Functional Properties of Cerebellar Interneuron Networks – INNET

In recent years, a wealth of information has been gathered on the cellular and synaptic properties of the neurons participating in the cerebellar network. Paradoxically, global understanding of the functioning of the network is still largely derived from work carried out in the 1960's and 1970's, and has benefited only marginally from the dramatic increase in knowledge about relevant cellular mechanisms in the intervening period. This is particularly true as far as the functional role of the two major types of cerebellar interneurons is concerned, molecular layer interneurons and Golgi cells. The present project intends to help bridge this gap by using an interdisciplinary approach ranging from slice work to studies on active awake animals, and combining various cutting edge techniques (targeted infection of genetically-encoded calcium sensor in specific compartments of specific neurons; two-photon calcium imaging; use of caged neurotransmitters; paired whole cell recordings in slices; training mice for behavior studies). Several of these techniques will be used in parallel experiments in vitro and in vivo in order to facilitate transfer of results and concepts between these two levels of analysis.
The project will be carried out by four teams that have extensive experience with the cerebellar network, and that have practiced for a number of years a combination of electrophysiological and imaging approaches on this system. One of the teams will be responsible for establishing in vivo procedures; two teams will be responsible for experiments in slices, one on molecular layer interneurons and the other on Golgi cells; the last team will concentrate on the development of a new technique to use caged neurotransmitters in vivo.
Based on a series of preliminary results, interneurons will be stained using the genetically encoded calcium reporter GCaMP3. GCaMP3-expressing viral vectors will be engineered using an adenovirus approach. The modified virus will be injected in mice and the injected animals will be used for calcium measurements with two-photon imaging.
A first series of experiments will be performed in anesthetized mice. Interneuron activity will be stimulated by extracellular stimulation of parallel fibers. To help with the interpretation of these results, parallel experiments will be performed in slices infected with GCaMP3, and the results of minimal stimulations of parallel fibers and mossy fibers will be characterized. Based on these various experiments we will determine the input-output relationships in the two main types of cerebellar interneurons. Additional slice work will examine gap junction properties and the correlation between interneuron activity in various regimes of network activity, with the aim to better understand the role of electrical and chemical synapses in the network interactions.
A second series of experiments will involve two-photon calcium imaging in head restrained mice performing the repetitive task of rhythmic licking. The pattern of interneuron activity will be determined as a function of cycle phase. This pattern will be disrupted by using a novel approach consisting of photorelease of neurotransmitters from caged precursors using an optical fiber.
Altogether, these studies will help in delineating how elementary signals such as those arising in individual synapses are combined together to shape interneuron activity in a physiologically relevant environment. They will further help our understanding of how interneuron-interneuron interactions transform this activity at the network level.