An Ex Vivo Model of Paired Cultured Hippocampal Neurons for Bi-directionally Studying Synaptic Transmission and Plasticity

Overview

Journal Bio Protoc

Specialty Biology

Date 2023 Jul 27

PMID 37497454

Authors

Ruslan Stanika

Gerald J Obermair

Affiliations

Soon will be listed here.

Abstract

Synapses provide the main route of signal transduction within neuronal networks. Many factors regulate critical synaptic functions. These include presynaptic calcium channels, triggering neurotransmitter release, and postsynaptic ionotropic receptors, mediating excitatory and inhibitory postsynaptic potentials. The key features of synaptic transmission and plasticity can be studied in primary cultured hippocampal neurons. Here, we describe a protocol for the preparation and electrophysiological analysis of paired hippocampal neurons. This model system allows the selective genetic manipulation of one neuron in a simple neuronal network formed by only two hippocampal neurons. Bi-directionally analyzing synaptic transmission and short-term synaptic plasticity allows the analysis of both pre- and postsynaptic effects on synaptic transmission. For example, with one single paired network synaptic responses induced by both, a wild-type neuron and a genetically modified neuron can be directly compared. Ultimately, this protocol allows experimental modulation and hence investigation of synaptic mechanisms and thereby improves previously developed methods of studying synaptic transmission and plasticity in ex vivo cultured neurons. Key features Preparation of ex vivo paired cultured hippocampal neurons. Bi-directional electrophysiological recordings of synaptic transmission and plasticity. Genetic modulation of synaptic network formation (demonstrated by presynaptic viral overexpression of the auxiliary calcium channel αδ-2 subunit). Graphical overview.

References

Kaech S, Banker G . Culturing hippocampal neurons. Nat Protoc. 2007; 1(5):2406-15. DOI: 10.1038/nprot.2006.356. View

Geisler S, Schopf C, Obermair G . Emerging evidence for specific neuronal functions of auxiliary calcium channel α₂δ subunits. Gen Physiol Biophys. 2014; 34(2):105-118. PMC: 4487825. DOI: 10.4149/gpb_2014037. View

Benskey M, Manfredsson F . Lentivirus Production and Purification. Methods Mol Biol. 2015; 1382:107-14. DOI: 10.1007/978-1-4939-3271-9_8. View

Nasri M, Karimi A, Farsani M . Production, purification and titration of a lentivirus-based vector for gene delivery purposes. Cytotechnology. 2014; 66(6):1031-8. PMC: 4235939. DOI: 10.1007/s10616-013-9652-5. View

Deperrois N, Graupner M . Short-term depression and long-term plasticity together tune sensitive range of synaptic plasticity. PLoS Comput Biol. 2020; 16(9):e1008265. PMC: 7549837. DOI: 10.1371/journal.pcbi.1008265. View

Wang Y, Baudry M . Acute Cerebellar Slice Preparation Using a Tissue Chopper. Bio Protoc. 2021; 9(5):e3187. PMC: 7854164. DOI: 10.21769/BioProtoc.3187. View

Eroglu C, Allen N, Susman M, ORourke N, Park C, Ozkan E . Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell. 2009; 139(2):380-92. PMC: 2791798. DOI: 10.1016/j.cell.2009.09.025. View

Obermair G, Szabo Z, Bourinet E, Flucher B . Differential targeting of the L-type Ca2+ channel alpha 1C (CaV1.2) to synaptic and extrasynaptic compartments in hippocampal neurons. Eur J Neurosci. 2004; 19(8):2109-22. DOI: 10.1111/j.0953-816X.2004.03272.x. View

Ablinger C, Geisler S, Stanika R, Klein C, Obermair G . Neuronal αδ proteins and brain disorders. Pflugers Arch. 2020; 472(7):845-863. PMC: 7351808. DOI: 10.1007/s00424-020-02420-2. View

10.

Geisler S, Schopf C, Stanika R, Kalb M, Campiglio M, Repetto D . Presynaptic αδ-2 Calcium Channel Subunits Regulate Postsynaptic GABA Receptor Abundance and Axonal Wiring. J Neurosci. 2019; 39(14):2581-2605. PMC: 6445987. DOI: 10.1523/JNEUROSCI.2234-18.2019. View

11.

Bouteiller J, Allam S, Greget R, Ambert N, Hu E, Bischoff S . Paired-pulse stimulation at glutamatergic synapses - pre- and postsynaptic components. Annu Int Conf IEEE Eng Med Biol Soc. 2010; 2010:787-90. DOI: 10.1109/IEMBS.2010.5626491. View

12.

Ablinger C, Eibl C, Geisler S, Campiglio M, Stephens G, Missler M . αδ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions. Int J Mol Sci. 2022; 23(17). PMC: 9456004. DOI: 10.3390/ijms23179885. View

13.

Schopf C, Ablinger C, Geisler S, Stanika R, Campiglio M, Kaufmann W . Presynaptic αδ subunits are key organizers of glutamatergic synapses. Proc Natl Acad Sci U S A. 2021; 118(14). PMC: 8040823. DOI: 10.1073/pnas.1920827118. View

14.

Barral J, Reyes A . Optogenetic Stimulation and Recording of Primary Cultured Neurons with Spatiotemporal Control. Bio Protoc. 2017; 7(12). PMC: 5548145. DOI: 10.21769/BioProtoc.2335. View