Brain Micro-Biopsies for in Vivo Longitudinal Investigation of Compulsion in a Model of Addiction

Repetitive activation of the mesolimbic dopamine system by drugs or optogenetic dopamine neuron self-stimulation can lead to compulsive reinforcement-seeking behaviors. The most addictive substances such as cocaine/amphetamines lead to 10-20% of consumers eventually developing an addiction. However, little is known about individual vulnerability to addiction, mainly because investigations have focused on brain changes after drug exposure due to a lack of technology to study the biological underpinning of addiction in the same animal before and after its exposure to drugs/addictive protocols. Here we showed that this compulsive reinforcement, a characterizing symptom of addiction, is related to an alteration of the orbitofrontal cortex to Dorsal striatum transmission. Then we developed a methodological pipeline combining brain tissue imprinting and sequencing of that brain tissue. Such a method will allow future investigations aiming at identifying addiction risk factors by correlating the gene expression of each naïve mouse with its own behavior during oDASS.Les substances les plus addictives telles que la cocaïne/amphétamines conduisent 10 à 20 % des consommateurs à développer une dépendance à terme. Cependant, nous en savons encore peu sur la vulnérabilité individuelle à l’addiction, principalement car les recherches se concentrent sur les changements cérébraux après une exposition à la drogue en raison de l’absence de technologie permettant l’évaluation des mécanismes biologiques sous-tendant l’addiction sur le même animal avant et après son exposition à des drogues/protocoles addictifs. Nous montrons ici que ce renforcement compulsif, symptôme caractéristique de l'addiction, est lié à une altération de la transmission du cortex orbitofrontal au Dorsal striatum. Puis nous avons développé un pipeline méthodologique combinant le microéchantillonnage et le séquençage de ce tissu cérébral. Cette méthode permettra de futures recherches visant à identifier les facteurs de risque de l’addiction en corrélant l'expression génique de souris naïves avec leur propre comportement individuel au cours d’un protocol d’addiction

Periaqueductal efferents to dopamine and GABA neurons of the VTA

Neurons in the periaqueductal gray (PAG) modulate threat responses and nociception. Activity in the ventral tegmental area (VTA) on the other hand can cause reinforcement and aversion. While in many situations these behaviors are related, the anatomical substrate of a crosstalk between the PAG and VTA remains poorly understood. Here we describe the anatomical and electrophysiological organization of the VTA-projecting PAG neurons. Using rabies-based, cell type-specific retrograde tracing, we observed that PAG to VTA projection neurons are evenly distributed along the rostro-caudal axis of the PAG, but concentrated in its posterior and ventrolateral segments. Optogenetic projection targeting demonstrated that the PAG-to-VTA pathway is predominantly excitatory and targets similar proportions of Ih-expressing VTA DA and GABA neurons. Taken together, these results set the framework for functional analysis of the interplay between PAG and VTA in the regulation of reward and aversion

PAG afferents equally target VTA DA and GABA neurons.

<p><b>A,</b> Left, schematic of the injection protocol for patch clamp experiments. Right, example of image of ChR2-EYFP infection in the PAG. Scale bar, 500 μm. <b>B,</b> High magnification confocal images showing the colocalization or exclusion of mCherry and TH in DAT-Cre or GAD65-Cre mice, respectively. Scale bars, 50 μm. <b>C,</b> Mean amplitude of the light-evoked postsynaptic currents in VTA DA (<i>n</i> = 47) and GABA (<i>n</i> = 62) neurons plotted against the percentage of connected neurons (Mann Whitney <i>U</i> test: no difference in amplitudes, <i>p</i> > 0.05; Fisher’s exact test: no difference in connectivity, p > 0.05). Scale bars, 20 ms, 20 pA. <b>D,</b> Left, schematic of the patch clamp experiments: whole-cell recordings were performed from mCherry-expressing VTA neurons while PAG afferents inputs were light-stimulated (left). Right, excitatory currents were blocked with kynurenic acid (kyn), while kyn-resistant inhibitory currents were blocked with picrotoxin (PTX). Scale bars, 20 ms, 20 pA. <b>E,</b> Proportion of kyn-sensitive glutamate inputs and PTX-sensitive GABA inputs in VTA DA (<i>n</i> = 17) and GABA (<i>n</i> = 18) neurons (Fisher’s exact test: no difference between cell types, <i>p</i> > 0.05). <b>F,</b> Mean light-evoked current amplitude plotted against <i>I</i>_h amplitude (Spearman’s rank correlation: no correlation between the variables, <i>r</i> = 0.2702, <i>p</i> > 0.05) and comparison of <i>I</i>_h between connected and non-connected VTA DA (left) or GABA neurons (right) (Mann Whitney <i>U</i> test: * <i>p</i> < 0.05).</p

Social transmission of food safety depends on synaptic plasticity in the prefrontal cortex

When an animal is facing unfamiliar food, its odor, together with semiochemicals emanating from a conspecific, can constitute a safety message and authorize intake. The piriform cortex (PiC) codes olfactory information, and the inactivation of neurons in the nucleus accumbens (NAc) can acutely trigger consumption. However, the neural circuit and cellular substrate of transition of olfactory perception into value-based actions remain elusive. We detected enhanced activity after social transmission between two mice in neurons of the medial prefrontal cortex (mPFC) that target the NAc and receive projections from the PiC. Exposure to a conspecific potentiated the excitatory postsynaptic currents in NAc projectors, whereas blocking transmission from PiC to mPFC prevented social transmission. Thus, synaptic plasticity in the mPFC is a cellular substrate of social transmission of food safety

Spatial distribution of VTA-projecting PAG inputs.

<p><b>A,</b> Schematic of the unilateral rabies injection protocol. <b>B-C,</b> Confocal images at low (B) and high (C) magnification showing the expression of TVA-mCherry (magenta) and RVΔG-EGFP (green) in the VTA seven days after the last injection. Scale bars, 500 μm (B) and 50 μm (C). <b>D,</b> Representative confocal images showing confined starter cell infection (i.e. TVA-mCherry and RVΔG-EGFP co-expression) in coronal sections rostral (top) or caudal (bottom) to the VTA of a DAT-Cre mouse. Antero-posterior coordinates (in mm) from Bregma are shown on the top right corner. Scale bars, 500 μm. ml, medial lemniscus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. <b>E,</b> RVΔG-EGFP alone was not able to infect neurons without TVA-mCherry expression. Antero-posterior coordinates (in mm) from Bregma are shown on the top right corner. Scale bars, 500 μm. IPN, interpeduncular nucleus. <b>F,</b> RVΔG-EGFP-expressing retrogradely labeled input neurons across the rostral, central and caudal PAG segments. Scale bars, 200 μm. <b>G,</b> Average number of inputs to VTA DA (DAT, <i>n</i> = 5) and GABA (GAD, <i>n</i> = 5) neurons along the rostro-caudal axis of the PAG (two-way ANOVA: no interaction between the cell type factor and AP coordinate factor, <i>F</i>_11,120 = 0.7027, <i>p</i> > 0.05; main effect of cell type, <i>F</i>_1,120 = 18.2, <i>p</i> < 0.0001; main effect of AP coordinate, <i>F</i>_11,120 = 6.31, <i>p</i> < 0.0001; Bonferroni post-hoc test, * <i>p</i> < 0.05). Dashed lines denote the boundaries between rostral, central and caudal PAG. Inset shows the average number starter cells per ROI in DAT-Cre and GAD65-Cre mice (two-tailed <i>t</i> test: no difference between genotypes, <i>p</i> > 0.05). <b>H,</b> Relative contribution of different PAG subregions to the total inputs to VTA DA and GABA neurons (two-way ANOVA: no interaction between the cell type factor and subregion factor, <i>F</i>_5,48 = 0.5013, <i>p</i> > 0.05; no main effect of cell type, <i>F</i>_1,48 < 0.0001, <i>p</i> > 0.05; main effect of subregion, <i>F</i>_5,48 = 42.85, <i>p</i> < 0.0001; Bonferroni post-hoc test, * <i>p</i> < 0.05, **** <i>p</i> < 0.0001). Inset shows the degree of lateralization of the PAG inputs (two-tailed <i>t</i> test: no difference between genotypes, <i>p</i> > 0.05). <b>I,</b> Color-coded representation of the relative input contribution of ipsilateral and contralateral PAG subregions.</p

Stochastic synaptic plasticity underlying compulsion in a model of addiction

Activation of the mesolimbic dopamine system reinforces goal-directed behaviours. With repetitive stimulation-for example, by chronic drug abuse-the reinforcement may become compulsive and intake continues even in the face of major negative consequences. Here we gave mice the opportunity to optogenetically self-stimulate dopaminergic neurons and observed that only a fraction of mice persevered if they had to endure an electric shock. Compulsive lever pressing was associated with an activity peak in the projection terminals from the orbitofrontal cortex (OFC) to the dorsal striatum. Although brief inhibition of OFC neurons temporarily relieved compulsive reinforcement, we found that transmission from the OFC to the striatum was permanently potentiated in persevering mice. To establish causality, we potentiated these synapses in vivo in mice that stopped optogenetic self-stimulation of dopamine neurons because of punishment; this led to compulsive lever pressing, whereas depotentiation in persevering mice had the converse effect. In summary, synaptic potentiation of transmission from the OFC to the dorsal striatum drives compulsive reinforcement, a defining symptom of addiction