Automated Macro Approach to Quantify Synapse Density in 2D Confocal Images from Fixed Immunolabeled Neural Tissue Sections.

This chapter describes an ImageJ/Fiji automated macro approach to estimate synapse densities in 2D fluorescence confocal microscopy images. The main step-by-step imaging workflow is explained, including example macro language scripts that perform all steps automatically for multiple images. Such tool provides a straightforward method for exploratory synapse screenings where hundreds to thousands of images need to be analyzed in order to render significant statistical information. The method can be adapted to any particular set of images where fixed brain slices have been immunolabeled against validated presynaptic and postsynaptic markers.

Measurement of saturation processes in glutamatergic and GABAergic synapse densities during long-term development of cultured rat cortical networks.

The aim of this study was to clarify the saturation processes of excitatory and inhibitory synapse densities during the long-term development of cultured neuronal networks. For this purpose, we performed a long-term culture of rat cortical cells for 35 days in vitro (DIV). During this culture period, we labeled glutamatergic and GABAergic synapses separately using antibodies against vesicular glutamate transporter 1 (VGluT1) and vesicular transporter of γ-aminobutyric acid (VGAT). The densities and distributions of both types of synaptic terminals were measured simultaneously. Observations and subsequent measurements of immunofluorescence demonstrated that the densities of both types of antibody-labeled terminals increased gradually from 7 to 21-28 DIV. The densities did not show a further increase at 35 DIV and tended to become saturated. Triple staining with VGluT1, VGAT, and microtubule-associated protein 2 (MAP2) enabled analysis of the distribution of both types of synapses, and revealed that the densities of the two types of synaptic terminals on somata were not significantly different, but that glutamatergic synapses predominated on the dendrites during long-term culture. However, some neurons did not fall within this distribution, suggesting differences in synapse distribution on target neurons. The electrical activity also showed an initial increase and subsequent saturation of the firing rate and synchronized burst rate during long-term culture, and the number of days of culture to saturation from the initial increase followed the same pattern under this culture condition.

Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses.

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.