Abstract
During postnatal development of cortical circuits, mammals experience dramatic changes in inputs and explosive growth of synaptic connections. Homeostatic regulatory mechanisms are believed to be in charge of maintaining and stabilizing targeted levels of activity in inhibitory and excitatory neurons during these dramatic changes in input. In the mouse brain, blocking activity during this developmental stage causes epilepsy in vivo and hyperexcitability in vitro. There are three proposed homeostatic mechanisms that could explain the physiological effects of activity deprivation. We first explored the possibility that prolonged activity deprivation had an effect on the chloride concentration gradient by delaying the expression of KCC2 in pyramidal neurons. This could change the reversal potential and the function of their GABAergic inputs. We found through immunohistochemistry that KCC2 expression depends on the developmental age but not activity. Second, we investigated possibility that cells compensate for activity deprivation by increasing the number of excitatory synapses onto excitatory neurons. We observed no change in spine density between control cells and TTX-treated cells through semi-automatic spine density estimation of cortical layer V pyramidal cells. Lastly, in order to better understand the regulatory mechanisms of inhibition, we developed a high throughput method of analyzing excitatory synapses onto inhibitory neurons. Our method Gel-Embedded Minimal fixation Synaptic Staining (GEMSS) seems to overcome the limitations of a previously tried method, Array Tomography.