we observed by Gene Ontology the involvement of multiple biological processes in injured neurons such as signal transduction, cytoskeleton organization or stress responses. On contrast, functional overrepresentations in non-injured neurons were noted only in metabolic or developmentally related mechanisms. At the level of superficial laminae of the dorsal horn, we reported changes of immune and inflammatory processes in injured-related DH and changes associated with synaptic organization and transmission in DH corresponding to non-injured neurons. Further transcriptional analysis of Nav1s indicated several changes in injured neurons. Functional analyses of Nav1s have established no difference in tetrodotoxin-sensitive (TTX-S) current densities in both injured and non-injured neurons, despite changes in TTX-S Nav1s subunit mRNA levels. The tetrodotoxin-resistant (TTX-R) voltage dependence of steady state inactivation was shifted to more positive potentials in both injured and non-injured neurons, and the rate of recovery from inactivation of TTX-S and TTX-R currents was accelerated in injured neurons. These changes may lead to alterations in neuronal electrogenesis. Taken together, these findings suggest different mechanisms occurring in the injured neurons and the adjacent non-injured ones. Moreover, central changes after injury are probably driven in a different manner if they receive inputs from injured or non-injured neurons. Together, these distinct and complex modulations may contribute to NP.

Cilengitide: an integrin-targeting arginine-glycine-aspartic acid peptide with promising activity for glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM), a highly invasive and vascular cancer, responds poorly to conventional cytotoxic therapy. Integrins, widely expressed in GBM and tumor vasculature, mediate cell survival, migration and angiogenesis. Cilengitide is a potent alphavbeta3 and alphavbeta5 integrin inhibitor. OBJECTIVE: To summarize the preclinical and clinical experience with cilengitide for GBM. METHODS: Preclinical studies and clinical trials evaluating cilengitide for GBM were reviewed. RESULTS/CONCLUSIONS: Cilengitide is active and synergizes with external beam radiotherapy in preclinical GBM models. In clinical trials for recurrent GBM, single-agent cilengitide has antitumor benefits and minimal toxicity. Among newly diagnosed GBM patients, single-arm studies incorporating cilengitide into standard external beam radiotherapy/temozolomide have shown encouraging activity with no increased toxicity and have led to a planned randomized Phase III trial.

Screening for foetal malformations: performance of routine ultrasonography in the population of the Swiss Canton of Vaud.

OBJECTIVE: To determine the sensitivity of ultrasonography in screening for foetal malformations in the pregnant women of the Swiss Canton of Vaud. STUDY DESIGN: Retrospective study over a period of five years. METHOD: We focused our study on 512 major or minor clinically relevant malformations detectable by ultrasonography. We analysed the global sensitivity of the screening and compared the performance of the tertiary centre with that of practitioners working in private practice or regional hospitals. RESULTS: Among the 512 malformations, 181 (35%) involved the renal and urinary tract system, 137 (27%) the heart, 71 (14%) the central nervous system, 50 (10%) the digestive system, 42 (8%) the face and 31 (6%) the limbs. Global sensitivity was 54.5%. The lowest detection rate was observed for cardiac anomalies, with only 23% correct diagnoses. The tertiary centre achieved a 75% detection rate in its outpatient clinic and 83% in referred patients. Outside the referral centre, the diagnostic rate attained 47%. CONCLUSIONS: Routine foetal examination by ultrasonography in a low-risk population can detect foetal structural abnormalities. Apart from the diagnosis of cardiac abnormalities, the results in the Canton of Vaud are satisfactory and justify routine screening for malformations in a low-risk population. A prerequisite is continuing improvement in the skills of ultrasonographers through medical education.

Oxygen tension controls the expression of the monocarboxylate transporter MCT4 in cultured mouse cortical astrocytes via a hypoxia-inducible factor-1α-mediated transcriptional regulation.

The monocarboxylate transporter MCT4 is a high capacity carrier important for lactate release from highly glycolytic cells. In the central nervous system, MCT4 is predominantly expressed by astrocytes. Surprisingly, MCT4 expression in cultured astrocytes is low, suggesting that a physiological characteristic, not met in culture conditions, is necessary. Here we demonstrate that reducing oxygen concentration from 21% to either 1 or 0% restored in a concentration-dependent manner the expression of MCT4 at the mRNA and protein levels in cultured astrocytes. This effect was specific for MCT4 since the expression of MCT1, the other astrocytic monocarboxylate transporter present in vitro, was not altered in such conditions. MCT4 expression was shown to be controlled by the transcription factor hypoxia-inducible factor-1α (HIF-1α) since under low oxygen levels, transfecting astrocyte cultures with a siRNA targeting HIF-1α largely prevented MCT4 induction. Moreover, the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced MCT4 expression in astrocytes cultured in presence of 21% oxygen. In parallel, glycolytic activity was enhanced by exposure to 1% oxygen as demonstrated by the increased lactate release, an effect dependent on MCT4 expression. Finally, MCT4 expression was found to be necessary for astrocyte survival when exposed for a prolonged period to 1% oxygen. These data suggest that a major determinant of astrocyte MCT4 expression in vivo is likely the oxygen tension. This could be relevant in areas of high neuronal activity and oxygen consumption, favouring astrocytic lactate supply to neurons. Moreover, it could also play an important role for neuronal recovery after an ischemic episode.

Six new loci associated with body mass index highlight a neuronal influence on body weight regulation.

Common variants at only two loci, FTO and MC4R, have been reproducibly associated with body mass index (BMI) in humans. To identify additional loci, we conducted meta-analysis of 15 genome-wide association studies for BMI (n > 32,000) and followed up top signals in 14 additional cohorts (n > 59,000). We strongly confirm FTO and MC4R and identify six additional loci (P < 5 x 10(-8)): TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2 and NEGR1 (where a 45-kb deletion polymorphism is a candidate causal variant). Several of the likely causal genes are highly expressed or known to act in the central nervous system (CNS), emphasizing, as in rare monogenic forms of obesity, the role of the CNS in predisposition to obesity.

The monocarboxylate transporter MCT4 : mechanism of regulation in astrocytes and its putative role in cerebral ischemia

Despite its small fraction of the total body weight (2%), the brain contributes for 20% and 25% respectively of the total oxygen and glucose consumption of the whole body. Indeed, glucose has been considered the energy substrate par excellence for the brain. However, evidence accumulated over the last half century revealed an important role for the monocarboxylate lactate in fulfilling the energy needs of neurons. This is particularly true during physiological neuronal activation and in pathological conditions. Lactate transport into and out of the cell is mediated by a family of proton-linked transporters called monocarboxylate transporters (MCTs). In the central nervous system, only three of them have been well characterized: MCT2 is the predominant neuronal isoform, while the other non¬neuronal cell types of the brain express the ubiquitous isoform MCT1. Quite recently, the MCT4 isoform has been described in astrocytes. Due to its high transport capacity compared to the other two isoforms, MCT4 is particularly adapted for glycolytic cells. Because of its recent discovery in the brain, nothing was known about its regulation in the central nervous system. Here we show that MCT4 is regulated by oxygen levels in primary cultures of astrocytes in a time- and concentration-dependent manner via the hypoxia inducible factor-la (HIF-la). Moreover, we showed that MCT4 expression is essential for astrocyte survival under low oxygen conditions. In parallel, we investigated the possible implication of the pyruvate kinase isoform Pkm2, a strong enhancer of glycolysis, in its regulation. Then we showed that MCT4 expression, as well as the expression of the other two MCT isoforms, is altered in a murine model of stroke. Surprisingly, neurons started to express MCT4, as well as MCT1, under such conditions. Altogether, these data suggest that MCT4, due to its high transport capacity for lactate, may be the isoform that enables cells to operate a major metabolic adaptation in response to pathological situations that alter metabolic homeostasis of the brain. -- Le cerveau représente 2% du poids corporel total, mais il contribue pour 20% de la consommation totale d'oxygène et 25% de celle de glucose au repos. Le glucose est considéré comme le substrat énergétique par excellence pour le cerveau. Néanmoins, depuis un demi- siècle maintenant, de plus en plus de travaux ont démontré que le lactate joue un rôle majeur dans le métabolisme cérébral et est capable du subvenir aux besoins énergétiques des neurones. Le lactate est tout particulièrement nécessaire pendant l'activation neuronale ainsi qu'en situation pathologique. Le transport du lactate à travers la barrière hématoencéphalique ainsi qu'à travers les membranes cellulaires est assuré par la famille des transporteurs aux monocarboxylates (MCTs). Dans le système nerveux central, uniquement trois d'entre eux ont été décrits: MCT2 est considéré comme le transporteur neuronal, alors que les autres types cellulaires qui constituent le cerveau expriment l'isoforme ubiquitaire MCT1. Récemment, l'isoforme MCT4 a été rapportée sur les astrocytes. Dû à sa grande capacité de transport pour le lactate, MCT4 est tout particulièrement adapté pour soutenir le métabolisme des cellules hautement glycolytiques, comme les astrocytes. En raison de sa toute récente découverte, les aspects comprenant sa régulation et son rôle dans le cerveau sont pour l'instant méconnus. Les résultats exposés dans ce travail démontrent dans un premier temps que l'expression de MCT4 est régulée par les niveaux d'oxygène dans les cultures d'astrocytes corticaux par le biais du facteur de transcription HIF-la. De plus, nous avons démontré que l'expression de MCT4 est essentielle à la survie des astrocytes quand le niveau d'oxygénation baisse. En parallèle, des résultats préliminaires suggèrent que l'isoforme 2 de la pyruvate kinase, un puissant régulateur de la glycolyse, pourrait jouer un rôle dans la régulation de MCT4. Dans la deuxième partie du travail nous avons démontré que l'expression de MCT4, ainsi que celle de MCT1 et MCT2, est altérée dans un modèle murin d'ischémie cérébrale. De façon surprenante, les neurones expriment MCT4 dans cette condition, alors que ce n'est pas le cas en condition physiologique. En tenant compte de ces résultats, nous suggérons que MCT4, dû à sa particulièrement grande capacité de transport pour le lactate, représente le MCT qui permet aux cellules du système nerveux central, notamment les astrocytes et les neurones, de s'adapter à de très fortes perturbations de l'homéostasie métabolique du cerveau qui surviennent en condition pathologique.

No association between ApoE polymorphism and febrile seizures.

Seizures associated with fever are a common pediatric problem, affecting about 2-7 % of children between 3 months and 5 years of age. Differentiation of febrile seizures from acute symptomatic seizures secondary to central nervous system infections or seizures associated with fever in children with epilepsy is essential to provide appropriate treatment and follow-up care. Here, we tested the hypothesis that children who exhibit simple febrile seizures during early childhood, but do not develop epileptic seizures later in life, might preferentially carry the ApoE2 allele of the gene coding for the apolipoprotein E. We did not find any differences in the distribution of ApoE alleles or genotypes between individuals who exhibited simple febrile seizures (n = 93) and age-matched, typically developing subjects (n = 80). We found that the observed allele and genotype frequencies did not deviate from Hardy-Weinberg equilibrium, which suggests that the frequencies of ApoE alleles and genotypes are stable in the Swiss population from which our samples were derived. Across both groups of subjects (n = 173), we found an ApoE2 allele frequency of 0.064, an ApoE3 frequency of 0.829 and an ApoE4 frequency of 0.107. Our findings are consistent with previous reports of the distribution of ApoE polymorphism for European subjects free of any neurological disorders, and show that the different alleles of the gene coding for the apolipoprotein E are not associated with the occurrence of simple febrile seizures.

A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia.

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.

Information processing in sleep-onset insomnia : a test of the neurocognitive model /

The present thesis study is a systematic investigation of information processing at sleep onset, using auditory event-related potentials (ERPs) as a test of the neurocognitive model of insomnia. Insomnia is an extremely prevalent disorder in society resulting in problems with daytime functioning (e.g., memory, concentration, job performance, mood, job and driving safety). Various models have been put forth in an effort to better understand the etiology and pathophysiology of this disorder. One of the newer models, the neurocognitive model of insomnia, suggests that chronic insomnia occurs through conditioned central nervous system arousal. This arousal is reflected through increased information processing which may interfere with sleep initiation or maintenance. The present thesis employed event-related potentials as a direct method to test information processing during the sleep-onset period. Thirteen poor sleepers with sleep-onset insomnia and 1 2 good sleepers participated in the present study. All poor sleepers met the diagnostic criteria for psychophysiological insomnia and had a complaint of problems with sleep initiation. All good sleepers reported no trouble sleeping and no excessive daytime sleepiness. Good and poor sleepers spent two nights at the Brock University Sleep Research Laboratory. The first night was used to screen for sleep disorders; the second night was used to investigate information processing during the sleep-onset period. Both groups underwent a repeated sleep-onsets task during which an auditory oddball paradigm was delivered. Participants signalled detection of a higher pitch target tone with a button press as they fell asleep. In addition, waking alert ERPs were recorded 1 hour before and after sleep on both Nights 1 and 2.As predicted by the neurocognitive model of insomnia, increased CNS activity was found in the poor sleepers; this was reflected by their smaller amplitude P2 component seen during wake of the sleep-onset period. Unlike the P2 component, the Nl, N350, and P300 did not vary between the groups. The smaller P2 seen in our poor sleepers indicates that they have a deficit in the sleep initiation processes. Specifically, poor sleepers do not disengage their attention from the outside environment to the same extent as good sleepers during the sleep-onset period. The lack of findings for the N350 suggest that this sleep component may be intact in those with insomnia and that it is the waking components (i.e., Nl, P2) that may be leading to the deficit in sleep initiation. Further, it may be that the mechanism responsible for the disruption of sleep initiation in the poor sleepers is most reflected by the P2 component. Future research investigating ERPs in insomnia should focus on the identification of the components most sensitive to sleep disruption. As well, methods should be developed in order to more clearly identify the various types of insomnia populations in research contexts (e.g., psychophysiological vs. sleep-state misperception) and the various individual (personality characteristics, motivation) and environmental factors (arousal-related variables) that influence particular ERP components. Insomnia has serious consequences for health, safety, and daytime functioning, thus research efforts should continue in order to help alleviate this highly prevalent condition.

A comparison of cognitive processes and components of neurological maturation in 3-and-11 year-old children

The relationship between the child's cogni tive development and neurological maturation has been of theoretical interest for many year s. Due to diff iculties such as the lack of sophisticated techniques for measur ing neurolog ical changes and a paucity of normative data, few studies exist that have attempted to correlate the two factors. Recent theory on intellectual development has proposed that neurological maturation may be a factor in the increase of short-term memory storage space. Improved technology has allowed reliable recordings of neurolog ical maturation.. In an attempt to correlate cogni tive development and neurological maturation, this study tested 3-and II-year old children. Fine motor and gross motor short-term memory tests were used to index cogni tive development. Somatosensory evoked potentials elici ted by median nerve stimulation were used to measure the time required for the sensation to pass along the nerve to specific points on the somatosensory pathway. Times were recorded for N14, N20, and P22 interpeak latencies. Maturation of the central nervous system (brain and spinal cord) and the peripheral nervous system (outside the brain and spinal cord) was indi~ated by the recorded times. Signif icant developmental di fferences occurred between 3-and ll-year-olds in memory levels, per ipheral conduction velocity and central conduction times. Linear regression analyses showed that as age increased, memory levels increased and central conduction times decreased. Between the ll-year-old groups, there were no significant differences in central or peripheral nervous system maturation between subjects who achieved a 12 plus score on the digit span test of the WISC-R and those who scored 7 or lower on the same test. Levels achieved on the experimental gross and fine motor short-term memory tests differed significantly within the ll-year-old group.

Effects of intracerebroventricular bombesin administration on local cerebral glucose utilization in the restrained and unrestrained rat

Intracerebroventricular (ICV) administration of bombesin (BN) induces a syndrome characterized by stereotypic locomotion and grooming, hyperactivity and sleep elimination, hyperglycemia and hypothermia, hyperhemodynamics, feeding inhibition, and gastrointestinal function changes. Mammalian BN-like peptides (MBNs), e.g. gastrin-releasing peptide (GRP), Neuromedin C (NMC), and Neuromedin B (NMB), have been detected in the central nervous system. Radio-labeled BN binds to specific sites in discrete cerebral regions. Two specific BN receptor subtypes (GRP receptor and NMB receptor) have been identified in numerous brain regions. The quantitative 2-[14C]deoxyglucose ([14C]20G) autoradiographic method was used to map local cerebral glucose utilization (LCGU) in the rat brain following ICV injection of BN (vehicle, BN O.1Jlg, O.5Jlg). At each dose, experiments were conducted in freely moving or restrained conditions to determine whether alterations in cerebral function were the result of BN central administration, or were the result of BN-induced motor stereotypy. The anteroventral thalamic nucleus (AV) (p=O.029), especially its ventrolateral portion (AVVL) (pcentral administration upon functioning of the cerebral regions influenced by BN administration. The restraint effects seen in LO, including LOOM and LOVL, are suggested to be the result of altered behavioral expression. The restraint effects seen in LG is suggested to be the result of reduced locomotion.

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.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Les mécanismes synaptiques et intrinsèques qui sous-tendent l’activité des cellules réticulospinales (RS) en réponse à une stimulation sensorielle de type cutané chez la lamproie

Chez diverses espèces animales, les informations sensorielles peuvent déclencher la locomotion. Ceci nécessite l’intégration des informations sensorielles par le système nerveux central. Chez la lamproie, les réseaux locomoteurs spinaux sont activés et contrôlés par les cellules réticulospinales (RS), système descendant le plus important. Ces cellules reçoivent des informations variées provenant notamment de la périphérie. Une fois activées par une brève stimulation cutanée d’intensité suffisante, les cellules RS produisent des dépolarisations soutenues de durées variées impliquant des propriétés intrinsèques calcium-dépendantes et associées à l’induction de la nage de fuite. Au cours de ce doctorat, nous avons voulu savoir si les afférences synaptiques ont une influence sur la durée des dépolarisations soutenues et si l’ensemble des cellules RS partagent des propriétés d’intégration similaires, impliquant possiblement les réserves de calcium internes. Dans un premier temps, nous montrons pour la première fois qu’en plus de dépendre des propriétés intrinsèques des cellules réticulospinales, les dépolarisations soutenues dépendent des afférences excitatrices glutamatergiques, incluant les afférences spinales, pour perdurer pendant de longues périodes de temps. Les afférences cutanées ne participent pas au maintien des dépolarisations soutenues et les afférences inhibitrices glycinergique et GABAergiques ne sont pas suffisantes pour les arrêter. Dans un deuxième temps, nous montrons que suite à une stimulation cutanée, l’ensemble des cellules RS localisées dans les quatre noyaux réticulés possèdent un patron d’activation similaire et elles peuvent toutes produire des dépolarisations soutenues dont le maintien ne dépend pas des réserves de calcium internes. Enfin, les résultats obtenus durant ce doctorat ont permis de mieux comprendre les mécanismes cellulaires par lesquels l’ensemble des cellules RS intègrent une brève information sensorielle et la transforment en une réponse soutenue associée à une commande motrice.