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.
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Data Availability
Original raw data will be available upon reasonable request from the corresponding author for academic/non-commercial purposes.
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
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Acknowledgements
The authors would like to thank the Georgetown Microscopy & Imaging Shared Resource which is partially supported by NIH/NCI grant P30-CA051008.
Funding
This work was supported by grants to DTSP: National Institutes of Health (RF1 AG056603-01) and SD: National Center for Advancing Translational Sciences of the National Institutes of Health (TL1TR001431).
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Conceptualization, SD and DTSP; Methodology SD, CRR and DTSP; Experiments, SD and CRR; Data Analysis, SD, CRR, MAG, and AKS; Writing, all authors; Funding Acquisition, DTSP and SD; Supervision, DTSP and SD.
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All animals used in experimental procedures were approved (IACUC protocol #: 15-026-100232) and performed in accordance with the Georgetown University Animal Care and Use Committee (GUACUC).
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Djemil, S., Ressel, C.R., Abdel-Ghani, M. et al. Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures. Cell Mol Neurobiol 41, 1787–1799 (2021). https://doi.org/10.1007/s10571-020-00948-6
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DOI: https://doi.org/10.1007/s10571-020-00948-6