Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures
- PDF / 2,370,650 Bytes
- 13 Pages / 595.276 x 790.866 pts Page_size
- 31 Downloads / 147 Views
ORIGINAL RESEARCH
Central Cholinergic Synapse Formation in Optimized Primary Septal‑Hippocampal Co‑cultures Sarra Djemil1 · Claire R. Ressel2 · Mai Abdel‑Ghani1 · Amanda K. Schneeweis1 · Daniel T. S. Pak1,3 Received: 23 June 2020 / Accepted: 14 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague–Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses. Keyword Septal · Cholinergic · Septal-hippocampal co-culture · Basal forebrain cholinergic neurons · Cholinergic synapse · Primary culture Abbreviations αBTX α-Bungarotoxin AChE Acetylcholinesterase AD Alzheimer’s Disease BFCN Basal forebrain cholinergic neurons bFGF Basic fibroblast growth factor BMP9 Bone morphogenetic protein 9 ChAT Choline acetyltransferase CHT1 High-affinity choline transporter CNS Central nervous system DIV Day in vitro GABA Gamma-aminobutyric acid
GAD65 Glutamate decarboxylase 65 Geph Gephyrin MAP2 Microtubule associated protein 2 MLA Methyllycaconitine nAChR Nicotinic acetylcholine receptor NGF Nerve growth factor nMDP Normalized mean deviation product PSD-93 Postsynaptic density 93 PSD-95 Postsynaptic density 95 VAChT Vesicular acetylcholine transporter VGAT Vesicular GABA transporter
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10571-020-00948-6) contains supplementary material, which is available to authorized users. * Daniel T. S. Pak [email protected] 1
Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, D.C., USA
2
Department of Biology, Georgetown University, Washington, D.C., USA
3
Interdisciplinary Program in Neuroscience, Georgetown University Medical Center, Washington, D.C., USA
Basal forebrain cholinergic neurons (BFCNs) are essential for proper memory formation within the hippocampus (Coyle et al. 1983; Luchicchi et al. 2014; Ballinger et al. 2016; Hampel et al. 2017). A great deal of information has accrued regardi
Data Loading...
