Role of calcium sensors in differential alignment of synaptic nanomachinery during neurotransmission

Abstract

Some of the brain's most remarkable feats such as learning and memory, are thought to emerge from elementary properties of chemical synapses. The distinctive feature of these synapses is that the action potential in the pre-synaptic terminal leads to the release of a chemical transmitter at a specialised site called the active zone (AZ) on the pre-synaptic membrane. The release of neurotransmitter from the pre-synaptic terminal is broadly classified into three types: synchronous, asynchronous, and spontaneous. Synchronous and asynchronous neurotransmitter release is a part of evoked release which is action potential dependent. Synchronous and asynchronous release depend on calcium sensors Synaptotagmin 1 (Syt1) and Synaptotagmin 7 (Syt7) respectively in the pre-synapse. The neurotransmitter released into the synaptic cleft is initially sensed by AMPA (alpha- amino- 3- hydroxy- 5 -methyl- 4 isoxazolepropionic acid) receptors which form functional nanodomains on the post- synaptic membrane. The main purpose of the current study is to understand how spatial alignments of different release events on the presynapse influence the organization of AMPAR nanodomains on the postsynapse. To address this, we evaluated spatial organization of these molecules on the pre and postsynapse when all evoked release is blocked tetrodotoxin (TTX). Absence of all evoked release triggers a type of plasticity known as homeostatic plasticity where in synapses it takes the form of synaptic scaling. It involves a negative feedback process by which neurons adjust (scale) synaptic strength to compensate for increased or decreased overall input preventing dysfunction of neuronal network. Homeostatic synaptic scaling by activity deprivation during different stages of synapse development modulates a switch between post and presynaptic strengthening. This study showed how presynaptic mechanisms are employed during synaptic scaling in mature synapses whereas postsynaptic scaling was utilised during synaptic scaling in young synapses. Previous observations have shown how CaV2.1 VGCC are important for presynaptic homeostatic plasticity. VGCCs are a crucial functional component of the AZ whose localization at the AZ depends on the structural scaffolding protein Bassoon. In the first part of this study, we wanted to understand the nanoscale organization of Bassoon and VGCC when subjected to synaptic scaling at young and mature synapses. Using super resolution microscopy, we looked at the propensity of cluster formation for Bassoon and VGCC when homeostatic scaling was applied at young (DIV7) and mature (DIV14) synapses. Our results showed that both Bassoon and VGCC form nanodomains and the propensity of cluster formation is higher at younger synapses compared to mature ones. Additionally, there is an increase in free Bassoon and VGCC molecules when homeostatic scaling is applied at mature synapses which is in agreement with previous literature. Our data also revealed CaV2.1 VGCC as a potential presynaptic player for multiplicative scaling. For the second part of the project, we looked at the transsynaptic alignment of synchronous and asynchronous calcium sensors along with postsynaptic GluA2 (subunit of AMPAR). Calcium sensors also formed nanodomains on the presynapse and their alignment changed when homeostatic scaling is applied at different developmental stages of a synapse. Homeostatic scaling in young synapses reduced levels of both synchronous calcium sensor (Syt1) and its associated GluA2 whereas it does not have any effect on asynchronous calcium sensor (Syt7) levels, but it significantly reduced the GluA2 levels associated with it on the post-synapse. When homeostatic scaling was applied at mature synapses, it offset the alignment of synchronous calcium sensor (Syt1) and its associated GluA2 by 100nm. On the other hand, it increased the alignment of asynchronous calcium sensor (Syt7) and its associated GluA2. After looking at homeostatic scaling in both young and mature synapses, we identified that the increase in clustering of asynchronous calcium sensor is concomitant with increased GluA2 clustering only in mature synapses. This increased alignment could be a part of the potential mechanism by which the neuron adjusts the rate and amplitude of synaptic transmission in response to synaptic scaling in a mature neuron. After evaluating the effect of blocking all evoked neurotransmitter release, we wanted to probe the effects of over expressing the calcium sensors important for evoked neurotransmitter release. Therefore, for the third part of the study, we overexpressed both Syt1 and Syt7 in young and mature neurons and looked at their localization in association with GluA2. Our data showed that there were no nanodomains of Syt1 and associated GluA2 within 100nm distance of each other in a young neuron. Same was observed for Syt7 and GluA2. Mature neurons on the other hand showed nanodomains of Syt1 with GluA2 both within and outside 100nm distance from each other, Syt7 and its associated GluA2 also showed similar spatial distribution. Mature neurons also showed increased amount of Syt1 and Syt7 within nanodomains. These observations summarize that formation of compact transsynaptic alignment between the over expressed calcium sensors and their associated GluA2 is manifested in mature synapses alone, consistent with alteration of synaptic frequency changes in mature cultures in presence of TTX. This implies that over expression calcium sensors in mature neurons behave akin to TTX induced homeostatic scaling. In conclusion the project allowed us to understand how the nanoscale organization of pre and postsynaptic proteins influences the fidelity of information transfer across a synapse. We demonstrate the role of differential synaptic nanoscale alignment resulting in discrete regulation of either frequency or amplitude of synaptic transmission.