Connectivité synaptique et intégration du signal de direction de la tête dans le présubiculum chez la souris

Le présubiculum (PrS) est une structure clé pour le codage de l’orientation spatiale ; il est localisé entre l’hippocampe et le cortex entorhinal. Il fait partie du circuit de direction de la tête, et ses neurones sont sensibles à la direction de la tête. Le presubiculum est stratégiquement positionné pour intégrer des entrées sensorielles vestibulaires et des informations sur les repères visuels de l’environnement, relayées respectivement par le noyau thalamique antérieur (ATN) et le cortex rétrosplenial (RSC). Au cours de mon doctorat, j’ai étudié la connectivité fonctionnelle entre ces deux régions afférentes et le microcircuit du PrS composé de neurones pyramidaux et d’interneurones inhibiteurs. J’ai utilisé des techniques de traçages anatomiques, d’optogénétique et d’enregistrements électrophysiologiques en patch-clamp dans des tranches de cerveau de souris. Mes expériences de traçage rétrogrades ont montré que les neurones de l’ATN et du RSC constituaient les entrées principales du PrS. L’utilisation de vecteurs viraux antérogrades a permis l’expression d’opsines fusionnées à une protéine rapportrice fluorescente dans l’ATN et le RSC, ainsi que la photostimulation des projections axonales de ces régions dans le presubiculum. J’ai pu ainsi montrer que les axones thalamiques ciblaient les couches I et III du PrS, sur l’ensemble de l’axe dorso-ventral. Les axones du RSC se ramifiaient également dans les couches I et III mais uniquement dans la portion dorsale du PrS. La photostimulation des axones ATN et RSC initiaient des évènements postsynaptiques excitateurs dans les neurones pyramidaux de la couche III avec de courtes latences monosynaptiques. Ces évènements étaient médiés par des récepteurs AMPA et NMDA et persistaient en présence de TTX et 4-AP. Les propriétés intrinsèques de ces neurones étaient similaires, indiquant que les afférences ATN et RSC ciblent une même population neuronale. Pour étudier directement la convergence des afférences ATN et RSC sur des neurones uniques, j’ai exprimé des opsines sensibles à la lumières bleue et à la lumière rouge, Chronos et Chrimson, respectivement dans l’ATN et le RSC. La stimulation optique combinée des axones afférents a montré que les neurones de la couche III reçoivent des entrées à la fois de l’ATN et du RSC. Une activation simultanée des afférences a évoqué une sommation supra-linéaire dans ces neurones. Cette non-linéarité pourrait résulter d’une amplification des PPSEs par l’activation de courants intrinsèques ou d’un changement de la balance excitation-inhibition dans les neurones post synaptiques. J’ai cherché à savoir si les interneurones VIP du PrS pouvaient favoriser l’association des signaux de direction de la tête provenant de l’ATN avec les informations de repères visuels du RSC par des motifs de désinhibition. Les propriétés des interneurones VIP ont montré qu’il en existait deux sous-populations. J’ai trouvé que certains de ces interneurones sont connectés de manière directe par les afférences ATN et RSC, ce qui pourrait suggérer qu’ils jouent bien un rôle dans la mise à jour du signal de direction de la tête. Enfin, j’ai enregistré des neurones pyramidaux de la couche IV. Des marquages rétrogrades ont montré qu’ils projetaient vers le noyau mamillaire latéral (LMN). Leur profil de décharge en bouffées de potentiels d’action est dépendant des canaux calciques de type T. Ces neurones étaient principalement connectés de manière indirecte par l’ATN et le RSC, probablement via les neurones de la couche III. Ma thèse de doctorat a permis de démontrer que le microcircuit du PrS est adapté pour l’intégration des signaux de direction de la tête d’origine vestibulaire provenant de l’ATN et des informations visuelles du RSC. Les neurones à décharge en bouffée de la couche IV pourraient transmettre les informations sur les repères visuels pour l’actualisation du signal de direction de la tête en amont, au LMN.The presubiculum is a key structure for spatial orientation coding, located between the hippocampus and the entorhinal cortex. It is part of the brain wide head direction circuit, and its neurons are sensitive to an animals’ head direction. The presubiculum is well positioned to integrate vestibular sensory input and visual landmark information, conveyed via the anterior thalamic nuclei (ATN) and the retrosplenial cortex (RSC), respectively. During my PhD, I investigated the functional connectivity between these two afferent regions and the presubicular microcircuit, including principal pyramidal cells and inhibitory neurons. I used anatomical tracing, optogenetics and electrophysiological patch clamp recordings in mouse brain slices. Retrograde tracing experiments showed that neurons in ATN and in RSC provide major inputs to the presubiculum. Anterograde viral vectors were used to express opsins fused to a fluorescent reporter protein in the ATN or the RSC, in order to specifically stimulate transfected axons in the presubiculum with light. I show that thalamic axons target the superficial layers I and III of the presubiculum, across the entire ventral-to-dorsal axis. RSC axons ramify in layers I and III of the dorsal portion of the presubiculum only. Photostimulation of either ATN or RSC axons initiated excitatory postsynaptic events in presubicular layer III neurons, with short, monosynaptic latencies. Light-evoked EPSPs were mediated by AMPA and NMDA receptors, and persisted in the presence of TTX and 4-AP. The intrinsic properties of the recorded postsynaptic neurons were similar, indicating that thalamic and retrosplenial inputs target a same population of pyramidal neurons in layer III. To directly investigate the convergence of ATN and RSC inputs onto single neurons, I expressed blue and red shifted opsins, Chronos and Chrimson, in the ATN and the RSC, respectively. Combined, double wavelength stimulation of opsin-expressing axons in the presubiculum showed that pyramidal layer III neurons receive synaptic inputs from both ATN and RSC. Simultaneous activation of ATN and RSC fibers showed supralinear summation, for 8 out of 11 cells tested. Non-linear EPSP amplification was dependent on NMDA receptor activation and voltage dependent intrinsic currents, and facilitated in the presence of GABA blockers. I asked whether VIP interneurons in the PrS could favor the association of head directional input form ATN and landmark input from the RSC, via disinhibition. VIP interneurons were highly excitable neurons that fell in two subpopulations according to their electrophysiological properties. At least some VIP interneurons were directly connected by ATN and RSC inputs supporting the idea that they could gate the updating of the head direction signal. Finally, I recorded from layer IV pyramidal neurons in the presubiculum. Retrograde labeling showed that they project to the lateral mammillary nucleus (LMN). Their intrinsic burst firing pattern is supported by t-type Calcium currents. Layer IV neurons were mostly indirectly connected by ATN and RSC, presumably via layer III neurons. My PhD thesis has demonstrated that the presubicular microcircuit is well suited for the integration of vestibular based head direction signals from ATN with visual information from RSC. Layer IV intrinsically bursting neurons may transmit visual landmark updating of the head direction signal to the upstream LMN

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

International audienceKnowledge of cell type specific synaptic connectivity is a crucial prerequisite for understanding brain wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional, electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light sensitive ion channels allows to selectively stimulate axons coming from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterodorsal thalamic nuclei, and also in other subcortical or cortical areas throughout the brain. Here we describe a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the anterodorsal thalamus, followed by photostimulation of their axon terminals in hippocampal slices. In combination with whole-cell patch clamp recording from a postsynaptically connected presubicular neuron, photostimulation of thalamic axons allows the detection of functional synaptic connections, their pharmacological characterization, and the evaluation of their strength in the brain slice preparation. We demonstrate that axons originating in the anterodorsal thalamus ramify densely in presubicular layers 1 and 3. The photostimulation of Chronos expressing thalamic axon terminals in presubiculum initiates short latency postsynaptic responses in a presubicular layer3 neuron, indicating a monosynaptic connection. In addition, biocytin filling of the recorded neuron and posthoc revelation confirms the layer localization and pyramidal morphology of the postsynaptic neuron. Taken together, the optogenetic stimulation of long-range inputs in ex vivo brain slices is a useful method to determine the cell-type specific functional connectivity from distant brain regions

Oligodendrocyte secreted factors shape hippocampal GABAergic neuron transcriptome and physiology

International audienceOligodendrocytes form myelin for central nervous system axons and release factors which signal to neurons during myelination. Here, we ask how oligodendroglial factors influence hippocampal GABAergic neuron physiology. In mixed hippocampal cultures, GABAergic neurons fired action potentials (APs) of short duration and received high frequencies of excitatory synaptic events. In purified neuronal cultures without glial cells, GABAergic neuron excitability increased and the frequency of synaptic events decreased. These effects were largely reversed by adding oligodendrocyte conditioned medium (OCM). We compared the transcriptomic signature with the electrophysiological phenotype of single neurons in these three culture conditions. Genes expressed by single pyramidal or GABAergic neurons largely conformed to expected cell-type specific patterns. Multiple genes of GABAergic neurons were significantly downregulated by the transition from mixed cultures containing glial cells to purified neuronal cultures. Levels of these genes were restored by the addition of OCM to purified cultures. Clustering genes with similar changes in expression between different culture conditions revealed processes affected by oligodendroglial factors. Enriched genes are linked to roles in synapse assembly, AP generation, and transmembrane ion transport, including of zinc. These results provide new insight into the molecular targets by which oligodendrocytes influence neuron excitability and synaptic function