Systematic Quantification of Synapses in Primary Neuronal Culture

Abstract

Summary Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

Comparative Milestones in Rodent and Human Postnatal Central Nervous System Development

“…Presently, this type of analysis is done using a raft of synaptic markers, both pre-and postsynaptic. 24,25 The combination of a pre-and postsynaptic marker can be used to identify synaptic contacts. This can be achieved using immunohistochemistry 26 or in mice via transgenic approaches that allow large scale labeling of the entire brain.…”

Synapsin III Controls Early Phases of Dopaminergic Neurons Development in Fishes and Mammals by Acting Upstream of BDNF and Cdk5 Signaling

“…Therefore, although we cannot fully rule out the possibility that that a decrease of synaptic activity related to the Syn III absence may also reduce BDNF expression, these observations suggest that the impact of Syn III deletion on neuronal activity may not be su cient to sustain a reduction of BDNF production at very early developmental stages. This is further supported by the fact that although Syn III ko mouse primary dopaminergic neurons at 1 div exhibited an improved BDNF response that appears indicative of receptors hypersensitivity in response to BDNF decrease, the rodent primary neurons in culture do not display mature synapses till 7 div [86][87][88][89].…”

Neuron–Microglia Contact-Dependent Mechanisms Attenuate Methamphetamine-Induced Microglia Reactivity and Enhance Neuronal Plasticity

“…The acquired images were analyzed using ImageJ, as described elsewhere [28][29][30]. The puncta were analyzed for number, area, and intensity in the selected region, per dendritic length.…”

“…PSD95 is a post-synaptic marker and the most abundant scaffold protein of excitatory synapses, while VGlut1 is a presynaptic maker, highly expressed in the mouse hippocampus, specifically in excitatory synapses. Both proteins display a punctate distribution, and their co-localization is defined as a synapse [29,30]. Compared with the ACM Ctrl condition, when ACM Meth…”

“…PSD95 is a postsynaptic marker and the most abundant scaffold protein of excitatory synapses, while VGlut1 is a presynaptic maker, highly expressed in the mouse hippocampus, specifically in excitatory synapses. Both proteins display a punctate distribution, and their co-localization is defined as a synapse [29,30]. Compared with the ACM Ctrl condition, when ACM Meth was added to the axonal/microglial compartment, we observed an increase in PSD95 puncta number (F (1254) = 11.92, p < 0.001), area (F (1252) = 29.38, p < 0.0001), and intensity (F (1251) = 51.97, p < 0.0001) (Figure 5A-D) and a decrease in VGlut1 puncta numbers (F (1258) = 5.71, p < 0.05) (Figure 5E,F).…”