Spinal cord injury, acute pain, and morphine tolerance are important issues in the clinical practice. A primary injury to the spinal cord causes both morphological and biochemical changes with initiation of the devastating secondary pathophysiological pathways that ultimately destroy CNS cells and cause degeneration of nerve fibers. Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central neurons, known as central sensitization. Nociceptor sensitization and central sensitization are believed to underlie the development of primary and secondary hyperalgesia, respectively. The most efficacious drugs used to relieve pain are the opioid analgesics. Chronic administration leads to the development of tolerance. Tolerance is manifested as a decreased potency of the drug, so that progressively larger doses must be administered to achieve a given level of analgesia. The processes underlying opioid tolerance still need to be elucidated. Recently, it is found calpain-Cdk5 (cyclin-dependent kinase-5)-p35 pathway modulation implicated in neuroprotection, acute nociceptive response, and morphine analgesia. In this thesis, we evaluate calpain inhibitor-MDL28170 and Cdk5 inhibitor-roscovitine against rat spinal cord hemisection, formalin-induced acute nociceptive responses, and chronic morphine tolerance. We found calpain-Cdk5-p35 pathway inhibition could protect spinal cord hemisection and subsequent neurodegeneration, inhibit formalin-induced flinch response involving DARPP-32 (dopamine and c-AMP regulated phosphoprotein, MW=32 kDa) phosphorylation, and reverse right shifted morphine dose-response curve with upregulated ED50 (50% of effective dose) reduction. Taken together, calpain-Cdk5-p35 pathway inhibition is useful in the management of spinal cord injury, acute inflammatory pain, and attenuate morphine tolerance development with further clinical application.

Cyclin-dependent kinase-5 (Cdk5) was identified as a serine/threonine kinase that plays an important role in neuronal development. Association with one of the neuronal activators, p35 or p39, is required for Cdk5 to elicit its diverse effects in the nervous system, such as neurite outgrowth. In addition to these, increasing evidence suggests that Cdk5 also plays an important role in cocaine addiction, neurotransmitter release, NMDA receptor phosphorylation. This thesis is divided into three parts which deals with the effects of Cdk5 inhibitor¡Ðroscovitine on the morphine tolerance development, acute inflammatory pain, and pilocarpine-induced seizure respectively. The first part explored the effect of Cdk5 inhibitior¡Ðroscovitine on the morphine antinociceptive tolerance development. Delta FosB activation is involved in morphine tolerance. Cyclin-dependent kinase- 5 (Cdk5) is found to be the downstream target of delta FosB. We examined the effects of the potent selective Cdk5 inhibitor¡Ðroscovitine on the development of antinociceptive tolerance of morphine. Tolerance was induced by continuous infusion of morphine 5 µg/hr intrathecally (i.t.) for 5 days. The effect of co-administration of roscovitine 1 µg/hr i.t. for 5 days was also examined. Roscovitine co-administration enhanced the antinociceptive effect of morphine in morphine tolerant rats. It also shift the morphine antinociceptive dose¡Ðresponse curve to the left during morphine tolerance induction, and reduced the increase in the ED50 of morphine two-fold. Collectively, these findings suggest Cdk5 modulation may be involved in the development of morphine tolerance and its inhibitor will enhance antinociceptive effect. The second part discussed the roscovitine effect on acute inflammatory pain. Formalin injected into the rat hind paw will evoke flinching (consisting of an elevation and shrinking back of the injected paw), a reliable parameter of pain behavior. The nociceptive response to formalin occurs in a biphasic pattern: there isan initial acute period (phase 1), and after a short period of remission, phase 2 begins and consists of a longer period (1 hour) of sustained activity. The initial response was initially attributed to a direct algogenic effect of formalin, whereas phase 2 was associated with the central sensitization. In this study, the Cdk5 inhibitor¡Ðroscovitine was injected intrathecally to elucidate the mechanism of Cdk5 activation during formalin-induced hyperalgesia. The 50 ul of 5% formalin solution was used as the noxious stimulant. The rats were injected with 0, 50, 100, and 200ug roscovitine intrathecally thirty minutes before hind paw formalin injection. Intrathecal 200ug roscovitine injection attenuates the phase I flinch response. And intrathecal 50, 100, and 200ug roscovitine injection suppress phase II flinch response effectively. Roscovitine administration could effectively suppress the formalin-induced flinch behavior. This implies the activation of Cdk5 plays an important role in the sensitization after nociceptive stimulation. The third part focus on the roscovitine effect on the pilocarpine induced seizure. Pilocarpine temporal lobe epilepsy model is widely used. Chronic electroconvulsive therapy could upregulate Cdk5 activity. Cdk5 inhibitor¡Ðroscovitine could suppress NMDA induced long-term potentiation in hippocampal slice. Intracerebroventricular injection of 100£gg roscovitine 30 min before pilocarpine-induced epilepsy could significantly decrease the seizure-induced mortality ( 11% in roscovitine group VS 77% in control group). The escape latency, spatial memory impairment, in the pilocarpine-induced seizure group is significant longer than the roscovitine pretreatment group in the Morris water maze test after one month (p¡Õ0.05). It is concluded Cdk5 may play an important in the pathogenesis of epilepsy. Therefore, Cdk5 inhibition may become another way for the epilepsy treatment.

Each year, over 50 million Americans suffer from persistent pain, including debilitating headaches, joint pain, and severe back pain. Although morphine is amongst the most effective analgesics available for the management of severe pain, prolonged morphine treatment results in decreased analgesic efficacy (i.e., tolerance). Despite significant headway in the field, the mechanisms underlying the development of morphine tolerance are not well understood. The midbrain ventrolateral periaqueductal gray (vlPAG) is a primary neural substrate for the analgesic effects of morphine, as well as for the development of morphine tolerance. A growing body of literature indicates that activated glia (i.e., microglia and astrocytes) facilitate pain transmission and oppose morphine analgesia, making these cells important potential targets in the treatment of chronic pain. Morphine affects glia by binding to the innate immune receptor toll-like receptor 4 (TLR4), leading to the release of proinflammatory cytokines and opposition of morphine analgesia. Despite the established role of the vlPAG as an integral locus for the development of morphine tolerance, to date, no studies have examined the role of glia activation within this region. Additionally, the role of TLR4 in the development of tolerance has not been elucidated. This dissertation seeks to address the lack of knowledge regarding the role of vlPAG glia and TLR4 in the development of morphine tolerance by (1) Characterizing the effects of chronic morphine and peripheral inflammatory pain on vlPAG glial cell activity; (2) Investigating the role of glia activation within the vlPAG in the development of morphine tolerance; (3) Characterizing the role of the glial receptor TLR4 within the vlPAG in the development of morphine tolerance; and (4) Characterizing the glia to neuron signaling mechanisms involved in the development of morphine tolerance. These experiments, together, provide novel information about the mechanism by which central nervous system glia regulate morphine tolerance, and identify a potential therapeutic target for the enhancement of analgesic efficacy in the clinical treatment of chronic pain.

In non-pathological states the central nervous system maintains a degree of immunological privilege. When illness or injury occur, this privilege can be lost and the immune system drives pathology in the brain and spinal cord. More so, resident immune cells, the microglial, act as major effectors of this response. Cerebral ischemia, or stroke, is the fourth leading cause of death in developed nations. After the initial ischemia, the inflammatory response propagates further injury and cell death. Another affliction of the central nervous system, chronic pain and persistent use of the opioid analgesic, morphine, leads to tolerance and ineffectiveness of the drug. Currently, only one in three patients receive adequate pain relief from their pharmacological regiment. This loss of efficacy in morphine is also driven by an inflammatory response. Thus, a way to quell inflammation in both disease states could lead to better treatments for both disorders. The endogenous cannabinoid system has two known receptors, CB1 and CB2. Both of these receptors have been intimately linked to inflammation and the activation or antagonism of the receptors can impart desired outcomes in modulating the immune response. Primarily the CB1 receptor expression is on presynaptic terminals of neurons to modulate neuronal firing. The CB2 receptor's expression predominates on immunological cells including microglial. However, some degree of expression exists with reports of neuronal CB2 receptors and immunological CB1 receptors. This makes pharmacological therapies targeted at both receptors ideal candidates in treating not only stroke and but also preventing the induction of morphine tolerance. In the studies described here, we sought to investigate the role of the endogenous cannabinoid system in both stroke and as a way to prevent the induction of morphine tolerance. The results showed that CB1 -/- CB2 -/- receptor mice were able to maintain greater blood flow during cerebral ischemia. More so, CB1 antagonism in a permanent occlusion of cerebral vessels showed a protective effect independent of the serotonin receptor. Lastly, a CB2 agonist was able to limit the degree of tolerance that developed from chronic morphine therapy and also prevent hyperalgesia in addition to showing a reduction in pro-inflammatory cytokines. Acutely, this same agonist was found to antagonize the morphine receptor but this could be avoided if morphine was administered before the CB2 agonist. In brief, the studies at hand show that the endogenous cannabinoid system can attenuate inflammation in central nervous system injury and shows great promise as a future therapeutic for clinical use. / Physiology

Bien que la régulation des canaux calciques par les récepteurs couplés aux protéines G soit connue depuis une trentaine d'année, ce n'est que récemment qu'il a été découvert que ce phénomène pouvait passer par une interaction directe entre ces deux partenaires. Les RCPGs sont les senseurs d'un grand nombre de paramètres (des simples photons aux molécules odorantes en passant par des hormones, acides aminés et nucléotides) et ils contrôlent un grand nombre de processus cellulaires en fonction de ces différents stimuli, ce qui en fait une cible thérapeutique majeure. Une de leurs cibles est l'activité des canaux calciques voltage dépendants qui est responsable d'un grand nombre de processus tels que le contrôle du potentiel de membrane, le relargage de neurotransmetteurs, la contraction musculaire ou, bien sûr, le contrôle du taux de calcium intracellulaire qui est lui-même un second messager impliqué dans de nombreuses voies de régulations.Il nous a donc paru intéressant de se pencher plus en avant sur ces interactions et de trouver une méthode nous permettant de cribler ces interactions potentielles avec des RCPG ciblés pouvant intervenir dans une thématique de contrôle de la douleur. Pour cela nous avons développé une stratégie de FRET en temps résolu utilisant les cryptates de terres rares à déactivation lente couplés aux ligands du tag SNAP comme donneurs de fluorescence, les canaux calciques étudiées étant fusionnés avec cet épitope et l'eGFP fusionnée aux RCPGs en tant qu'accepteur. Ce test nous a permis de confirmer l'interaction entre CaV2.2 et ORL1 le récepteur de la nociceptine. Nous avons ensuite cherché à caractériser plus précisément cette interaction et nous avons déterminé quelles en étaient les séquences peptidiques responsables au sein des domaines C-terminaux de ces deux protéines grâce à des expériences de GST-pull down. Nous avons synthétisé un peptide reproduisant la séquence d'interaction d'ORL1 que nous avons couplé à la séquence TAT, le rendant ainsi capable de pénétrer les membranes cellulaires. Lorsque nous ajoutons ce peptide leurre dans les expériences de TR-FRET, l'augmentation de fluorescence observée en présence de CaV2.2-SNAP et ORL1-GFP disparait totalement alors que l'ajout d'un peptide contrôle composé des mêmes acides aminés mais présentés dans le désordre n'a aucun effet. Nous avons ensuite cherché à étudier les effets de ce peptide in vivo lors d'un protocole de tolérance à la morphine étant donné que les souris K.O. pour le gène d'ORL1 sont résistantes à l'apparition de cette tolérance. Cette stratégie de découplage CaV2.2 :: ORL1 abolit complètement le phénomène de tolérance aux effets analgésiques de la morphine par une action au niveau spinal. Ce travail peut conduire à l'utilisation d'une telle approche dans une perspective thérapeutique visant à améliorer l'utilisation de morphiniques lors du traitement des douleurs chroniques. / The regulation of the calcium channels by GPCRs has been known for almost thirty years but the direction interaction between those two proteins is a recent breakthrough. GPCRs are sensors for a great number of parameters (photons, smell molecules, hormones, amino acids, nucleotides…) and they control numerous cellular functions according to those parameters making them a major target for pharmacology. One of the GPCR's targets is the calcium channel activity which is responsible for a great number of cellular processes like control of the membrane potential, neurotransmitters or hormonal secretion, muscular contraction and, of course, control of the intracellular calcium level which is a second messenger of numerous cell-regulation pathways.It appears to us that it would be interesting to study more closely those interactions and find a way to screen the GPCR/calcium channels interactions that may occur in pain regulation. We developed a strategy of time resolved FRET, using rare earth cryptate coupled to the ligand of the SNAP tag which is fused to the calcium channel as fluorescence donor and eGFP fused GRPRs as acceptors. That test confirmed the interaction between CaV2.2 and ORL1, the nociceptin receptor. We characterized more precisely the peptide sequence of the carboxy-terminal domain of the two proteins which is responsible for the interaction using GST-pull down experiments. We synthesized a peptide reproducing the ORL1 interaction sequence coupled to the TAT sequence allowing to go through the cell membranes. When we add this decoy peptide to ours TR-FRET experiments we lose all the increase of fluorescence that we see in presence of CaV2.2-SNAP and ORL1-GFP but the adding of a control peptide made of the same peptides but scrambled didn't affect the experiment. Then we look for the effects of this peptide in vivo, during a morphine tolerance protocol as it was reported that the ORL1 knock-out mice were insensitive to this phenomenon. This strategy of uncoupling CaV2.2 and ORL1 leads to a complete suppression of the tolerance to the analgesic effects of the morphine by an action at the spinal level. This work could lead to a therapeutic use of this approach which could enhance the use of morphinic compounds in treatment of chronics pains.