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 Electronic supplementary material The online version of this article (

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OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previous published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons in the co-culture attained up to ~5-7%, depending on conditions such as embryo age at dissection or ratio of septal to hippocampal cells. We also report on cholinergic synapse structure by examining postsynaptic markers (excitatory and inhibitory) and staining for nicotinic acetylcholine receptor subunits. DISCUSSION/SIGNIFICANCE OF IMPACT: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.