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

Summary

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Abstract

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm²), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Introduction

Growing hippocampal neurons under in vitro conditions enables observation and experimental manipulation of these neurons that are otherwise not possible in vivo. This experimental approach is widely used to reveal neuronal mechanisms of growth, polarity, neurite specification, trafficking and subcellular localization of proteins, synapse formation and functional maturation¹. These in vitro cultured hippocampal neurons, when harvested from late embryonic stages, are relatively pure (>90%) glutamatergic cells of pyramidal morphology². Because neurons were grown in a 2-D surface under in vitro conditions, this....

Protocol

All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the University of Arizona, and conformed to NIH guidelines.

1. Tissue Source for Hippocampal Neuron Culture

1. To generate prenatal mouse pups for hippocampal neuron culture, use time-pregnant mice (C57Bl6/J) that are bred in house¹⁷. The day with vaginal plug detection is designated as E0.5. The planned harvest time for culture is E16.5-E17.5.
   Note: This protocol describes cultures of two 24-well plates of two high density neurons co-culturing with low-density neurons (48 coverslips total). For more plat....

Results

The protocol described here enables successful ultra-low density, long-term culture of pure glutamatergic neurons without the need of glia cells serving as a feeder layer. The protocol is diagramed in Figure 1, which involves preparation of high density (on poly-D-lysine coated 24 wells) and low-density neurons (on poly-D-lysine coated glass coverslips) separately, and subsequent co-culture that can be maintained up to three months.

Discussion

We present a detailed protocol for long-term culture of ultra-low density hippocampal glutamatergic neurons under serum free conditions. At ~2000 neurons/cm², the density is at least two fold lower than most 'low density' culture preparations with or without glia support reported by the existing literature^2,3,11,13,14. In addition to being ultra-low density, this protocol is novel and significant in two more ways. First, no glia feeder layer is needed as the low density neurons obtain trophi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal medium	Life Technologies	21103-049	Protect from light
B27 supplement	Life Technologies	17504-044	aliquot, store in 0.6ml size
GlutaMAX-I	Life Technologies	35050-061	dilute 100X
antibiotic-antimycotic (AA)	Life Technologies	15240-096	dilute 100X
Complete Culture medium			Neurobasal medium with 1X B27, 1X AA, 1X GlutaMAX-I
Wash medium			same as 'Neurobasal medium'
Feed medium			Neurobasal with 1X B27 supplement
DNAse I	Sigma-Aldrich	D5025	prepare 100X stock at 0.6mg/ml
poly-D-lysine	Sigma-Aldrich	P6407	M.W. 70000-150000
borate buffer	Sigma-Aldrich	B6768 (boric acid); 71997(borax)	1.24g boric acid & 1.9g borax in 400ml H2O, pH to 8.5 use HCl
12-mm round glass coverslips	Glasswarenfabrik Karl Hecht GmbH	1001/12	No. 1 glass, purchase from Carolina Biological Supply
proFection transfection kit	Promega	E1200	see  protocol for details
2X HEPES buffered saline (HBS)	Promega	E1200	see  protocol for details
Syringe filter	Pall Corporation	4192	0.2um pore size
Endofree plasmid prep kit	Qiagen	12362	for preparation of transfection grade plasmid DNA
anti-MAP2 antibody	Millipore	MAB3418	mouse antibody, clone AP20
anti-p-Tau antibody	Millipore	AB10417	rabbit polyclonal antibody
anti-NR1 antibody	Millipore	MAB1586	mouse antibody, clone R1JHL
anti-GluR1 antibody	Millipore	AB1504	rabbit polyclonal antibody
Hank's balanced salt solution	ThermoFisher	14025092	500ml size