Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

Abstract

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

Neuronal megalin mediates synaptic plasticity—a novel mechanism underlying intellectual disabilities in megalin gene pathologies

“…This indicates that megalin is probably mainly required for dendritic spine maturation, and not for the formation of novel dendritic spines. Up on that, we evaluated if the effects at dendritic spines were associated to changes in synaptic activity, by analysing the expression levels of synaptic proteins in the hippocampus (pre-synaptic-VGLUT1 and post-synaptic-PSD95), as well as the number of synapses, defined as the co-localization of pre- and post-synaptic proteins that appear at a punctate distribution ( McLeod et al , 2017 ). In total hippocampal extracts, VGLUT1 levels are decreased in Meg +/− mice compared to Meg +/+ littermates, and PSD95 shows a tendency to decrease ( P = 0.08) ( Fig.…”

Synapse development is regulated by microglial THIK-1 K+channels

“…Size exclusion (>1.2 μm 2 , <0.05 μm 2 ) was applied to exclude any objects unlikely to represent synaptic puncta. Synapses were defined by the presence of overlapping presynapticpostsynaptic puncta (30). Presynaptic colocalization with microglia was analysed as the total area of Bassoon puncta within the Iba1-stained cell area after thresholding.…”

“…Deficits in microglial phagocytosis could result in impaired pruning of synapses during development (43), so we tested the effects of THIK-1 KO on hippocampal synapse numbers. Using P17-P19 mice, when synapse pruning in the hippocampus is near its peak (12, 13), we assessed the labeling of presynaptic (Bassoon) and postsynaptic glutamatergic (Homer1) markers in the stratum radiatum of the CA1 hippocampal region by immunohistochemistry (30). A colocalization of both markers was taken to indicate an excitatory synapse (see Fig 2A).…”

Altered synaptic connectivity in anin vitrohuman model of STXBP1 encephalopathy

“…Slices were fixed in 4% paraformaldehyde (PFA)/4% sucrose in phosphate buffered saline (PBS; 0.12 M, pH 7.4; PBS) for 20 minutes at room temperature (RT). Staining was performed using the protocol optimised specifically for acute slices 15 . In brief, slices were incubated in blocking solution (10% donkey serum, 1% Triton X-100 in PBS) for ~ 4-6 hours at RT on a vibrating shaker.…”