Facteurs neurobiologiques associés à l'addiction à la cocaïne : etude au moyen d'un modèle d'addiction chez le rat

De nombreux individus consomment une, ou plusieurs, substances psychoactives au cours de leur vie. Alors que la majorité des consommateurs maintient une utilisation épisodique et contrôlée, certains perdent le contrôle sur l’usage de la substance ; symptôme majeur d’une addiction. Comprendre les mécanismes psychobiologiques, qui sous-tendent cette vulnérabilité à passer d’un usage contrôlé à un usage compulsif, constituerait une étape décisive dans la compréhension de la pathologie et l’identification de cibles thérapeutiques pertinentes. En effet, malgré un intense effort de recherche au cours des 40 dernières années, les thérapies disponibles se révèlent d’une efficacité limitée. Il faut probablement en chercher la cause dans la complexité de la pathologie, mais aussi dans l’inadéquation des préparations expérimentales à la définition clinique de l'addiction. Dans ce contexte, une étape décisive a été franchie il y a 5 ans lorsque des chercheurs de notre équipe ont développé le premier modèle pluri-symptomatique d’addiction à la cocaïne chez le rat. Grâce à ce modèle, il est possible d’identifier des animaux qui développent un comportement similaire à l’addiction, alors que d’autres maintiennent un usage contrôlé, et ce, malgré une consommation préalable de drogue équivalente. S'appuyant sur ce modèle d'addiction chez le rat, le but de nos travaux de thèse était d’identifier des différences neurobiologiques entre usagers addicts et non-addicts et d’aborder les mécanismes neurobiologiques qui sous-tendent la transition vers l’addiction. Pour ce second point, nous avons considéré les différences majeures identifiées et étudié leur évolution de l’usage précoce de cocaïne (avant l'addiction) à l'usage tardif (après développement de l’addiction) en fonction des sujets (addicts versus non-addicts). Nous avons utilisé une approche multidisciplinaire associant les principales méthodes des neurosciences comportementales, moléculaires et des systèmes. Nous avons comparé addicts et non-addicts au moyen d’une stratégie ciblée et d’une stratégie non ciblée. La stratégie non ciblée a consisté à évaluer l'expression des gènes à grande échelle, dans des structures cérébrales clés, au moyen d’une technique de gene profiling (environ 28 000 gènes testés simultanément). L'approche ciblée a consisté à analyser des facteurs connus pour être modifiés par l'usage ou l’administration chronique de cocaïne. Nous nous sommes concentrés sur une structure majeure, le noyau accumbens, et avons étudié : i. l'expression de gènes cibles au moyen de la qPCR, ii. plusieurs formes de plasticité synaptique à l'aide de l’électrophysiologie in vitro, iii. l'activité des neurones dopaminergiques de l’aire tegmentale ventrale (VTA) (projetant vers le noyau accumbens) au moyen de l’électrophysiologie in vivo, iv. la libération de dopamine dans le noyau accumbens au moyen de la microdialyse in vivo. Nous avons montré que les rats addicts et non-addicts s'adaptent très différemment à la cocaïne. Les usagers non-addicts paraissent en mesure de contrecarrer les changements neurobiologiques précoces provoqués par la cocaïne, tandis que les addicts ne le sont pas. Ils présentent des réponses neurobiologiques similaires à celles d'animaux naïfs vis-à-vis de la cocaïne ou d'animaux ayant été exposés à la drogue sur une très courte période. En résumé, les données obtenues au cours de ce travail de thèse modifient drastiquement la perception commune de la psychopathologie de l’addiction. L'addiction résulterait moins de modifications produites par la drogue (comme on le pense depuis 40 ans) que de l'incapacité à lutter contre ces modifications.Numerous individuals consume one, or several, psychoactive substances during their lifetime. Although most consumers make only occasional and controlled use of a substance, some lose control of their use, which constitutes a major symptom of addiction. Understanding the psychobiological mechanisms which underlie this vulnerability to the transition from controlled drug use to addiction would constitute a decisive step forward in our understanding of the pathology and in our identification of the relevant therapeutic targets. Indeed despite intense research efforts during the last 40 years, the therapies available are of limited efficacy. This is probably related to the complexity of the pathology, as well as to the unsuitability of experimental preparations to the clinical definition of addiction. In such a context, there was a significant breakthrough 5 years ago when our research team developed the first pluri-symptomatic model of cocaine addiction in the rat. Thanks to this model it is possible to single out animals that develop an addiction-like behavior from others that, despite equivalent prior drug consumption, keep their use under control. The aim of our doctoral research, based on this rat addiction model, was to identify the neurobiological differences between addicted and non-addicted users, and then to start investigating neurobiological mechanisms that could underly transition to addiction. For this latter point we considered the major differences identified between addicts and non-addicts, and we studied their evolution from early (before addiction) to late cocaine use (after addiction development). We used a multidisciplinary approach associating behavioral, molecular and systems neuroscience. We compared addicts and non-addicts by means of a targeted strategy and a non-targeted strategy. The non-targeted strategy consisted in evaluating large-scale gene expression by means of a gene–profiling technique (approximately 28,000 genes tested simultaneously). The targeted approach consisted in analysing factors known to be modified by the use or chronic administration of cocaine. Concentrating on one main structure, the nucleus accumbens, we studied: (i) the expression of targeted genes by means of qPCR; (ii) several forms of synaptic plasticity using in vitro electrophysiology; (iii) the activity of the dopaminergic neurons of the ventral tegmental area (VTA) (projecting towards the nucleus accumbens) using in vivo electrophysiology; (iv) the liberation of dopamine in the nucleus accumbens by means of in vivo microdialysis. We showed that addict and non-addict rats adapt to cocaine in very different ways. Non-addict users seem able to counteract the early neurobiological changes triggered by cocaine, while addict users are not. Addict users present neurobiological responses to cocaine that are similar to those of naive animals or of animals having been exposed to the drug for a very short period. In conclusion, the data obtained during this PhD work radically modify the general perception of the psychopathology of addiction. Addiction would be less due to modifications produced by the drug (as thought for the last 40 years) than to the inability to fight against those modifications

Facteurs neurobiologiques associés à l'addiction à la cocaïne : etude au moyen d'un modèle d'addiction chez le rat

De nombreux individus consomment une, ou plusieurs, substances psychoactives au cours de leur vie. Alors que la majorité des consommateurs maintient une utilisation épisodique et contrôlée, certains perdent le contrôle sur l’usage de la substance ; symptôme majeur d’une addiction. Comprendre les mécanismes psychobiologiques, qui sous-tendent cette vulnérabilité à passer d’un usage contrôlé à un usage compulsif, constituerait une étape décisive dans la compréhension de la pathologie et l’identification de cibles thérapeutiques pertinentes. En effet, malgré un intense effort de recherche au cours des 40 dernières années, les thérapies disponibles se révèlent d’une efficacité limitée. Il faut probablement en chercher la cause dans la complexité de la pathologie, mais aussi dans l’inadéquation des préparations expérimentales à la définition clinique de l'addiction. Dans ce contexte, une étape décisive a été franchie il y a 5 ans lorsque des chercheurs de notre équipe ont développé le premier modèle pluri-symptomatique d’addiction à la cocaïne chez le rat. Grâce à ce modèle, il est possible d’identifier des animaux qui développent un comportement similaire à l’addiction, alors que d’autres maintiennent un usage contrôlé, et ce, malgré une consommation préalable de drogue équivalente. S'appuyant sur ce modèle d'addiction chez le rat, le but de nos travaux de thèse était d’identifier des différences neurobiologiques entre usagers addicts et non-addicts et d’aborder les mécanismes neurobiologiques qui sous-tendent la transition vers l’addiction. Pour ce second point, nous avons considéré les différences majeures identifiées et étudié leur évolution de l’usage précoce de cocaïne (avant l'addiction) à l'usage tardif (après développement de l’addiction) en fonction des sujets (addicts versus non-addicts). Nous avons utilisé une approche multidisciplinaire associant les principales méthodes des neurosciences comportementales, moléculaires et des systèmes. Nous avons comparé addicts et non-addicts au moyen d’une stratégie ciblée et d’une stratégie non ciblée. La stratégie non ciblée a consisté à évaluer l'expression des gènes à grande échelle, dans des structures cérébrales clés, au moyen d’une technique de gene profiling (environ 28 000 gènes testés simultanément). L'approche ciblée a consisté à analyser des facteurs connus pour être modifiés par l'usage ou l’administration chronique de cocaïne. Nous nous sommes concentrés sur une structure majeure, le noyau accumbens, et avons étudié : i. l'expression de gènes cibles au moyen de la qPCR, ii. plusieurs formes de plasticité synaptique à l'aide de l’électrophysiologie in vitro, iii. l'activité des neurones dopaminergiques de l’aire tegmentale ventrale (VTA) (projetant vers le noyau accumbens) au moyen de l’électrophysiologie in vivo, iv. la libération de dopamine dans le noyau accumbens au moyen de la microdialyse in vivo. Nous avons montré que les rats addicts et non-addicts s'adaptent très différemment à la cocaïne. Les usagers non-addicts paraissent en mesure de contrecarrer les changements neurobiologiques précoces provoqués par la cocaïne, tandis que les addicts ne le sont pas. Ils présentent des réponses neurobiologiques similaires à celles d'animaux naïfs vis-à-vis de la cocaïne ou d'animaux ayant été exposés à la drogue sur une très courte période. En résumé, les données obtenues au cours de ce travail de thèse modifient drastiquement la perception commune de la psychopathologie de l’addiction. L'addiction résulterait moins de modifications produites par la drogue (comme on le pense depuis 40 ans) que de l'incapacité à lutter contre ces modifications.Numerous individuals consume one, or several, psychoactive substances during their lifetime. Although most consumers make only occasional and controlled use of a substance, some lose control of their use, which constitutes a major symptom of addiction. Understanding the psychobiological mechanisms which underlie this vulnerability to the transition from controlled drug use to addiction would constitute a decisive step forward in our understanding of the pathology and in our identification of the relevant therapeutic targets. Indeed despite intense research efforts during the last 40 years, the therapies available are of limited efficacy. This is probably related to the complexity of the pathology, as well as to the unsuitability of experimental preparations to the clinical definition of addiction. In such a context, there was a significant breakthrough 5 years ago when our research team developed the first pluri-symptomatic model of cocaine addiction in the rat. Thanks to this model it is possible to single out animals that develop an addiction-like behavior from others that, despite equivalent prior drug consumption, keep their use under control. The aim of our doctoral research, based on this rat addiction model, was to identify the neurobiological differences between addicted and non-addicted users, and then to start investigating neurobiological mechanisms that could underly transition to addiction. For this latter point we considered the major differences identified between addicts and non-addicts, and we studied their evolution from early (before addiction) to late cocaine use (after addiction development). We used a multidisciplinary approach associating behavioral, molecular and systems neuroscience. We compared addicts and non-addicts by means of a targeted strategy and a non-targeted strategy. The non-targeted strategy consisted in evaluating large-scale gene expression by means of a gene–profiling technique (approximately 28,000 genes tested simultaneously). The targeted approach consisted in analysing factors known to be modified by the use or chronic administration of cocaine. Concentrating on one main structure, the nucleus accumbens, we studied: (i) the expression of targeted genes by means of qPCR; (ii) several forms of synaptic plasticity using in vitro electrophysiology; (iii) the activity of the dopaminergic neurons of the ventral tegmental area (VTA) (projecting towards the nucleus accumbens) using in vivo electrophysiology; (iv) the liberation of dopamine in the nucleus accumbens by means of in vivo microdialysis. We showed that addict and non-addict rats adapt to cocaine in very different ways. Non-addict users seem able to counteract the early neurobiological changes triggered by cocaine, while addict users are not. Addict users present neurobiological responses to cocaine that are similar to those of naive animals or of animals having been exposed to the drug for a very short period. In conclusion, the data obtained during this PhD work radically modify the general perception of the psychopathology of addiction. Addiction would be less due to modifications produced by the drug (as thought for the last 40 years) than to the inability to fight against those modifications

Anti-nociceptive effect of peripheral serotonin 5-HT2B receptor activation on neuropathic pain.

International audienceSerotonin is critically involved in neuropathic pain. However, its role is far from being understood owing to the number of cellular targets and receptor subtypes involved. In a rat model of neuropathic pain evoked by chronic constriction injury (CCI) of the sciatic nerve, we studied the role of 5-HT(2B) receptor in dorsal root ganglia (DRG) and the sciatic nerve. We showed that 5-HT(2B) receptor activation both prevents and reduces CCI-induced allodynia. Intrathecal administration of 5-HT(2B) receptor agonist BW723C86 significantly attenuated established mechanical and cold allodynia; this effect was prevented by co-injection of RS127445, a selective 5-HT(2B) receptor antagonist. A single application of BW723C86 on the sciatic nerve concomitantly to CCI dose-dependently prevented mechanical allodynia and significantly reduced cold allodynia 17 days after CCI. This behavioral effect was accompanied with a marked decrease in macrophage infiltration into the sciatic nerve and, in the DRG, with an attenuated abnormal expression of several markers associated with local neuroinflammation and neuropathic pain. CCI resulted in a marked upregulation of 5-HT(2B) receptor expression in sciatic nerve and DRG. In the latter structure, it was biphasic, consisting of a transient early increase (23-fold), 2 days after the surgery and before the neuropathic pain emergence, followed by a steady (5-fold) increase, that remained constant until pain disappeared. In DRG and sciatic nerve, 5-HT(2B) receptors were immunolocalized on sensory neurons and infiltrating macrophages. Our data reveal a relationship between serotonin, immunocytes, and neuropathic pain development, and demonstrate a critical role of 5-HT(2B) receptors in blood-derived macrophages

Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis

Image texture parameter “granularity” for analysis of early mHTT aggregation.

<p>(<b>A</b>) Image analysis strategy for early aggregation parameter “granularity”. The area of cell nuclei was detected based on the DAPI signal. The spatial pattern of pixel intensities of nuclear EM48-ir signal was analyzed and a granularity index indicating formation of small EM48-ir inclusion species was calculated using the SER (Spots) texture algorithm (Acapella, PerkinElmer). Brain sections from 2–12 months old zQ175 heterozygote mice were immunolabelled for DARPP-32 and EM48. The “granularity” index in the striatum and cortex was used to detect and monitor early changes in mHTT distribution and signal clustering. (<b>B</b>) A significant increase in the nuclear EM48 “granularity” in the striatum was observed from 2 to 6 months of age followed by a significant decrease from 6 to 12 months. (<b>C</b>) In the cortex, a significant increase in EM48 granularity was observed from 6 to 12 months. Data are displayed as dot plots with mean +/-SD. Statistical analysis performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every age an n of 8 animals with 6 sections per animal were used for quantitation; *p<0.05; **p<0.01; ***p<0.001.</p

Time course of mHTT inclusions appearance in the striatum and cortex of zQ175 mice.

<p>Brain samples from 3–12 months old zQ175 heterozygous mice were stained for mHTT inclusions (bright spots) by EM48-ir and imaged on the Opera high content microscope. A progressive increase in EM48 signal in both striatal and cortical brain regions was clearly visible with age. mHTT inclusions as indicated by EM48-ir puncta appeared earlier and with higher abundance in the striatum as compared to cortex.</p

Modulation of HTT levels, as evidenced by MAB2174 and EM48 immunoreactivity in the striatum of zQ175 heterozygous mice by AAV2 viruses expressing HTT-targeting shRNAs.

<p>AAV2 viruses encoding GFP and shRNAs directed against HTT were injected in the right ventricle of neonate zQ175 heterozygous mice. At 4 months of age, mice were euthanized and analysed for HTT inclusions and HTT cytoplasmic levels. Representative images showing DARPP-32, mHTT IHC staining (<b>A</b>) or DARPP-32, HTT (MAB2174) IHC staining (<b>B</b>) in the GFP positive striatal region transduced with AAV2 encoding shRNA against HTT (mHtt-sh#2 and mHtt-sh#4) or non-target control shRNAs (shC004). Quantitative analysis of mHTT inclusions (<b>C</b>) and MAB2174-ir intensity (<b>D</b>) in GFP positive cells of the striatum: mHtt targeting shRNA led to a significant decrease in the number of nuclear HTT EM48-ir inclusions and MAB2174-ir HTT cytoplasmic levels in comparison to the control AAV/shC004. Data are displayed as bar graphs with mean +/-SD. Statistical analysis was performed using standard ANOVA and Sidak’s multiple comparisons’ test. For every group an n of 4 animals with 3 sections per animal were used for quantitation; p<0.05; **p<0.01; ***p<0.001.</p

Illustration of inclusion quantification in the striatum of zQ175 mice.

<p>Micrographs showing image segmentation strategy for the automated analysis of mHTT inclusion numbers, localization and size in the striatum. (<b>A</b>) Coronal brain sections were co-immunostained for detection of DAPRPP-32 and mHTT and image acquisition of striatal and cortical regions was performed with the Opera (PerkinElmer Inc.). (<b>B</b>) Multi-field images were acquired using 40x objective lens corresponding to the region of interest for subcellular resolution. (<b>C</b>) Area of cell nuclei was determined based on the DAPI signal using a sliding parabola filter for background correction. (<b>D</b>) Medium spiny neurons were identified using nuclear intensity of DARPP-32 staining (green). (<b>E</b>) An extranuclear region was defined with 10 pixels spacing to the nuclear region. Numbers of mHTT nuclear and extranuclear inclusions in medium spiny neurons (MSNs) were quantified based on EM48 signal.</p

Workflow of high content imaging for <i>ex vivo</i> phenotypic characterization.

<p>Mouse brain sections fluorescently stained for up to three target proteins were aligned and mounted in glass-bottom multi-well plates suitable for high content imaging. Using an automated imaging setup (Opera, PerkinElmer) up to 600 high resolution confocal images per brain section were acquired. Automated multi-parametric analysis was applied to every single image to generate comprehensive quantitative data sets for each section describing numbers, subcellular structures, morphology and intensities for stained subpopulation of cells.</p

Inclusion appearance in various cortical and striatal regions in zQ175 heterozygous mice.

<p>(<b>A</b>) Using an automated microscope, whole mouse brain sections were scanned by high resolution multi-image acquisition. Individual images were assigned to distinct areas within the cortex including the cingulate cortex (ccx) and motor cortex (mcx), or within the striatum, including dorsal (d)/ventral (v) and medial (m)/lateral (l) parts to allow region-specific automated multiparametric analysis. (<b>B</b>) Region-specific analysis in the striatum of nuclear mHTT inclusions in MSNs. Inclusion number was found to be significantly higher in lateral quadrants (ld and lv) than in medial ventral quadrant at 8 and 12 months old zQ175 mice. (<b>C</b>, <b>D</b>) Subregion specific analysis in the cortex showing quantification of the number of nuclear (<b>C</b>) and extranuclear (<b>D</b>) mHTT inclusions in the cingulate and motor cortex over time. A significantly higher number of inclusions were detected in the ccx compared to mcx region in zQ175 heterozygous mice at 12 months of age. Data are displayed as mean +/-SD. Statistical analysis was performed by two-way ANOVA and Sidak’s multiple comparisons’ test. Mean values were calculated for every age and region using an n of 8 animals with 6 sections per animal; *p<0.05; **p<0.01; ***p<0.001.</p