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

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.

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

RIM1 and RIM2 redundantly determine Ca2+ channel density and readily releasable pool size at a large hindbrain synapse

Zsolt Norbert Babai, Yunyun Han, Ralf Schneggenburger

The localization and density of voltage-gated Ca2+ channels at active zones are essential for the amount and kinetics of transmitter release at synapses. RIM proteins are scaffolding proteins at the active zone that bind to several other presynaptic proteins, including voltage-gated Ca2+ channel alpha-subunits. The long isoforms of RIM proteins, which contain NH2 -terminal Rab3- and Munc13-interacting domains, as well as a central PDZ domain and two COOH-terminal C2 domains, are encoded by two genes, Rim1 and Rim2. Here, we used the ideal accessibility of the large calyx of Held synapse for direct presynaptic electrophysiology to investigate whether the two Rim genes have redundant, or separate, functions in determining the presynaptic Ca2+ channel density, and the size of a readily releasable vesicle pool (RRP). Quantitative PCR showed that cochlear nucleus neurons, which include calyx of Held generating neurons, express both RIM1 and RIM2. Conditional genetic inactivation of RIM2 at the calyx of Held led to a subtle reduction in presynaptic Ca2+ current density, whereas deletion of RIM1 was ineffective. The release efficiency of brief presynaptic Ca2+ "tail" currents and the RRP were unaffected in conditional single RIM1 and RIM2 knockout (KO) mice, whereas both parameters were strongly reduced in RIM1/2 double KO mice. Thus, despite a somewhat more decisive role for RIM2 in determining presynaptic Ca2+ channel density, RIM1 and RIM2 can overall replace each other's presynaptic functions at a large relay synapse in the hindbrain, the calyx of Held.

American Physiological Society2015

Imaging of AMPA-type glutamate receptors in hippocampal neurons

Michel Kropf

Synaptic plasticity is the ability of the connections between nerve cells, called synapses, to change in strength. The presence of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPAR) at the synapse controls the strength of excitatory transmission and their number can be modulated. Controlled surface insertion of AMPA-type glutamate receptors and their lateral diffusion into synapses are proposed to be major mechanisms for the regulation of synaptic plasticity. Here we applied the recently established Acyl-Carrier-Protein-labeling technique in combination with lentiviral expression in hippocampal neurons to label individually ACP-tagged AMPA receptor subunits at the surface of neurons. This technique uses a carrier protein from the fatty acid synthesis and a mutant therof as a tag, which were fused by genetic engineering to the AMPAR subunits. The labeling procedure is specific for proteins, AMPARs in our case, which are present at the plasma membrane of the cell. We show that this technique allows to differentially label two subunits of the receptor. Moreover, these subunits are integrated into heteromeric receptors together with endogenous subunits, and these labeled receptors are targeted to functional synapses. The diffusion coefficient of labeled GluR2 at the surface of living neurons is significantly higher in GluR2/GluR3-infected neurons compared to GluR1/GluR2-infected neurons suggesting a higher mobility of GluR2/3 receptors compared to GluR1/2 receptors. Finally, in sequential labeling experiments we detected newly inserted receptor subunits colocalized with presynaptic markers. The GluR1 subunit displayed a faster kinetics of insertion compare to the other subunits. Internalized GluR2 receptors have been shown to accumulate in perinuclear late endosomes in HEK 293T cells. Moreover, we expressed and labeled surface tagged-GluR2 receptors in hippocampal slices, using similar labeling technique as ACP. High-resolution images, sub-difraction limited, of hippocampal neurons stained for GluR2 were acquired with an experimental scanning near-field optical microscopy (SNOM). Together our results strongly indicate that receptor insertion is faster for GluR1 and that insertion occurs at or in close vicinity of synapses in mature hippocampal neuron cultures.