Department of Biological Sciences, Auburn University
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Seibenhener, M. L., Wooten, M. W. Isolation and Culture of Hippocampal Neurons from Prenatal Mice. J. Vis. Exp. (65), e3634, doi:10.3791/3634 (2012).
Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.
1. Set-up Prior to Harvest
2. Tissue Harvest
3. Tissue Dissociation
4. Neuron Trituration
5. Cell Plating
6. Representative Results
The ability to grow and culture primary neuronal cells has become an indispensible part of neuroscience. Primary cultures allow the researcher to analyze specific cellular pathways, chemical modification and treatment, target localization and growth patterns in a controlled environment. Many of these procedures utilize sophisticated methodology to visualize specific changes in cell responses. In this case, hippocampal neurons are used to study specific neuronal pathways that would prove difficult, if not impossible to analyze in the intact brain. Preparation of near homogeneous populations of neurons from specific areas of the brain is critical for studying brain function. Molecular effects in individual neurons can be instrumental in delineating higher order pathways such as memory or learning. As this protocol yields relatively pure cultures of hippocampal neurons, without the need of a feeder layer of glial cells, these neurons are easily utilized for immunofluorescence studies. However, as with all primary culture from organs containing multiple cell types, some contamination by less desired cells can occur. In isolation of neuronal cells, contamination by glial cells can be a common problem. Glial cells can be easily detected upon microscopic visualization of the culture as their morphology differs significantly from the target neurons (Figure 6). The impact of glial cell contamination will depend on the planned use of the cultures. If cells are being used for immuno-fluorescence examination, glial contamination can be nothing more than an inconvenience when trying to photograph individual neurons. However, if the neuronal cultures are to be used for biochemical analysis, any significant contamination by glial cells could cause major changes in the results. Ways to address glial cell contamination are outlined further in the Discussion.
Once neurons have been successfully isolated and grown in culture, one typical application is to examine cellular processes immuno-fluorescence techniques. As illustrated in Figure 7, organelles, such as the mitochondria, can be stained using vital dyes added to the culture media prior to fixation. Endogenous cellular proteins can be visualized from fixed cells using standard immuno-fluorescence techniques (Figure 8). Once neuronal cells are fixed, specific antibodies for proteins of interest can be introduced to the cell and these proteins can be visualized using a fluorescence microscope. Cultured neurons also provide the researcher with the means to examine individual protein effects on neuronal functions. Using a variety of techniques including DNA transfections, electroporation or viral transduction, proteins can be overexpressed in neuronal cells (Figure 9). How neural cells respond to the effects of over-expressed proteins can have direct inferences on how the brain may respond and offers the possibility of identifying cellular targets for drug treatments. The details of these types of experiments go beyond the scope of this paper but they do illustrate that cultures prepared by this technique are suitable for a wide array of down-stream applications. However, the overall simplicity of this protocol, as well as, the short time period required to prepare these neuronal cultures make this an ideal method for use in today's neuroscience laboratory.
Figure 1. Dissection of the prenatal mouse brain. The first incision is down the midline of the brain separating it into two hemispheres.
Figure 2. Location of the hippocampus in the prenatal mouse brain. The striatum is moved aside to visualize the hippocampus and is noted by the curved "kidney bean" type structure in the distal region of each hemisphere.
Figure 3. Dissociation of hippocampal tissue in trypsin solution.
Figure 4. Pasteur pipette tips used in trituration of hippocampal tissue. (a) Normal Pasteur pipette tip. (b) Fire-polished Pasteur pipette tip. Take note of the rounded edges and the approximate 50% decrease in pipette opening size.
Figure 5. Hippocampal neurons isolated using this procedure and plated in NB Media. (a) Cell growth 1 day post-plating. Neuronal processes begin to be visible during Day 1. (b) Cell growth 10 day post-plating, neurites are branched and overlapping.
Figure 6. Hippocampal neurons contaminated with glial cells grown for 7 days and stained with the organelle marker MitoTracker Red CM-H2XRos (Invitrogen #M7515) and transfected with GFP-LC3 using Lipofectamine 2000 (Invitrogen #11668019). Mitochondria are visible in all cells however only a single neuron was successfully transfected with the fluorescent construct. Contamination with glial cells makes analysis of GFP-LC3 expression in the neuronal processes difficult to visualize.
Figure 7. Hippocampal neurons grown for 7 days and stained with the organelle marker MitoTracker Red CM-H2 XRos (Invitrogen #M7513). This vital dye is used to stain active mitochondria in tissue culture cells. The cells were fixed in 4% paraformaldehyde/PBS and visualized by fluorescent microscopy. The dye itself is non-fluorescent until oxidized in the mitochondria. Active mitochondria can be seen throughout the neuronal processes.
Figure 8. Hippocampal neurons grown for 7 days, fixed with 4% paraformaldehyde/PBS and immuno-stained with monoclonal anti-tubulin β antibody (Sigma #T0198). Following primary antibody, Oregon Green labeled goat-anti-mouse secondary antibody (Invitrogen #O11033) was added and fluorescence visualized by microscopy.
Figure 9. Hippocampal neuronal cultures were grown for 5 days and transfected with GFP-LC3β DNA construct using Lipofectamine 2000 (Invitrogen #11668019). At Day 7, cells were fixed using 4% paraformaldehyde/PBS and aggresomes with GFP tagged LC3β incorporated into their outer membrane were visualized using fluorescent microscopy. Aggresomes are located throughout the cell body and neurites and are denoted with arrows.
Hippocampal cultures have been used in molecular biology for more than 20 years. While in principle, neuronal cultures can be made from any part of the brain, hippocampal cultures have proven to be the most popular due to the relatively simple architecture of the nerve cell population in the hippocampus7. Hippocampal cultures are typically made from late-stage embryonic tissue. This tissue is easier to dissociate and contains fewer glial cells than does mature brain tissue1. Isolation of hippocampal neurons from embryonic tissue also decreases shearing damage to axons and dendrites due to fewer adhesion contacts3. While hippocampal cultures are most often generated from rats due to the relatively easier isolation of the hippocampus, mice can also be used with the same protocols if appropriate care is taken during tissue isolation. Once neurons are cultured, the ability to use advanced molecular techniques to analyze subcellular localization and trafficking can be employed. This can be especially advantageous when analyzing embryonic lethal transgenic mice as it provides the ability to study protein interactions that would result in the death of the embryo.
The hippocampus has been implicated in both spatial and contextual learning5 and memory6. Growth of primary cultures from the hippocampus can allow a correlation between subcellular biological events and their effects on the brain's ability to learn and remember.
As with all neural cells, neurons grown from hippocampal cultures require critical growth factors, hormones and amino acids. In the brain, these factors are provided by glial cells. This symbiotic relationship can also be carried into a culture environment by growing a "feeder" layer of glial cells along with the cultured neurons. However, glial cells will also produce cytotoxic factors during their lifespan11 which can be toxic to cultured neurons. To circumvent this, neurons have been grown in serum-free media such as Neurobasal medium supplemented with B27. The B27 supplement is optimized for survival of hippocampal neurons but will support growth of other neuronal cultures as well4. L-glutamine is an essential amino acid for energy production and protein synthesis in cell culture. However, glutamine can be labile over time, degrading into ammonia and carboxylic acid byproducts once added to culture media. Glutamax, a cell culture supplement from Invitrogen, can be used as a direct substitute for L-glutamine if desired. Glutamax is more stable in media but slightly more expensive. Growth of neurons in serum-free media allows the study of effects of growth factors and hormones on neuronal growth and differentiation.
We submit a protocol for the rapid isolation of hippocampal neurons from mouse prenatal embryos using Neurobasal media and the B27 supplement for growth of neurons in a serum free environment without the use of feeder cells. As with all cultured primary cell protocols, it is advantageous to minimize the growth of non-desired cell types (i.e. glial cells). This is most readily accomplished by careful dissection of the hippocampus from surrounding regions of the brain. Prenatal cells also contain a comparatively small number of glial cells aiding in this goal. However, glial cell contamination can still occur. It is possible to reduce glial cell contamination by treatment with cytosine arabinoside (AraC) at early time points in the culture8,9. AraC can be used as an anti-mitotic agent to reduce the population of non-neuronal cells capable of DNA synthesis. However, to avoid possible toxic effects of treatment on neurons, it should be used at its lowest effective does (5 μM) and not added after 3-4 days of culture11. Another option would be the use of 5-fluoro-2'-deoxyuridine (FUdR) treatment to decrease the proportion of fibroblastic-reactive microglial cells10.
Once harvested, hippocampal tissue can be treated with a dilute trypsin solution to dissociate/disaggregate adherent cells. However, prolonged exposure to higher concentrations of trypsin can be detrimental to cell subculture so time in trypsin solution is most often limited to between 3-5 minutes. The diluted concentration of trypsin used in this protocol does allow longer times of enzyme incubation to increase individual cellular disassociation but care should be taken to strictly adhere to the time points provided. Papain can be used as an alternative enzyme and has been proven to be more effective and less destructive with certain tissues such as retinal neurons 10.
Cell dissociation is followed by trituration of the tissue. This has proven to be the most important step in consistent neuronal culture and is highlighted by two important points. First, two sterile Pasteur pipettes are used during this process. The first is used to grossly disrupt tissue/cellular association. The opening size of this first pipette should be between 1.0-1.5 mm. Careful selection of pipettes directly from the vendor package can usually result in appropriate pipettes for this first trituration. The second Pasteur pipette used is "fire-polished" over a Bunsen burner to reduce the size of the opening to approximately 0.5 mm in diameter. This also results in "polishing" the glass ring of the opening to a smoother surface reducing mechanical damage to cells as they pass through. Secondly, the speed/force of trituration through the pipette is of major importance. As the cells dissociate from the whole tissue, they are of course more fragile. Thus, "rough" treatment as this point would be detrimental to survival of the neuronal cells. Trypsinized tissue should be passaged through the pipette at a consistent but firm flow rate. A common mistake by the researcher is the idea of having to completely disassociate the tissue until there is no discernible structure remaining. In most cases, this will result in massive neuronal death as the cells will be too damaged by the repeated mechanical manipulation. The indicated number of times to pass the tissue through the pipette will result in sufficient numbers of neurons without resulting in gross damage to the population.
Once the cells have been dissociated and pooled, a Trypan Blue stain of an aliquot of cells will provide a ratio of live to dead cells. Typically, 75-80% of the cells should survive the harvest process and can be used for plating. Trypan Blue staining can also be used to obtain an accurate cell count when done on a hemocytometer. In practice, once a total cell count is achieved, add 20% to the total and use that cell number for plating to account for the approximate amount of cell death. Numbers provided in the above protocol are a good starting point to achieve good densities of neurons. If neuron density is too low at plating, cell growth will likely not be sufficient as plating density is an important variable in propagation of the cell population. Cells should be fed every four days with B27/Neurobasal Feed media. Neuronal growth under these conditions can easily be carried out to 10-14 days in culture and has been used to propagate neurons2 out to 30 days in culture when seeded at 80 cells / mm2.
No conflicts of interest declared.
We thank Dr. Michael Wooten for his help in preparing the manuscript. This work was supported by NIH 2RO1NS033661 (MWW).
|Rat Tail Collagen 1||BD Biosciences||354236|
|Hanks Balanced Salt Solution||Invitrogen||14175-095|
|Trypsin Solution (1X) 0.25%, liquid||Invitrogen||15050-065|
|NeuroBasal Medium (1X) liquid||Invitrogen||21103-049|
|B27 Supplement (50X) liquid||Invitrogen||17504-044|
|L-Glutamine 200 mM (100X) liquid||Invitrogen||25030-149|
|Penicillin (10,000 units/ml) / Streptomycin (10,000 μg/ml)||Invitrogen||15140-148|
|HI-Donor Horse Serum||Atlanta Biologicals||S12150H|