Name: Isolation and Culture of Hippocampal Neurons from Prenatal Mice (Video) | JoVE Journal
Uploaded: 2012-07-26T12:00:00.000+00:00
Duration: 10 min 27 s
Description: Learn to isolate and culture highly purified hippocampal neurons from prenatal mice using dissection, trypsinization, and neuro basal media with B27 supplement. This method enables molecular techniques like immunofluorescence.

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5 minutes

We thank Dr. Michael Wooten for his help in preparing the manuscript. This work was supported by NIH 2RO1NS033661 (MWW).

1. Set-up Prior to Harvest
<ol>
 <li>To generate prenatal pups for neuron harvest, schedule breeding between adult mice 19 days prior to the day of neuron isolation. (C57BL/6 mice ages 2-8 months were used in matings for the purposes of developing this protocol). Successful mating can be confirmed by detection of a vaginal plug in the female, palpitation or visual confirmation of pregnancy.</li>
 <li>The day prior to neuron isolation:
 <ol class="ualpha">
 <li>For immunofluorescence applications, coat glass coverslips in a 24-well plate with a light coating of 3:1 Collagen 1, Rat Tail:poly-D-Lysine solution.</li>
 <li>For cell culture applications, coat appropriate size tissue culture plastic ware with a light coating of 3:1 Collagen 1, Rat Tail:poly-D-Lysine solution.</li>
 </ol>
 </li>
 <li>Rest the plates uncovered in a tissue culture hood under a UV light overnight.</li>
 <li>Wash the plates with sterile Hank's Balance Salt Solution (HBSS) prior to use. Coated plates can be filled with HBSS and stored up to one week at 4 °C in the dark.</li>
</ol>
2. Tissue Harvest
<ol>
 <li>Sterility is always a factor when growing primary cell cultures and as such, the greatest caution should be exercised to ensure the most sterile environment possible. With careful attention to sterile technique, initial dissection and harvest of neural tissue for this protocol can be completed outside of a laminar flow hood with minimal risk of contamination. After initial harvest, all subsequent steps should be conducted under maximum sterile conditions within a hood rated for cell culture.</li>
 <li>Euthanize a pregnant mouse at approximately 19 days post-fertilization by decapitation. Use of anesthesia to euthanize the pregnant female is not recommended as anesthesia is known to cause brain cell death (Stratmann, et al., 2010). Using sterile dissecting scissors and forceps, create an opening in the mid-ventral side of the mouse to completely reveal body cavity. Instruments can be sterilized using alcohol and an open flame.</li>
 <li>Prenatal pups will be located towards the posterior of the mouse's body cavity and should be easily visible in the uterus. With autoclaved sterile forceps, open the uterus and remove pups. Decapitate pups with fresh sterile scissors and place removed head on sterile gauze under a dissecting microscope. Sterile, autoclaved instruments can be flame cleaned using alcohol and an open flame prior to and during use. </li>
 <li>Using sterile scissors or scalpel, open cranium of pup from back of the neck to the nose. This procedure can normally be completed by inserting one tip of the scissors into the vertebral foramen and then proceeding anteriorly. Carefully remove the entire brain with forceps. Place the brain on sterile gauze. Using a sterile scalpel, remove the cerebellum and incise down the midline of the brain to separate it into two hemispheres (Figure 1).</li>
 <li>Grasp a small section of meninges surrounding the hippocampus with sterile forceps and pull it gently away. Although it is not strictly necessary to remove the meninges before isolating the hippocampus, the presence of the meninges can make the dissection of the hippocampus more difficult due to the toughness of the membrane. In either case, the hippocampus will be more clearly visible after the meninges have been removed. The hippocampus is a curved structure that starts in the distal part of the hemisphere and bends ventrally (Figure 2). As the inner, concave, side (caudal) is facing a ventricle, it is already free. Therefore, to isolate the hippocampus, one needs to cut along the convex outer side. After the dissection, gently lift each hippocampi with sterile tissue forceps and transfer into a small tissue culture dish with warmed (37 °C) HBSS under a cell culture hood. Brain tissue can be combined from multiple pups.</li>
</ol>
3. Tissue Dissociation
<ol>
 <li>Using a sterile scalpel, gently mince brain tissue in 3 ml of sterile HBSS in a 100 mm Tissue Culture dish.</li>
 <li>Transfer the minced tissue and HBSS to a 15 ml conical tube. Add 1.5 ml of HBSS and 0.5 ml of 0.25% Trypsin solution to a total volume of 5 ml.</li>
 <li>Cap and gently invert the tube 4-5 times to mix. Try to avoid producing bubbles as DNA released from the digested tissue will adhere to the bubbles and cause the minced tissue to float instead of settling to the bottom of the tube (Figure 3).</li>
 <li>Incubate hippocampal tissue at 37 °C for 15 minutes, inverting tube as above every 5 minutes.</li>
 <li>Allow the tissue to settle to the bottom of the tube.</li>
 <li>Carefully remove excess solution using a sterile pipette, leaving tissue undisturbed at the bottom of the tube.</li>
 <li>Wash tissue pellet with 5 ml of HBSS at 37 °C for 5 minutes. Repeat a total of 3 times. Allow tissue to completely settle to the bottom of the tube each time before proceeding to the next wash step.</li>
 <li>Remove the final wash from the tissue pellet and replace with 2 ml of fresh HBSS.</li>
</ol>
4. Neuron Trituration
<ol>
 <li>Before beginning trituration steps, you will need to prepare fire polished Pasteur pipettes. Using a Bunson burner, hold a sterile 9 inch Pasteur pipette tip (Figure 4a) in the flame until diameter of the pipette opening is approximately 0.5 mm in size and edges of the pipette opening have been rounded slightly (Figure 4b). Allow pipette to completely cool before beginning the trituration process.</li>
 <li>Using a normal sterile 9 inch Pasteur pipette, gently triturate the tissue a total of 7 times. Larger tissue pieces are normal at this point and should be allowed to settle to the bottom of the tube prior to moving to the next step.</li>
 <li>Transfer the supernatant to a fresh sterile 50 ml conical tube.</li>
 <li>To the remaining tissue, add 2 ml of sterile HBSS and triturate a total of 5 times using the fire polished Pasteur pipette.</li>
 <li>Allow all remaining larger tissue pieces to settle to the bottom of the tube and combine supernatant with the previous supernatant for a total of 4 ml dissociated neuronal cells.</li>
</ol>
5. Cell Plating
<ol>
 <li>Count the dissociated cells using a hemocytometer.</li>
 <li>As a general rule, once cell numbers have been determined, subtract 20% from that final number to account for any cell death that may occur after plating.</li>
 <li>Cells can be plated using the recommendations below: 
 For coverslips in a 24 well plate - 6 x 104 cells/well in 0.5 ml 
 For 60 mm Tissue Culture plates - 4 x 105 cells/plate in 3 ml 
 For 100 mm Tissue Culture plates - 6 x 106 cells/plate in 6 ml</li>
 <li>Mix appropriate cell numbers with indicated volume of Neurobasal Plating Media (Neurobasal Media containing B27 Supplement [1 ml / 50 ml], 0.5 mM Glutamine Solution, 25 μM Glutamate (Mr 147.13 g / mol), Penicillin (10,000 units / ml)/Streptomycin (10,000 μg / ml) [250 μl / 50 ml], 1mM HEPES (Mr 238.3 g / mol), 10% Heat Inactivated Donor Horse Serum) and add cells to plates. Swirl plates gently to distribute cells evenly. HI-Donor Horse Serum is added to the Plating Media to enrich the cells during the first 24 hours of growth. The cells are subsequently weaned from the serum and returned to a Serum-Free environment by serial reduction of the serum at each media replacement. It should also be noted that while glutamate at higher concentrations is toxic to neuronal cell cultures, at the lower concentrations added here, it will inhibit the attachment of non-neuronal cells11. However, it should only be added to the plating media for the first 24 hours in culture and subsequently left out of any Feeding Media to prevent neurotoxicity of the cells.</li>
 <li>Place neurons in a 37 °C, 5% CO2 incubator overnight.</li>
 <li>Remove half the volume of media from the cells and replace with same volume of Neurobasal Feeding Media (Neurobasal Media containing B27 Supplement [1 ml / 50 ml], 0.5 mM Glutamine Solution, Penicillin (10,000 units / ml) / Streptomycin (10,000 μg / ml) [250 μl / 50 ml], 1 mM HEPES (Mr 238.3 g / mol).</li>
 <li>Neurons should be fed every 4 days by removing half of the old media and replacing it with the same volume of fresh Neurobasal Feeding media. Neuronal processes should begin to be visible on Day 1 (Figure 5a) and become prevalent by Day 10 (Figure 5b).</li>
</ol>
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.
<img alt="Striatum identification, labeled anatomy, diagram, neuroscience study, brain dissection." src="/files/ftp_upload/3634/3634fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
Figure 1. Dissection of the prenatal mouse brain. The first incision is down the midline of the brain separating it into two hemispheres.
<img alt="Rat brain section diagram with labeled hippocampus and striatum; neuroscience study setup." src="/files/ftp_upload/3634/3634fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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.
<img alt="Laboratory experiment setup with test tubes in a rack, illustrating sample handling and analysis." src="/files/ftp_upload/3634/3634fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Dissociation of hippocampal tissue in trypsin solution.
<img alt="Microscopy of capillary diameter measurement; illustrated comparative analysis in physical process." src="/files/ftp_upload/3634/3634fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
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.
<img alt="Neuron development process; Fig. 5a Day 1, Fig. 5b Day 10; microscope image in cell biology study." src="/files/ftp_upload/3634/3634fig5.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
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.
<img alt="Confocal fluorescence microscopy image showing neuronal cells with red and green fluorescence." src="/files/ftp_upload/3634/3634fig6.jpg" data-altupdatedat="1787630408000" data-alt="Figure 6"> 
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.
<img alt="Neuronal structure under fluorescence microscopy, showing dendritic branching." src="/files/ftp_upload/3634/3634fig7.jpg" data-altupdatedat="1787630408000" data-alt="Figure 7"> 
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.
<img alt="Neural structure fluorescence microscope image; neuron network visualization; cell communication study." src="/files/ftp_upload/3634/3634fig8.jpg" data-altupdatedat="1787630408000" data-alt="Figure 8"> 
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.
<img alt="Neuronal cell under fluorescence microscope, highlighting dendrites with arrows, cellular imaging." src="/files/ftp_upload/3634/3634fig9.jpg" data-altupdatedat="1787630408000" data-alt="Figure 9"> 
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.

<ol><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+hippocampal+neurons+in+dispersed+cell+culture%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126, 397-442 (1977).</li>
<li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serum-free+B27%2Fneurobasal+medium+supports+differentiated+growth+of+neurons+from+the+striatum%2C+substantia+nigra%2C+septum%2C+cerebral+cortex%2C+cerebellum%2C+and+dentate+gyrus%5BTitle%5D)+AND+%22J.+Neurosci.+Res%22%5BJournal%5D)">Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus.</a> J. Neurosci. Res. 42, 674-683 (1995).</li>
<li>Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+adult+rat+hippocampal+neurons%5BTitle%5D)+AND+%22J.+Neurosci.+Methods%22%5BJournal%5D)">Isolation and culture of adult rat hippocampal neurons.</a> J. Neurosci. Methods. 71, 143-155 (1997).</li>
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<li>Burwell, R. D., Saddoris, M. P., Bucci, D. J., Wiig, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Corticohippocampal+contributions+to+spatial+and+contextual+learning%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Corticohippocampal contributions to spatial and contextual learning.</a> J. Neurosci. 24, 3826-3836 (2004).</li>
<li>Gluck, M. A., Myers, C., Meeter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortico-hippocampal+interaction+and+adaptive+stimulus+representation%3A+a+neurocomputational+theory+of+associative+learning+and+memory%5BTitle%5D)+AND+%22Neural+Netw%22%5BJournal%5D)">Cortico-hippocampal interaction and adaptive stimulus representation: a neurocomputational theory of associative learning and memory.</a> Neural Netw. 18, 1265-1279 (2005).</li>
<li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+hippocampal+neurons%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Culturing hippocampal neurons.</a> Nat. Protoc. 1, 2406-2415 (2006).</li>
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<li>Price, P. J., Brewer, G. J. Serum -free media for neural cell cultures. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPrice%2C+PJ+%22Protocols+for+Neural+Cell+Culture%22">Protocols for Neural Cell Culture</a>. Federoff, S., Richardson, A. , Humana Press. (2001).</li>
<li>Shen, J., Watanabe, S., Kaneko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+dissociation+with+papain+reduces+the+density+of+cGMP-activated+channels+of+the+retinal+rod%5BTitle%5D)+AND+%22Jpn.+J.+Physiol%22%5BJournal%5D)">Cell dissociation with papain reduces the density of cGMP-activated channels of the retinal rod.</a> Jpn. J. Physiol. 45, 151-164 (1995).</li>
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</ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td>Rat Tail Collagen 1</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Poly-D-lysine Solution</td><td>Chemicon</td><td>A-003-E</td><td></td></tr><tr><td>Hanks Balanced Salt Solution</td><td>Invitrogen</td><td>14175-095</td><td></td></tr><tr><td>Trypsin Solution (1X) 0.25%, liquid</td><td>Invitrogen</td><td>15050-065</td><td></td></tr><tr><td>NeuroBasal Medium (1X) liquid</td><td>Invitrogen</td><td>21103-049</td><td></td></tr><tr><td>B27 Supplement (50X) liquid</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>L-Glutamine 200 mM (100X) liquid</td><td>Invitrogen</td><td>25030-149</td><td></td></tr><tr><td>Penicillin (10,000 units/ml) / Streptomycin (10,000 &amp;mu;g/ml)</td><td>Invitrogen</td><td>15140-148</td><td></td></tr><tr><td>HI-Donor Horse Serum</td><td>Atlanta Biologicals</td><td>S12150H</td><td></td></tr></tbody></table>

isolation and culture of hippocampal neurons from prenatal mice

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

The overall goal of this procedure is to isolate and culture. Highly purified hippocampal neurons from prenatal mice. This is accomplished by first isolating and dissecting the hippocampus from embryonic mouse tissue.The second step of the procedure is to dissociate individual cells by mechanical force, followed by trypsin, ization, and careful tation of the brain tissue. Finally, the dissociated cells are counted and cultured using neuro basal media and B 27 supplement. Ultimately purified hippocampal neurons in culture can be used with various molecular techniques such as immunofluorescence.Visual demonstration of this method is critical in that it's difficult to visualize the iteration process. The tendency is to be too vigorous when separating the individual cells from the brain tissue. Successful general cell ation is required to complete the neuronal culture procedure To generate prenatal pups for neuron harvest.Schedule breeding between adult mice 19 days prior to the day of neuron isolation. Successful mating can be confirmed by detection of a vaginal plug in the female palpitations or visual confirmation of pregnancy the day prior to neuron isolation. For immunofluorescence applications, coat glass cover slips in a 24 well plate with a light coating of three to one rat tail collagen, one poly de lysine solution spread the solution evenly over the cover slip, rest the coated plates uncovered in a tissue culture hood under a UV light overnight.The next day, wash the plates with sterile HBSS or store the plates for up to a week at four degrees Celsius in the dark. Soaking an HBSS critical to this operation. Sterilize all the instruments and use only sterile gauze with careful attention to technique.The dissection and harvest of neural tissue can be completed outside of a laminar flow hood. Begin the tissue harvest by decapitating the pregnant mouse at approximately 19 days Post fertilization. Using dissecting scissors and forceps, create an opening in the mid ventral side of the mouse to completely reveal the mouse's body cavity.Prenatal pups will be located toward the posterior of the mouse's body cavity and should be easily visible in the uterus with forceps. Open the uterus and remove pups. Decapitate the pups with scissors and place the head on gauze under a dissecting microscope.To open the cranium, carefully slice through the skin and cranium using a sharp sterile scalpel or dissecting scissors carefully remove the entire brain with forceps and place the brain on gauze. Using a scalpel, remove the cerebellum and incise down the midline of the brain. To separate it into two hemispheres, grasp a small section of meninges surrounding the hippocampus with forceps and pull it gently away to make dissection of the hippocampus easier.The hippocampus is a curved structure that starts in the distal part of the hemisphere and bends ventrally. The inner concave side is facing a ventricle, is the codal side and is already free. To isolate the hippocampus, gently lift each hippo campi with tissue forceps and transfer them to a 100 millimeter tissue culture dish.With three milliliters of warmed HBSS under a cell culture hood, brain tissue can be combined from multiple pups using all the sterile precautions possible. Under a cell culture rated laminar flow hood, use a scalpel to gently mince the brain tissue in HBSS. Transfer the mince tissue and HBSS to a 15 milliliter conical tube.Add 1.5 milliliters of HBSS and 0.5 milliliters of 0.25%tripsin solution to a total volume of five milliliters. Cap and gently invert the tube four or five times. Avoid producing bubbles or the tissue may become trapped on the bubble and not settle to the bottom of the tube.Incubate the tissue at 37 degrees Celsius for 15 minutes. Inverting tube as before every five minutes after the incubation, carefully pipet away the solution, leaving the tissue at the bottom undisturbed. Now wash the tissue with five milliliters of HBSS.Three to times allow tissue to completely settle to the bottom of the tube between each wash and carefully remove the wash solution after the final wash. Resuspend the tissue in two milliliters of HBSS Before beginning ation steps. Prepare fire polished pasture pipettes.Hold a sterile nine inch pasture pipette tip in a flame until diameter of the pipette. Opening is approximately 0.5 millimeters and the edges are slightly rounded. Two large.An opening will make tation inefficient and too small. An opening will damage the cells, so be careful using a normal sterile nine inch pasture pipette. Gently tri the tissue a total of seven times.If this process is performed too vigorously, the cells can be damaged and die. Allow larger tissue pieces to settle to the bottom of the tube prior to moving to the next step. Transfer the S supernatant to a fresh sterile 50 milliliter conical tube to the remaining tissue.Add two milliliters of sterile HBSS and tritrate a total of five times. Using a fire polish pasture pipette. Allow all remaining larger tissue pieces to settle to the bottom of the tube and combine supernatant with the previous supernatant for a total of four milliliters.Dissociated neuronal cells count the dissociated cells using a hemo cytometer. As a general rule, once cell numbers have been determined, subtract 20%from that final number to account for any cell death that may occur. After plating, mix the cells with neuro basal plating media for 24.Well plates prepare to add 60, 000 cells per well in 0.5 milliliters of media for 60 millimeter culture plates. Prepare to add 400, 000 cells in three milliliters of media for 100 millimeter culture plates. Prepare to add 6 million cells in six milliliters of media swirl plates gently to distribute cells evenly.Place neurons in a 37 degree Celsius 5%carbon dioxide incubator overnight. The next day, remove half the volume of media from the cells and replace it with the same volume of neuro basal feeding media. Unlike plating media, this media lacks glutamate and high donor horse.Serum neurons should be fed every four days by removing half of the old media and replacing it with the same volume of fresh neuro basal feeding.Media. Neuronal processes should begin to be visible on day one and become extensive and branched by day 10. Sometimes contamination by glial cells can occur.They're easily distinguished from neurons in culture and in this instance, they make analysis of A GFP lc three expressing neuronal process difficult to visualize to reduce glial cell contamination. Treat the culture with cytosine aide before the fourth day of culture at its lowest effective dose. If the culture is contaminated by fibroblastic reactive microglial cells, treat it with FRDR to decrease their proportion.Hippocampal neurons were grown for seven days and stained with a vital dye to mark active mitochondria and tissue culture cells. The cells were fixed in 4%paraform aldehyde, PBS, and visualized by fluorescent microscopy. The diet itself is non fluorescent until oxidized in the mitochondria.Active mitochondria can be seen throughout the neuronal processes. Cells from the seven day culture were also fixed with 4%paraform aldehyde, PBS and immuno stain with monoclonal anti tubulin antibody followed by Oregon green labeled goat anti-US secondary antibody and fluorescence visualized by microscopy. Hippocampal neuronal cultures were grown for five days and transfected with G FP lc three beta DNA construct using lipectomy in 2000.At day seven, the cells were fixed using 4%Paraform Aldehyde, PBS and MICROSOMES with GFP tagged lc three beta incorporated into their outer membrane were visualized using fluorescent microscopy. Acrosome are located throughout the cell body and neurites and are denoted with arrows While attempting to perform this protocol, it's important to remember to perform the procedure quickly but carefully and to be gentle with cells during the iteration process. Paying attention to these variables will greatly enhance your ability to successfully culture.Mouse hippocampal neurons.

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Research

JoVE Journal

Neuroscience

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons.  Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

The video demonstrates a method for culturing neurons from murine hippocampi. Initially, the hippocampi undergo enzymatic digestion to break down the extracellular tissue matrix. Subsequently, the digested tissue is mechanically disrupted to isolate individual neurons. Finally, these neurons are cultured on glial feeder cells.

A Technique to Culture Mouse Hippocampal Neurons

This video demonstrates the dissociation and culture of hippocampal neurons from hippocampal tissue samples using enzymatic and mechanical dissociations. The treatment with Ara-C inhibits the growth of non-neuronal cells, facilitating the selective growth of hippocampal neurons.

Dissociating and Culturing Neurons from Hippocampal Tissue Samples

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

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.

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

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Low-Density Primary Hippocampal Neuron Culture

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

The present protocol describes how to isolate and purify primary hippocampal microglia from adult mice, followed by instructions for conducting whole-cell patch-clamp recordings on these acutely isolated cells.

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice

Learn to isolate and culture highly purified hippocampal neurons from prenatal mice using dissection, trypsinization, and neuro basal media with B27 supplement. This method enables molecular techniques like immunofluorescence.

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

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