Non-nociceptive roles of opioids in the CNS: opioids' effects on neurogenesis, learning, memory and affect.

Mortality due to opioid use has grown to the point where, for the first time in history, opioid-related deaths exceed those caused by car accidents in many states in the United States. Changes in the prescribing of opioids for pain and the illicit use of fentanyl (and derivatives) have contributed to the current epidemic. Less known is the impact of opioids on hippocampal neurogenesis, the functional manipulation of which may improve the deleterious effects of opioid use. We provide new insights into how the dysregulation of neurogenesis by opioids can modify learning and affect, mood and emotions, processes that have been well accepted to motivate addictive behaviours

Analyse cellulaire et intégrée de l'implication du neurostéroïde déhydroépiandrostérone dans la régulation des processus nociceptifs

Les douleurs chroniques rebelles constituent un problème majeur de santé publique car elles sont résistantes aux analgésiques actuels et provoquent une souffrance persistante ainsi que des difficultés socio-économiques telles qu une baisse de productivité et des dépenses élevées. La découverte de nouvelles molécules capables de traiter efficacement les douleurs rebelles est donc aujourd hui une nécessité absolue pour la recherche biomédicale. Sont incluses dans la catégorie des douleurs rebelles, les douleurs neuropathiques qui sont liées à des lésions du système nerveux ou à des perturbations de l équilibre fonctionnel des structures spinales et supraspinales contrôlant la nociception. Il est donc évident que les composés à découvrir pour la thérapie des douleurs neuropathiques devraient nécessairement être de puissants modulateurs de l activité des neurones sensoriels spinaux et supraspinaux. Diverses pistes sont explorées parmi lesquelles les neurostéroïdes qui sont des modulateurs des récepteurs GABAA, NMDA et P2X intervenant dans la nociception. Les médias présentent souvent le neurostéroïde déhydroépiandrostérone (DHEA) comme étant une molécule miracle capable de réduire les déficits physiologiques liés au vieillissement. La DHEA est considérée comme un neuromodulateur endogène dont la biosynthèse diminue avec l âge. Paradoxalement, les preuves scientifiques démontrant les effets bénéfiques de la DHEA sont rares. Contrairement aux humains, les concentrations plasmatiques de DHEA sont quasiment indétectables chez les rongeurs adultes puisque l enzyme de synthèse de la DHEA, le cytochrome P450c17 (P450c17), est présente dans les glandes surrénales humaines mais n est pas exprimée dans les surrénales de rongeurs. Par conséquent, même si l administration de DHEA synthétique contrôle plusieurs processus neurophysiologiques chez les rongeurs adultes, le rôle de la DHEA comme neuromodulateur endogène n est possible que si la DHEA est produite dans le système nerveux central. Pour obtenir des renseignements utiles sur la DHEA, notamment sur son rôle régulateur de processus importants tels que la nociception et la douleur, nous avons élaboré un projet permettant d intégrer des données depuis les niveaux moléculaire et cellulaire jusqu à l échelle de l organisme entier. Nous avons étudié chez le rat adulte l éventualité de la biosynthèse de DHEA dans la moelle épinière (ME), une structure cruciale dans la nociception et la douleur. Nous avons ensuite déterminé les modifications intervenant dans l expression du gène et de l activité enzymatique du P450c17 dans la ME au cours du développement de la douleur chronique neuropathique induite par ligature lâche du nerf sciatique. Nos travaux ont été complétés par une évaluation comportementale des effets de la DHEA et du kétoconazole (un inhibiteur pharmacologique du P450c17) sur la sensibilité à la douleur.La PCR quantitative en temps réel après transcription inverse a révélé l existence d ARNm codant le P450c17 dans tous les segments de la ME. La présence de la protéine enzymatique a été mise en évidence dans la ME par Western Blot avec un anticorps dirigé contre le P450c17. Des études immunohistochimiques ont permis de localiser de nombreux neurones et cellules gliales P450c17-positifs dans la corne dorsale de la ME. Des expériences de pulse-chase combinées à l analyse HPLC et à la détection en flux continu ont montré que les tranches de ME sont capables de convertir la [ H]prégnènolone en [ H]DHEA. Lorsque les rats sont soumis à une douleur neuropathique, les analyses moléculaires et biochimiques ont révélé une diminution de la biosynthèse de DHEA provoquée par la répression du gène et de l activité du P450c17 dans la ME. Les études comportementales ont démontré que la DHEA induit une action bi-phasique sur la sensibilité à la douleur à savoir un effet pro-nociceptif à court terme et une action antalgique tardive liée à la conversion de la DHEA en métabolites androgéniques. L effet propre de la DHEA semble effectivement être pro-nociceptif puisque le blocage in vivo de la synthèse de DHEA dans la ME par injection intrathécale de kétoconazole induit une analgésie chez les rats neuropathiques. Contrairement au kétoconazole, l injection intrathécale de DHEA potentialise l hyperalgie thermique et l allodynie mécanique caractérisant la douleur neuropathique.Parallèlement au travail principal de thèse sur la DHEA, j ai participé à un projet satellite sur les interactions entre la neurostéroïdogenèse spinale et les neurotransmetteurs impliqués dans la transmission des messages douloureux. Nos résultats montrent que la substance P, en agissant par les récepteurs neurokinine-1, diminue de façon dose-dépendante la biosynthèse du neurostéroïde alloprégnanolone (un puissant activateur des récepteurs GABAA) dans la corne dorsale de la ME.Compte tenu de la nature endogène des neurostéroïdes et de leur capacité à interagir avec l ensemble des systèmes majeurs de la neurotransmission, nous espérons qu à terme nos travaux ouvriront des perspectives sérieuses pour le développement de médicaments efficaces contre les douleurs rebelles.Stubborn chronic pain constitutes a major public health concern because this category of pain is refractory to the currently available analgesics and provokes persistent suffering in several thousands of patients with socio-economical problems such as substantial costs due to disability and decreased productivity. Therefore, development of novel therapeutic strategies against stubborn pain has become a real challenge for biomedical research. Included in the category of stubborn pain is neuropathic pain generated by lesions or injuries of the nervous system or by disturbances in the activity of spinal and supraspinal neural networks controlling nociception. Thus, it appears that the compounds to be characterized for the treatment of stubborn neuropathic pain must necessarily be capable of modulating the activity of spinal and supraspinal neuronal pathways. Various family of molecules are currently explored among which are neurosteroids that strongly modulates GABAA, NMDA and P2X receptors intervening in nociception and pain control. Since several years, the neurosteroid dehydroepiandrosterone (DHEA) is excessively advertised as a miraculous pill capable of reducing various physiological deficits associated with aging. DHEA is considered as an endogenous neuromodulator the synthesis of which strongly decreases with aging. Paradoxically, scientific proofs supporting beneficial effects of DHEA are rare. Unlike in humans, plasma concentrations of DHEA are undetectable in adult rodents concurrently with the fact that cytochrome P450c17 (P450c17), the key DHEA-synthesizing enzyme, is expressed by the adrenals in humans but not in rodents. Consequently, even though the administration of synthetic DHEA controls several processes in the central nervous system of adult rodents, the role of DHEA as endogenous neuromodulator remains possible only if DHEA is produced by nerve cells. To obtain fundamental and useful results on DHEA, particularly on its possible role in the regulation of important neurobiological processes such as nociception and pain, we developed a multidisciplinary project allowing integration of data from genomic, molecular and cellular levels to behavioral components of the whole individual. In a first step, we investigated the occurrence of DHEA biosynthesis in the adult rat spinal cord (SC), a crucial structure involved in nociceptive transmission and pain sensation. Afterwards, we identified changes occurring in P450c17 gene expression and enzymatic activity in the SC of rats submitted to chronic neuropathic pain provoked by sciatic nerve ligature. Our studies were completed with behavioral analysis allowing in vivo assessments of the effects of DHEA and ketoconazole (a pharmacological inhibitor of P450c17) on pain sensitivity.Quantitative real-time PCR after reverse transcription revealed P450c17 gene expression in all SC segments. Western blot analyses allowed identification of a specific P450c17 protein in the SC and immunohistochemical studies localised P450c17 in neurons and glial cells. Pulse-chase experiments combined with HPLC and continuous radioactive flow detection showed that SC slices converted [3H]pregnenolone into [3H]DHEA, a conversion markedly reduced by ketoconazole. When the animals were submitted to chronic neuropathic pain situation, molecular and biochemical analyses revealed a significant decrease of DHEA synthesis which resulted from the down-regulation of P450c17 gene expression and bioactivity in the SC. Behavioral investigations showed that DHEA induced a biphasic action on pain sensitivity: a short term pro-nociceptive effect followed by a late analgesic action due to DHEA conversion into androgenic metabolites. The proper action of DHEA seems effectively to be pro-nociceptive because in vivo blockade of DHEA biosynthesis in the SC by intrathecal injection of ketoconazole induced analgesia in neuropathic rats. Unlike ketoconazole, intrathecal administration of DHEA potentiated both thermal hyperalgesia and mechanical allodynia characterizing the neuropathic pain.In addition to the main PhD program on DHEA, I have also participated to a side-project aiming to investigate anatomic and functional interactions between spinal neurons secreting neurosteroids and classical neurotransmitters involved in painful message transmission. We demonstrated that substance P, acting through neurokinin-1 receptors, inhibits the production of the neurosteroid allopregnanolone (a potent stimulator of GABAA receptors) in the SC dorsal horn.Owing to the fact that neurosteroids are endogenous compounds capable of interacting with main systems of the central neurotransmission, we hope that our project, at full completion, may open new perspectives for the development of efficient therapeutic drugs against stubborn pain.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Non-nociceptive roles of opioids in the CNS: opioids' effects on neurogenesis, learning, memory and affect.

Mortality due to opioid use has grown to the point where, for the first time in history, opioid-related deaths exceed those caused by car accidents in many states in the United States. Changes in the prescribing of opioids for pain and the illicit use of fentanyl (and derivatives) have contributed to the current epidemic. Less known is the impact of opioids on hippocampal neurogenesis, the functional manipulation of which may improve the deleterious effects of opioid use. We provide new insights into how the dysregulation of neurogenesis by opioids can modify learning and affect, mood and emotions, processes that have been well accepted to motivate addictive behaviours

Assessment of neuroactive steroid formation in diabetic rat spinal cord using high-performance liquid chromatography and continuous flow scintillation detection

The combination of pulse-chase experiments with high-performance liquid chromatography and continuous flow scintillation detection was used successfully to determine the effects of chronic diabetes on neurosteroid production in the adult rat spinal cord. The long-term diabetes was induced by treatment of adult rats with streptozotocin. In the first part, the review provides an extensive description of the HPLC combined with continuous flow scintillation detection method, its advantages and appropriateness for the question investigated. Afterwards, the paper shows that progesterone formation is up-regulated in the spinal cord of diabetic rats while the biosynthesis of tetrahydroprogesterone decreased. The downregulation of tetrahydroprogesterone appeared as a mechanism facilitating progesterone accumulation in the spinal cord of streptozotocin-treated rats. Progesterone is well known to be a potent neuroprotective steroid. Enhancement of its biosynthesis may be an endogenous mechanism triggered by neural cells in the spinal tissue to cope with degenerative effects provoked by chronic diabetes. Since steroid metabolism in the spinal cord is pivotal for the modulation of several neurobiological processes including sensorimotor activities, the data analyzed herein may constitute useful information for the development of efficient strategies against deleterious effects of diabetes on the nervous system

Effects of inflammatory pain on CB1 receptor in the midbrain periaqueductal gray.

IntroductionThe periaqueductal gray (PAG) mediates the antinociceptive properties of analgesics, including opioids and cannabinoids. Administration of either opioids or cannabinoids into the PAG induces antinociception. However, most studies characterizing the antinociceptive properties of cannabinoids in the PAG have been conducted in naive animals. Few studies have reported on the role of CB1 receptors in the PAG during conditions which would prompt the administration of analgesics, namely, during pain states.ObjectivesTo examine inflammatory pain-induced changes in CB1 receptor expression and function in the midbrain periaqueductal gray.MethodsIn this study, we used the Complete Freund Adjuvant model to characterize CB1 receptor expression and G-protein coupling during persistent inflammatory pain.ResultsInflammatory pain induced an upregulation in the expression of synaptic CB1 receptors in the PAG. Despite this pain-induced change in CB1 expression, there was no corresponding upregulation of CB1 mRNA after the induction of inflammatory pain, suggesting a pain-induced recruitment of CB1 receptors to the synaptic sites within PAG neurons or increased coupling efficiency between the receptor and effector systems. Inflammatory pain also enhanced ventrolateral PAG CB1 receptor activity, as there was an increase in CP55,940-stimulated G-protein activation compared with pain-naïve control animals.ConclusionThese findings complement a growing body of evidence which demonstrate pain-induced changes in brain regions that are responsible for both the analgesic and rewarding properties of analgesic pharmacotherapies. Because much of our understanding of the pharmacology of cannabinoids is based on studies which use largely pain-naïve male animals, this work fills in important gaps in the knowledge base by incorporating pain-induced adaptations and cannabinoid pharmacology in females