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Microscopic evaluation of synaptic connectivity and network formation in primary hippocampal neurons
Author(s) -
Verstraelen Peter,
Timmermans JeanPierre,
Detrez Jan R,
Pintelon Isabel,
Nuydens Rony,
Meert Theo,
De Vos Winnok
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.371.2
Subject(s) - neuroscience , neurite , dendritic spine , hippocampal formation , synaptophysin , synapse , calcium imaging , biological neural network , premovement neuronal activity , biology , neurodegeneration , chemistry , calcium , medicine , pathology , in vitro , immunohistochemistry , biochemistry , organic chemistry , immunology , disease
During brain development, orchestrated activation of genetic programs, as well as spontaneous electrical activity guide the correct wiring of neuronal networks. Mature neuronal networks are characterized by the expression of synaptic markers, synchronized electrical activity and the presence of dendritic spines, tiny protrusions from the dendritic shaft that compartmentalize single synapses to ensure optimal regulation of synaptic strength. While neurodevelopmental disorders such as schizophrenia are thought to result from defective neuronal network formation, neurodegenerative disorders like Alzheimer's disease are characterized by progressive synapse loss and associated mild cognitive impairment long before actual neurodegeneration occurs. We established an in vitro model based on primary hippocampal neurons that recapitulates features of mature neuronal networks. We optimized a set of microscopy workflows to quantitatively characterize both morphological (neurite outgrowth, synapse density, dendritic spine density) and functional (spontaneous electrical activity) aspects of the established neuronal networks (Cornelissen et al. 2013; Pani et al. 2014; Verstraelen et al. 2014). Neurite outgrowth and synapse density were quantified after immunostaining for β‐III‐tubulin and synaptophysin using an in‐house developed FIJI‐based image analysis script. Targeted photoconversion of mEos4b‐LifeAct in single neurons facilitated image analysis of dendritic spine density. Calcium fluctuations, serving as a proxy for electrical activity, were detected in neurons expressing the GCaMP6f reporter. A dedicated script was developed to obtain parameters describing both single neuron bursting patterns and correlated network events. Using calcium imaging, we pinpointed a critical period in which stochastic activity of individual neurons turned into robust, synchronized network activity, indicative of the formation of functional synapses. This synchronization coincided with an increase in neurite outgrowth and synapse density, while dendritic spine density increased mainly after synchronization of the activity, suggesting that the latter enables fine‐tuning of synaptic connections. We further showed that synchronized network activity is mediated by the NMDA receptor, and that interference with microtubule stability alters the bursting pattern. In brief, we have established a robust platform for pharmacological and/or genetic interrogation of synaptic connectivity in in vitro neuronal networks. Our approach is easily amenable to upscaling, which makes it an attractive model for high content screening in the context of neurodevelopmental or neurodegenerative disorders.