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Nanoscale organisation of axo-axonic synapses along the axon initial segment of cortical pyramidal neurons in health and disease.

Periodic Reporting for period 1 - Nano-axo-syn (Nanoscale organisation of axo-axonic synapses along the axon initial segment of cortical pyramidal neurons in health and disease.)

Reporting period: 2020-05-01 to 2022-04-30

Synapses in the central nervous system are highly specialized structures dedicated to the transfer of information from one neuron to another. Each neuron receives thousands of excitatory and inhibitory synaptic inputs along their dendrites, that are integrated at the axon initial segment (AIS) to generate a new electrical signal called an Action Potential. Regulation of this fundamental process is key for proper brain function and was shown to be altered in several neurodevelopmental pathologies such as schizophrenia and autism spectrum disorder. Interestingly, the AIS of pyramidal neurons (PNs) is innervated by a specific type of inhibitory interneuron, a Chandelier cell. Although these axo-axonic synapses are thought to be capable of tightly regulating AP initiation, their structural and functional properties remain largely unexplored. At classical synapses formed along dendrites, the molecular organisation of synaptic proteins at the nanoscale is a key factor in fine- tuning the efficiency of synaptic transmission. However, the precise nanoscale arrangement of molecules at axo-axonic synapses has not been previously characterised, nor its impact on the modulation of pyramidal neuron firing. Here, we have shown that key proteins such as Gephyrin, the main scaffolding protein of inhibitory synapses, forms subsynaptic domains (SSD) at axo-axonic synapses. These SSDs display different properties, in term of size and density of proteins, when compared to dendritic synapses, which are formed by different types of interneurons. In addition, using genetic tools to chronically increase neuronal activity of PNs in the somatosensory cortex in vivo, we have shown that the nanoscale organisation of gephyrin is plastic. We are currently assessing the functional impact of this intriguing plasticity. Our findings provide a potential mechanism by which Chandelier cells synapse tune their strength to control PN output.
To study axo-axonic synapse organisation, we first had to establish in the host lab a super-resolution microscopy technique named dSTORM. Such approach is commonly use to image single layer neuronal cultures. Since Chandelier cells are absent from neuronal cultures in vitro, we had to use intact brain tissue to investigate axo-axonic synapses. We first had to setup an experimental pipeline allowing us to perform dSTORM in brain slices to image synapses. We took advantage of genetic tools delivered by means of in vivo viral injection to label endogenous gephyrin at inhibitory synapse in the somatosensory cortex. Using this sparse labelling approach together with dSTORM we were able to establish (i) that gephyrin is organised in subsynaptic domains (SSDs) at axo-axonic synapses and (ii) that gephyrin SSDs at axo-axonic synapses have different properties from their dendrites counterparts, suggesting inhibitory synapses are organised differently along different the subcellular compartments of PNs. In addition, we implemented a chemo-genetic approach (DREADDs) to manipulate neuronal activity in vivo and establish whether axo-axonic synapses are plastic at the nanoscale. Two days of stimulation using the designer drug CNO (delivered twice daily) elevated the activity of PNs in the somatosensory cortex. We observed that following chronic increase of neuronal activity, Gephyrin SSDs along the AIS were reduced in size, suggesting a depression of axo-axonic synapses. This result suggest that Chandelier cell synapses can rearrange their molecular organisation in response to change of neuronal activity, probably to tune their control on PN firing. This work has been presented at international conferences and will eventually be submitted for peer-reviewed publication, but additional experiments are currently ongoing to assess the Impact on network activity.
In parallel, we also developed a method allowing us to assess the relationship between nanoscale organisation and functional properties of synapses. Synapses are remarkably heterogenous subcellular compartment both in terms of their function (neurotransmitter release probability, synaptic strength…) and molecular composition/organisation. However how these two properties are related remains unknown, mostly as the tools to investigate both at the same synapses were lacking. We decided to combine live imaging of SypHy-RGECO, a unique dual reporter that simultaneously measures presynaptic calcium influx and neurotransmitter release, with post hoc immunolabelling and multicolour dSTORM microscopy. This work published in Frontiers in Synaptic Neuroscience (Jackson*, Compans* and Burrone, Correlative Live-Cell and Super-Resolution Imaging to Link Presynaptic Molecular Organisation With Function. 2022 Frontiers in Synaptic Neuroscience) provided a proof of principle that measures of both nanoscale molecular organisation and function can be investigated at the level of individual boutons and will hopefully allow in future study to understand the structure-function relationship at individual synapse.
Overall, our study will provide new insights into the mechanisms that fine-tunes communication between chandelier cells and pyramidal neurons. Because chandelier cells are thought to be strong modulators of network activity, our study, upon completion, should provide a better understanding of how activity patterns can be regulated by this unique type of interneurons in the brain. Because it is known that several neurodevelopmental disorders such as autism spectrum disorders and schizophrenia are associated with a dysregulation of network excitability, our project could help better understand the pathophysiology of these diseases, as well as identify targets for future therapeutic approaches.
Nanoscale organisation of axo-axonic synapses