Abstract
Analyzing synaptic densities is an effective tool to investigate the properties of a neural circuit. However, visualizing synapses presents challenges which imaging systems solve differently. Here, we present a set of experiments which utilize super resolution confocal microscopy coupled with immunostaining with fluorescent-conjugated antibodies. Fluorescent antibodies bind to proteins of interest allowing us to find colocalization of synaptic puncta which represent presumptive synapses. Experiment 1 explores an instance in organotypic cultures from mice where homeostatic plasticity mechanisms are applied inappropriately after silencing via tetrodotoxin (TTX) leading to hyperexcitability of the circuit. Colocalization analysis shows that there is a decrease in excitatory synapses after prolonged silencing in these cultures and confirms that TTX does not cause cell death. Experiment 2 applies similar methods to postmortem tissue from ferrets. Staining with CUBIC-1 and DeepLabel solutions did not achieve visualization of VGluT2, which is a protein of interest specific to the thalamocortical (TC) terminals in layer IV of the primary visual cortex (V1) in ferrets. Colocalization analysis between two ferrets showed that fewer VGluT2 synapses are formed within V1 in adulthood onto inhibitory neurons than to excitatory cells. This work successfully extends the super resolution imaging techniques from experiment 1 and should be applied in the future to a full developmental study of TC connections onto layer IV in V1 in ferrets.