An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses.

RIM Determines Ca²+ Channel Density and Vesicle Docking at the Presynaptic Active Zone

Yunyun Han

Fast neurotransmitter release is essential for neuron-neuron communication and is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles at the presynaptic active zone. The high concentration of Ca2+ channels at the active zone is important to secure fast transmitter release. However, the mechanism that enriches Ca2+ channels at active zones is largely unknown, maybe because of the limited accessibility of most model synapses to direct measurements of Ca2+ signaling in the nerve terminal. RIM's (Rab3 interacting molecule) are scaffolding proteins in the active zone which interact with several presynaptic proteins. As revealed by previous studies in C. elegans and cultured neurons, RIM proteins affect synaptic transmission. However, their complex functions have not been clearly dissected, because direct access to the presynaptic nerve terminal has so far not been possible in synapses that are amenable to genetic manipulation. Here, we have established a Cre-lox based conditional KO approach at a presynaptically accessible CNS synapse, the calyx of Held. This has allowed us to use presynaptic recordings and Ca2+ uncaging, as well as electron microscopic analyses of synapses which have developed in vivo, to directly study the presynaptic functions of RIM proteins. We discovered three major functions of RIM proteins: First, RIM's hold Ca2+ channels in the presynaptic terminals to secure the high Ca2+ channel density at active zones. Removal of RIM proteins leads to a decrease of presynaptic Ca2+ current amplitude (by ∼ 50%) as well as to a decreased immunostaining signal using an antibody against a Ca2+ channel α-subunit (by ∼ 40%). Second, RIM proteins dock vesicles to the active zone and therefore control the size of readily releasable pool. In RIM1/2 cDKO synapses, the number of docked vesicles is reduced by ∼ 80% and the functional pool size is decreased by ∼ 75%. The good correlation of morphological and physiological data identified RIM as the first presynaptic protein in a CNS synapse with a clearly corresponding role in vesicle docking, and priming. Third, RIM proteins increase the release probability and speed up the release kinetics. This is carried out by two new functions: increasing the intrinsic Ca2+ sensitivity of release and tightly coupling docked vesicles to Ca2+ channels. Taken together, this study shows that RIM proteins function as the central organizers to coordinate active zone function: holding the Ca2+ channels in the presynaptic terminal, docking vesicles to the active zones, and regulating the release probability of any given readily-releasable vesicle. All the three functions together secure the fast vesicle release when the AP arrives in the presynaptic terminal.

EPFL2011

Functional and structural properties of strong inhibitory synapses in a binaural auditory circuit

Enida Gjoni

Neuronal circuits are defined in their function and computational outcome by the synaptic interplay of Excitation and Inhibition (E/I). One step further in understanding the role of these opposite interactors in neuronal computations consists in determining their relative contribution, in terms of input strength and convergence. In the auditory brainstem, neurons in the Lateral Superior Olive (LSO) rely upon binaural E/I integration for computing sound-source localization. We made use of this circuit as a model for understanding the role of synaptic connectivity in information processing. We characterized the functional connectivity of the inhibitory and excitatory inputs onto a given LSO neuron. We found that LSO neurons receive few but large inhibitory inputs with a unitary conductance of 9 nS (under physiological intracellular Cl- concentration), although the unitary strength of inputs scattered over a 10-fold range. We estimated that unitary inhibitory postsynaptic currents (IPSCs) were mediated by a large number of synchronously released glycine vesicles (on average: 48 ± 39, range 9 - 115). The large quantal content suggests that these IPSCs are generated by a high number of functional release sites. Electron-microscopy based reconstructions revealed the structural specialization of the inhibitory axons which contacted the soma and proximal dendrites of a LSO neuron multiple times via large varicosities (diameter range 1 ¿ 11 µm), each containing on average three active zones. In contrast to the strong unitary IPSCs, excitatory inputs were numerous but had a small unitary conductance (~ 0.7 nS). A computational model of E/I integration showed that the strength and convergence of excitatory inputs determines how spontaneous firing is transmitted from upstream neurons, whereas the strength and convergence of inhibitory inputs determines the dynamic range of the tuning curve. We then aimed to study mechanisms underlying the developmental acquisition of strong inhibitory inputs on LSO neurons. Input-output curves were gradual at post-natal days (P) 5 and 6 but showed, in most cases, clear steps from P7 onwards, indicative of a reduction in the number of synaptic inputs and a growth in the strength of the individual inputs. In addition, we wished to investigate whether Synaptotagmin1 (Syt1) represents the Calcium sensor that guarantees the co-release of glutamate which has been known to occur at the immature inhibitory MNTB - LSO synapses. Genetic deletion of Syt1 caused asynchronous glutamate release only during the first 2 ¿ 3 post-natal days, suggesting that Syt1 might gradually become redundant with another Calcium sensor after ~ P5. In conclusion, this study highlights the functional and structural specializations of inhibitory synapses optimized for fulfilling the computational properties of binaural neurons devoted to sound localization. It also suggests that the basic synaptic connectivity at this connection is laid down in the first week of postnatal development.

EPFL2017

RIM Determines Ca²+ Channel Density and Vesicle Docking at the Presynaptic Active Zone

Yunyun Han

EPFL2011

Functional and structural properties of strong inhibitory synapses in a binaural auditory circuit

Enida Gjoni

EPFL2017