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Thèse Stefano Zucca

Analysis of synaptic function of CA3 microcircuits in vivo using optogenetic tools

Le 20 décembre 2013

Vendredi 20 décembre directeur de thèse Christophe Mulle   IINS

Université Bordeaux Segalen, Salle des Thèses, à 15h00, 20 Décembre 2013

The mossy fibers axons of the granule cells of the dentate gyrus pass the information coming from the layer II of the entorhinal cortex to pyramidal neurons located in the CA3 area of the hippocampus. Leaving the hilus, the axons of granule cells gather in a narrow area called stratum lucidum, where large pre-synaptic terminals called Mossy Fiber Boutons (MFBs) form synaptic connections with the thorny excrescences of CA3 pyramidal neurons. A large number of in vitro studies showed that mf-CA3 synapses are particularly distinct from most of other synapses in the central nervous system for their unique physiological properties such as low probability of neurotransmitter release associated with a robust short term plasticity (paired pulse facilitation and frequency facilitation). This raises the question if these unique properties reflect a unique functional role of mf-CA3 synapses in hippocampal circuits. Unfortunately very little is known on how granule cells modulate the activity of CA3 neurons in the intact network in vivo. The paucity of information is due to the fact that classical manipulation of neuronal circuits using electrical, pharmacological and genetic approaches lack spatial and temporal precision in vivo. However the recent and fast growth of optogenetics in neuroscience has provided new probes with high spatial selectivity (cell specific optical activation) and temporal precision (at the millisecond scale), allowing the dissection and investigation of even deep neuronal circuits in vivo.

The aim of my thesis was to gain insight into the physiological operation of the CA3 region and characterize how activation of mossy fibers impacts on synaptic transmission and plasticity in the CA3 region in the intact mouse brain. To study the mossy fiber synapses in vivo I used a combined approach of optogenetics to selectively target and control the activity of granule cells, while characterizing the electrophysiological properties of CA3 pyramidal neurons. In the first part of my PhD I implemented optogenetic tools and optimized in vivo viral expression of opsins. To drive ChR2 expression in granule cells I used a Cre-LoxP recombinase approach in POMC-Cre mice. For my studies I chose a variant of ChR2 that carries a mutation in position H134R.

This ChR2 variant is widely used for in vivo applications and carries a gain-of-function mutation that increases photocurrents and sensitivity to light. I verified expression and functionality of H134R variant using ex vivo acute slices. I found that brief pulses of blue light reliably induced action potentials over a range of different frequencies of stimulation. I also found that optical stimulation can be used to trigger short-term plasticity at mf-CA3 synapses in vitro. In the second part of my studies I aimed at characterizing the synaptic properties of mf-CA3 in vivo. First I characterized the electrophysiological properties of granule cells and CA3 neurons performing extracellular and whole-cell recordings in anesthetized mice.

Subsequently I refined optogenetic stimulation methodology in vivo for non-invasive characterization of synaptic plasticity of the mf-CA3 microcircuit. I found that 1) frequency facilitation processes occur in vivo at mf-CA3 synapses and 2) the period of synaptic facilitation is correlated with an increase of gamma activity supporting the key role of gamma oscillations in regulating the flow of information among anatomically coupled networks. In conclusion it appears that increased gamma oscillations that occurs during granule cells activation may play a key role during the storage of information in the CA3 network.



Bruno Bontempi
DR CNRS Président
Jack Mellor
DR Université de Bristol Rapporteur
Jean Christophe Poncer
DR INSERM Rapporteur
Jérôme Baufreton
CR CNRS Examinateur
Jérôme Epsztein
CR INSERM Examinateur
Christophe Mulle
Directeur de thèse

Directeur de thèse

Christophe Mulle
Directeur de recherche - PhD
Responsable de l'équipe - Physiologie des synapses glutamatergiques