Abstract
Understanding how neuromodulators influence synaptic transmission and intrinsic excitability within the entorhinal cortex (EC) is critical to furthering our understanding of the molecular and cellular aspects of this region. Organotypic cultures can provide a cost-effective means to employ selective molecular biological strategies in elucidating cellular mechanisms of neuromodulation in the EC. We therefore adapted our acute slice model for organotypic culture applications and optimized a protocol for the preparation and biolistic transfection of cultured horizontal EC slices. Here, we present our detailed protocol for culturing EC slices. Using an n-methyl-d-glucamine (NMDG)-containing cutting solution, we obtain healthy EC slice cultures for electrophysiological recordings. We also present our protocol for the preparation of “bullets” carrying one or more constructs and demonstrate successful transfection of EC slices. We build upon previous methods and highlight specific aspects in our method that greatly improved the quality of our results. We validate our methods using immunohistochemical, imaging, and electrophysiological techniques. The novelty of this method is that it provides a description of culturing and transfection of EC neurons for specifically addressing their functionality. This method will enable researchers interested in entorhinal function to quickly adopt a similar slice culture transfection system for their own investigations.
Highlights
Background and discussionOrganotypic cultures are a well-established in vitro model to investigate the cytoarchitecture and neurophysiology of brain tissue [5,6]
Immunofluorescent staining for the neuronal marker NeuN (1:200, Milipore, #ABN78) demonstrated that entorhinal cortex (EC) layers were well-preserved in slice cultures and that neurons were detected after being in culture for several days (Fig. 2B)
Whereas such cultures comprised of hippocampal regions have been utilized by many labs, relatively fewer reports involve organotypic cultures consisting of entorhinal tissue
Summary
Surgical draping (4410-imc, IMCO products) 1 250 mL beaker 1 30 mL beaker 1 20 mL beaker 37 C incubator Biorad Gene-Gun Low-Pressure System (#1652451, Biorad) Polyvinylpyrrolidone (PVP, #PVP360-100G, Sigma) Spermidine (#S2501-1G, Sigma). Tefzel Tubing (#1652441, Biorad) Molecular biology grade ethanol Mini centrifuge (e.g. Daigger Sprout Mini-Centrifuge) Nitrogen tank Helium tank Serological pipettes Pipette aid Vacuum and syringe filters (0.22 mm). Large Surgical scissors (#RS-6818, Roboz) Fine Surgical Scissors (#RS-5914, Roboz) 2 Fine forceps (#RS-5041, Roboz) 2 Spatulas (#13523, Ted Pella) 2 pieces of filter paper (#09-803-5A, Fisherbrand) 1 brush (11860, Ted Pella) 1 mL syringe with hand-bent 0.5 in. Needle (we use this tool with the brush further dissect slices) Scalpel blade (RS-9861-21, Roboz). Biorad Helios Gene-Gun barrel liner with rubber gasket Modified diffusing screen (#9317T109, McMaster-Carr) [1] Biorad diffuser screen washer (#1652475, Biorad) Fine forceps.
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