{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,17]],"date-time":"2026-08-17T14:38:16Z","timestamp":1786977496429,"version":"build-2736575974"},"reference-count":22,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2023,10,26]],"date-time":"2023-10-26T00:00:00Z","timestamp":1698278400000},"content-version":"vor","delay-in-days":25,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2023,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high\u2010content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real\u2010world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384\u2010well plate format. \u00a9 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Culture of primary hippocampal rat neurons in 384\u2010well plates\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Lentiviral shRNA transduction of primary neuronal culture in 384\u2010well plates\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Immunostaining of the neuronal network and synaptic markers in 384\u2010well plates\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Image acquisition using a high\u2010throughput reader\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 5<\/jats:bold>\n                    : Image segmentation and analysis\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 6<\/jats:bold>\n                    : Synaptic density analysis\n                  <\/jats:p>","DOI":"10.1002\/cpz1.904","type":"journal-article","created":{"date-parts":[[2023,10,26]],"date-time":"2023-10-26T10:46:07Z","timestamp":1698317167000},"source":"Crossref","is-referenced-by-count":3,"title":["High\u2010Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures"],"prefix":"10.1002","volume":"3","author":[{"given":"Audrey","family":"Coulon","sequence":"first","affiliation":[{"name":"Universit\u00e9 de Lille, Inserm, CHU Lille, Institut Pasteur de Lille U1167 \u2010 RID\u2010AGE \u2010 Facteurs de risque et d\u00e9terminants mol\u00e9culaires des maladies li\u00e9es au vieillissement  Lille 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CHU Lille, Institut Pasteur de Lille U1167 \u2010 RID\u2010AGE \u2010 Facteurs de risque et d\u00e9terminants mol\u00e9culaires des maladies li\u00e9es au vieillissement  Lille France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandre","family":"Vandeputte","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille Center for Infection and Immunity of Lille (CIIL)  Lille France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Priscille","family":"Brodin","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille Center for Infection and Immunity of Lille (CIIL)  Lille France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiago","family":"Mendes","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, Inserm, CHU Lille, Institut Pasteur de Lille U1167 \u2010 RID\u2010AGE \u2010 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