Electrophysiological analysis of synaptic properties in inhibitory cortical neurons

The synaptic properties of central neurons have been widely studied over the past century through electrophysiological approaches including the patch clamp technique. In our study, primary cultured autapses of inhibitory cortical neurons from C57BL/6 mice have been used to better understand the properties of the vesicle pools released at presynaptic site. Specifically, the goal of this thesis is to quantify the size of the readily releasable pool (RRP) as well as the rate of recycling of synaptic vesicles using two different approaches distinguished by the ability to empty the RRP in a calcium-dependent and independent way. The first method focuses on the role of calcium entry at presynaptic membrane during high frequency stimulation (HFS), while the second one promotes vesicles release in a calcium-independent way through the administration of a hypertonic solution (500mM) of sucrose. By comparing these two methods, previous studies reached different conclusions. Some of them propose that through the two methods similar results can be obtained, while others indicate significantly different RRP size values. We observed that, similarly to what noticed in other neurons, in our experimental model the RRP size measured through hypertonic solution is 5-fold larger than the value obtained when measured through high frequency stimulation. Additionally, we focused on the synaptic vesicles recycling showing that this latter is accelerated by calcium. In particular, we found that upon sucrose administration an average amount of 90 vesicles are recycled each second, while during HFS the number of vesicles recycled during each second are approximately 6-fold larger. We concluded that in our experimental model, the two methodologies investigated produced very different results. Some hypotheses are thus proposed, which allow us to better understand the synaptic structure and related functions.