Mechanisms of transmitter release at GABAergic synapses

The mechanisms of transmitter release have been widely studied at a variety of glutamatergic synapses, including the calyx of Held in the auditory brainstem. However, very little is known about the mechanisms of exocytosis at GABAergic synapses. In our previous research on the output synapses of fast-spiking, parvalbumin-expressing GABAergic interneurons, we have found that the coupling distance between Ca2+ source and Ca2+ sensor is in the nanometer range and that the number of open Ca2+ channels required for transmitter release is small, presumably only two or three. Furthermore, we have shown that a Ca2+ sensor different from synaptotagmin 1 must be involved in fast GABA release. Thus, transmitter release at this GABAergic synapse differs substantially from that at glutamatergic synapses. Based on these surprising initial findings, we propose to systematically examine the mechanisms of transmitter release at the output synapses of different types of GABAergic interneuron in the hippocampus, with major focus on the coupling between Ca2+ channels and Ca2+ sensors of exocytosis. To address this fundamental question, we will combine cutting edge electrophysiology techniques (paired recordings between synaptically connected parvalbumin- and CCK-expressing interneurons and their target cells), confocal imaging, Ca2+ uncaging, and molecular techniques. We will focus on the following questions: First, we want to determine how the short action potential in GABAergic interneurons contributes to tight coupling between Ca2+ source and Ca2+ sensor. Second, we want to study the developmental regulation of the coupling distance between channels and sensors of exocytosis at GABAergic synapses. Third, we will quantitatively examine synapse-specific differences in the coupling configuration and explore the underlying cellular and molecular mechanisms. Fourth, we will determine the affinity and molecular identity of the Ca2+ sensor that triggers transmitter release at GABA synapses, which is probably different from synaptotagmin 1. The sensor affinity will be measured using caged Ca2+ (DM- nitrophen) loaded into presynaptic terminals. The identity of the Ca2+ sensor will be determined by immunohistochemistry combined with functional analysis of knockout synapses in paired recordings. Finally, we will examine how the coupling between Ca2+ channels and Ca2+ sensors is controlled at the molecular level. Specifically, we want to investigate the functional role of three presynaptic proteins: Rab3a-interacting molecules (RIMs), neurexins, and septins. These proteins have been suggested to regulate coupling at central synapses, but their role in identified GABAergic synapses is unclear. Our results will give a clear picture of the Ca2+ channel - sensor coupling and the mechanisms of transmitter release at GABAergic synapses, approaching the level of depth previously obtained at the calyx of Held. The results will have important implications for our understanding of the function of inhibition in neuronal networks and the dysfunction of inhibition in neurological and psychiatric diseases, including epilepsy and schizophrenia.

Inhibitory synapses, which release the transmitter gamma-aminobutyric acid (GABA), play a key role in several brain functions, such as inhibitory control of brain activity, network oscillations, and separation of input patterns. For all of these functions, the fast and efficient signaling at GABAergic synapses is of fundamental importance. However, the underlying molecular and subcellular mechanisms are poorly understood. To address this question, we examined synaptic signaling in the basket cellPurkinje cell synapse, a key inhibitory synapse in the cerebellum. To examine synaptic transmission with biophysical rigor, we performed simultaneous paired recordings from synaptically connected cells. This approach allowed us to measure transmitter release with microsecond temporal resolution. We found that the mechanisms of transmitter release were different from those of excitatory synapses in multiple ways. First, we discovered tight coupling between presynaptic calcium channels (in the plasma membrane) and release sensors (in the membrane of synaptic vesicles which contain the transmitter). The coupling distance between the two molecular elements was only ~10 nanometer, much less than that at many other synapses. Tight coupling reduces diffusional delays, contributing to fast signaling at GABAergic interneuron output synapses. Second, we discovered that the calcium sensor of exocytosis was highly specialized at these inhibitory synapses. Genetic elimination of synaptotagmin 2, one of the calcium sensor candidates, substantially reduced the transmitter release evoked by single action potentials, identifying synaptotagmin 2 as the major calcium sensor at this synapse. As synaptotagmin 2 shows the fastest binding and unbinding kinetics among all members of the synaptotagmin family, these results identified a mechanism that ensures the speed of inhibitory synaptic transmission.Third, we discovered that synaptotagmin 7 played an entirely unexpected role in the regulation of transmitter release at these synapses. Genetic elimination of synaptotagmin 7 did not affect transmitter release following single action potentials, but markedly reduced transmission during trains of stimuli. Thus, synaptotagmin 7 guarantees the stability of inhibitory synaptic transmission during high-frequency activity, as would occur in cerebellar interneurons under in vivo conditions. Finally, our results suggested several similarities between GABAergic synapses in cerebellum and hippocampus, suggesting that our conclusions apply more generally. In conclusion, the results from this project revealed the main molecular mechanisms underlying speed and efficacy at GABAergic synapses in the brain. They further suggested a division of labor between two calcium sensors at GABAergic synapses, with synaptotagmin 2 being responsible for the speed and synaptotagmin 7 being important for the maintenance of synaptic efficacy during repetitive activity. The results were published in three main papers (Arai et al., 2014, eLife; Chen et al., 2017a, Cell Reports; Chen et al., 2017b, Cell Reports). These publications attracted a lot of interest in the scientific community as well as in the public media. As inhibitory synapses are sites of several neurological and psychiatric diseases, the results may be also relevant for the understanding of brain disorders and the development of new therapeutic strategies.