Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Abstract

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (> 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function. Video LinkThe video component of this article can be found at https://www.jove.com/video/2373/ Protocol Preparation of Coverslips and Chambers1. Preparation of clean coverslips and chambers is essential for healthy cultures. Shortcuts should not be taken at any of these steps. 2. Wash the coverslips (12mm or 22mm round, German glass -Carolina Assistant Brand) overnight in concentrated nitric acid (HNO3) in a dedicated glass jar or beaker. 3. Remove the coverslips from nitric acid and wash extensively (5-7x) in deionized water. 4. Separate the coverslips and dry in a laminar flow hood or biosafety cabinet. When dry, sterilize them with UV light for 30 minutes. Place sterilized coverslips into a sterile petri dishes for storage. If plating neurons on 12mm coverslips directly place coverslips in a sterile 35mm dish and proceed to Section 2. 5. Imaging chambers are constructed by drilling a 15mm hole in the bottom of 35mm petri dishes (remove all burrs) and attaching the cleaned coverslips with a 3:1 mixture of paraffin and petroleum jelly. 6. Melt the paraffin/petroleum jelly mixture in a conical tube inside a boiling water bath. Use a small paint brush and coat the underside of the dish around the 15mm hole. Make sure to keep stirring the paraffin/petroleum jelly mixture as it will separate. This usually results in chambers glued together with a higher concentration of petroleum jelly, which will become viscous when dishes are placed in the incubator, resulting in coverslip detachment. The remaining paraffin/petroleum jelly can be stored at room temperature. 7. Place the dis...

Guided neuronal growth on arrays of biofunctionalized GaAs/InGaAs semiconductor microtubes

“…Dissected embryonic primary cortical E15.5 mouse neurons (prepared following a previously published protocol [15]) were plated without glia at a density of 5000 cells/cm 2 onto the samples together with control substrates where the PDMS coating was omitted. After incubation for 1 hour at 37°C and 5% CO 2 , the plating medium was exchanged with serum-free medium.…”

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

“…These cultured neurons are usually used at 1-4 weeks in vitro, with the exact age of the culture depending on the purpose of the experiment. It is of note that some neurons cultured on a glial feeder layer can survive for more than 10 weeks 34,68 .…”

“…Typically, in primary cultures of mammalian brain neurons, the cells are plated with a relatively high density, on coated coverslips without a glial feeder layer (e.g., [31][32][33][34][35][36][37][38][39] ). When the plating density of neurons is reduced using this method, the initial lack of glial sheet leads to poor neuronal growth and dendritic extension (panels B, C in Figures 8-10), as reported previously [40][41][42] , probably due to poor glial growth 43,44 .…”

“…In the third approach, neurons are plated on a pre-established glial feeder layer that supports neuronal growth (e.g., [50][51][52][53] ) (Figures 8-10). Specifically, mouse brain neurons are plated on a glial feeder layer prepared from mice 8,34,54 or rats 41,55,56 .…”

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

“…These in vitro cultured hippocampal neurons, when harvested from late embryonic stages, are relatively pure (>90%) glutamatergic cells of pyramidal morphology 2 . Because neurons were grown in a 2-D surface under in vitro conditions, this method allows easy observation, such as live imaging or immunocytochemistry (ICC) staining through a single focal plane 3 ; or manipulations, such as drug treatment and transfections [3][4][5][6] . When grown at high density, neurons tend to have high rates of survival because of higher concentrations of secreted growth factors in addition to alimentary support from the growth media, and also because of neurite contact-dependent mechanisms 7 .…”