Name: isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains
Uploaded: 2015-02-19T15:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

This work was performed using the Division of Brain Sciences, Department of Medicine, Imperial College London startup funds to K.N.A.

NOTE: The animals were maintained and handled in compliance with the institutional guidelines and all animal procedures were approved by the Imperial College&#39;s Animal Welfare and Ethical Review Body (AWERB) and the Home Office and Harvard University Institutional Animal Care and Use Committee (IACUC), in compliance with federal and state regulations.
1. Reagent and Equipment Setup
<ol>
	<li>Thaw, aliquot and store 1 mg/ml&#160;Laminin solution at &#8211;80 &#186;C. Dissolve 2 &#181;l in 1 ml DMEM/F12, resulting in a coating concentration of 1-2 &#181;g/cm2. 
	NOTE: Thaw Laminin slowly on ice and dissolve in cold DMEM/F12 and immediately add it to the plates/coverslips.</li>
	<li>Dilute 75 ml of 0.01% Poly-L-ornithine in 425 ml PBS and store at 4 &#186;C. 
	NOTE: The shelf life of the working solution is up to one month.</li>
	<li>Dissolve 25% (wt/vol) BSA in PBS, pH 7.4, and store at 4 &#186;C for up to a year.</li>
	<li>Prepare 50% FBS (in HBSS) deactivation media.</li>
	<li>Prepare complete medium by adding 50 U/ml penicillin and streptomycin, 1x N2 supplement, 5% (vol/vol) FBS, 0.36% D-(+)-Glucose (wt/vol), 0.25% BSA (wt/vol) to DMEM/F12 and store at 4 &#186;C. 
	NOTE: The shelf life is up to 10 days.</li>
	<li>Place coverslips in boiling 70% (vol/vol) ethanol for at least 30 min to remove any traces of grease and autoclave.</li>
	<li>Place coverslips in 24-well plates and add 500 &#181;l Poly-L-ornithine solution to each well. Incubate 1 hr under the tissue culture hood at RT and then, wash 3 times with 500 &#181;l water. Add 500 &#181;l laminin solution to each plate and incubate O/N in the humidified tissue culture incubator at 37 &#186;C. 
	NOTE: While no washes are necessary after laminin treatment, washing the poly-L-ornithine less than three times results in death of the cells 24 h after culturing.</li>
	<li>Polish the tips of glass pipettes over natural gas or torch flame. 
	NOTE: After this step the diameter of the tip should be about half of its normal size.</li>
	<li>Cut the caps of 1.5 ml microcentrifuge tubes, using an autoclaved scissor and autoclave.</li>
</ol>
2. Dissection of Embryonic Ventral Midbrain
<ol>
	<li>Narcotize timed-pregnant (E12.5) dams by CO2 inhalation and then euthanize by cervical dislocation.</li>
	<li>Spray the abdomen with 70% ethanol and make an abdominal incision until the uterine sacs are all exposed. Using forceps cut the vaginal attachment and remove uterus. Place the uterus in ice-cold HBSS, in a 100 mm Petri dish.</li>
	<li>Remove embryos from the uterus and amniotic sac, using forceps (Figure&#160;1C). Place embryos in fresh ice-cold HBSS. 
	NOTE: The rectangle in Figure 1D indicates the location of embryonic ventral midbrain.</li>
	<li>Place the embryos under a dissection microscope (10x magnification) and dissect the mesencephalic arch by cutting the brain at the isthmus and mesencephalic-diencephalic boundary region, using the bioscissor and forceps (Figure 1E, please refer to Figure 1A for lines of incision).</li>
	<li>After taking out the entire midbrain, make a cut in the mediodorsal midbrain (Figure 1F).</li>
	<li>Remove meninges, using two forceps (Figure 1H). 
	NOTE: CRITICAL STEP: This step is essential for optimal neuronal yield and survival. Since the culture conditions are optimal for brain cells, most of the cells from the surrounding tissue die after plating and the apoptotic signal from this tissue may damage integrity of the cultures.</li>
	<li>Flatten the midbrain tissue on the Petri dish to observe the butterfly shape and cut approximately half of each wing, using a sterilized shaving blade (Figure 1H,I). 
	NOTE: Figure 1J depicts the isolated ventral midbrain.</li>
	<li>Transfer the piece of ventral midbrain into ice-cold HBSS in a 15 ml conical tube and proceed with the next embryo. 
	NOTE: Steps 2.1-2.8 should not take more than 1 hr. Keeping the embryos beyond this time frame on ice decreases cell viability significantly.</li>
</ol>
3. Dissociation of Ventral Midbrain Cells
<ol>
	<li>Transfer the pieces of ventral midbrain under a laminar flow hood. Remove the HBSS and add 1 ml pre-warmed (37 &#186;C) 0.05% trypsin-EDTA into the conical tube (volumes enough for up to 12 pieces of ventral midbrain). Incubate the tissue at 37 &#186;C for 5-10 min.</li>
	<li>Remove trypsin-EDTA under the hood and add 1 ml de-activation medium (the serum will deactivate trypsin) to the tissue.</li>
	<li>Remove the deactivation medium and wash the tissue in 1 ml complete medium twice. 
	NOTE: Avoid removing all the medium from the tube and pipetting the tissue, to prevent discarding the dissociated cells.</li>
	<li>Add 1ml complete medium and triturate the tissue with a fire-polished glass pipette. Avoid formation of bubbles and continue triturating until single cell suspension is achieved. 
	NOTE: Usually, 8-10 passes through the restricted tip are required for complete dissociation.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at RT and remove the medium.</li>
	<li>Re-suspend the pellet in 1 ml complete medium by slowly pipetting up and down for up to four times.</li>
</ol>
4. Plating Ventral Midbrain Cells
<ol>
	<li>Mix 10 &#181;l of the cell suspension with 90 &#181;l Trypan blue solution and count the number of cells with a hemocytometer. 
	NOTE: Viability of the cells can also be checked at this stage.</li>
	<li>Place sterilized microcentrifuge tube caps in 100 mm Petri dishes and transfer the Poly-L-ornithine/Laminin coated coverslips, using forceps, one at a time on top of the caps. 
	NOTE: Do not wash the coverslips and avoid drying the laminin, since this may cause uneven cultures and decrease in survivability of the cells.</li>
	<li>Adjust the volume to 1,500 cells per 1 &#181;l by adding complete medium and add 100 &#181;l (total of 150,000 cells) of the cell suspension on top of each coverslip (the volume is optimal to cover the surface of the coverslip without spillage). Close the Petri dishes and incubate them for 1 hr in a humidified tissue culture incubator (37 &#176;C, 5% CO2). 
	NOTE: CRITICAL STEP: This step is required for enhancing the attachment and viability of the cells and for increasing the number of coverslips per embryo. Alternatively, the cells can be transferred directly into the wells (containing coverslips) but the number of cells should be increased to 350,000 per well (instead of 150,000). In other words, using this method, 8-10 coverslips can be acquired, while direct transfer yields about 3-4 coverslips because of the cells, which attach to the plates (beside or underneath the coverslips). The average viability of the cultures, using this method was around 90%.</li>
</ol>
5. Culture Growth and Maintenance
<ol>
	<li>After 1 hr of incubation, carefully transfer the coverslips including the medium into 24-well plates, containing 400 &#181;l pre-warmed (to 37 &#186;C) complete medium (total volume: 500 &#181;l). Incubate the cells O/N at 37 &#186;C.</li>
	<li>Within 24 hr after incubation of the wells, gently add 500 &#181;l complete medium into each well. 
	NOTE: The first few hours is the most critical stage for survival of the dopaminergic neurons and the number of surviving cells does not change significantly beyond this point.</li>
	<li>Do not add additional media to the cultures for the first two weeks or until the medium becomes yellow. If culturing for more than two weeks or medium color changes to yellow, exchange half of the media (500 &#181;l) with fresh complete pre-warmed media (typically every two weeks). 
	NOTE: Dopaminergic neurons within the cultures would survive for more than 6 weeks under these conditions.</li>
</ol>

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No conflict of interest declared.

Dopaminergic neurons in midbrain are the main source of dopamine in the central nervous system. They are divided into three groups, substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and retrorubral field (RRF) 10,11. The neurons in SNpc and VTA give rise to major dopaminergic pathways, mesocortical, mesolimbic and nigro-striatal, involved in functions such as control of emotion, motivation and motor behavior. Demise of the neurons in SNpc and functional disruption of the nigrostriatal system is the main pathological characteristic of the second most prominent neurodegenerative disorder, Parkinson&#8217;s disease (PD) 12,13. Current therapeutic approaches address symptoms of the disease and do not halt or slow down degeneration and the causes of the slow, progressive cell loss are still unknown 14-16. Therefore, development of in vivo and in vitro techniques for study of the developmental, biochemical, and physiological characteristics of this neuronal population is imperative to understanding the etiology of PD and to development of new therapeutic approaches.
Here we describe an optimized method for dissection, dissociation and culturing of primary neurons from embryonic mouse and rat ventral midbrain, which results in long-term in vitro survival of the dopaminergic neurons from this region, allowing study of the processes such as differentiation, axonal outgrowth and synapse formation. Several advantages of this protocol include the increased number of coverslips per embryo, independence from growth factors and the length of in vitro survival, maximizing development of neurons 4. This method may complement the in vivo studies in allowing for understanding the mechanisms underlying normal physiological functions of the midbrain dopaminergic neurons as well as progressive degeneration of this neuronal population in cytotoxic and genetic models of Parkinson&#8217;s disease.
One of the main disadvantages of this protocol is the lack of distinction between SNpc and VTA dopaminergic neurons. This problem exists because at the time of culturing (E12.5 in mice or E14.5 in rats) terminal differentiation of the neurons and their regionalization is not complete and while the use of older embryos is possible, it significantly reduces the survival of the neurons due to axotomy. Distinguishing the two populations would be possible by immunocytochemistry, using antibodies against specific markers for each population such as Girk2 (to identify SNpc neurons) and Calbindin (to identify VTA neurons)17.
The optimized plating described in section 4 decreases the amount of embryos per experiment by more than 50% and therefore contributes to reducing the use of animals in research. The most critical steps for survivability of the cultures are the time between euthanization of the dams and complete dissection of the embryonic midbrains (step 2.1 &#8211; 2.8), as well as the complete removal of the meninges (step 2.6, Figure&#160;1H).

Loss of dopaminergic neurons from the substantia nigra pars compacta leads to the cardinal motor symptoms of Parkinson&#8217;s Disease (PD), the second most prevalent neurodegenerative disorder. The underlying cause of the demise of this mesencephalic neuronal population is not known. To study the biochemical pathways responsible for the development and modulating neurophysiological properties and survival of mesDA neurons, several cell culture and animal model systems have been used. Immortalized cell lines, including the rat dopaminergic cell line 1RB3AN27(N27), the human dopaminergic neuroblastoma cell line SH-SY5Y, the mouse dopaminergic hybrid cell line MN9D and human mesencephalic LUHMES cells have been used for biochemical and limited mechanistic studies 1-5. For the study of the specific loss of mesDA neurons, several neurotoxin-based and genetic models have been developed 6-8. Primary ventral midbrain cultures, provide an indispensable tool for studying the neuronal and synaptic properties of the dopaminergic neurons and the pathways involved in pathogenesis of this common disorder.
Here we present a detailed protocol for the isolation of mesencephalic dopaminergic neurons, which contains modifications resulting in higher survivability and increased yield of coverslips per embryo. Use of pre-mature E12.5 mouse mesencephalon (E14.5 in rat) enhances survivability. At this age neurons have not developed axons yet, which leaves cells intact during dissection and minimizes the stress thereby significantly increasing viability. In addition, careful dissection of the ventral midbrain, as described in section 2 of this protocol, further enhances survivability. To increase the numbers of coverslips per embryo, an alternative plating method is presented in section 4 of this protocol. This leads to a yield of up to 10 coverslips per embryo as compared to 4 coverslips under standard plating conditions thus reducing the amount of animals per experiment.
Neurons in culture exhibit outgrowth of axons and dentrites, form synaptic connections and reveal the presence of neuronal and synaptic markers making these cultures suitable for live cell imaging, immunocytochemical and electrophysiological studies. Furthermore, the use of neuronal cultures facilitates genetic and pharmacologic manipulation. Outgrowth of neurites from day 2 in vitro allows for developmental studies. Furthermore, the long-term survival of cultures (up to six weeks) makes them suitable for study of the slow, progressive degeneration of these neurons.

<table><tbody><tr><td>Dulbecco&#039;s modified Eagle medium nutrient mixture F-12</td><td>Invitrogen</td><td>11330</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) (1X), liquid</td><td>Invitrogen</td><td>24020-117</td><td></td></tr><tr><td>Fetal bovine serum, heat-inactivated (FBS)</td><td>Invitrogen</td><td>16140</td><td></td></tr><tr><td>N2 Supplement (100X), liquid</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>D-(+)-Glucose solution (45% (wt/vol) in water</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>Bovine serum albumin BSA</td><td>Sigma</td><td>A9430</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Invitrogen</td><td>15070</td><td></td></tr><tr><td>Trypsin (0.05% (wt/vol)</td><td>Invitrogen</td><td>25300</td><td></td></tr><tr><td>Bovine serum albumin (BSA) cell culture tested</td><td>Sigma&amp;nbsp;</td><td>A9418</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma</td><td>P3813</td><td></td></tr><tr><td>Anti-Tyrosine hydroxylase (Th) antibody</td><td>Pel-Freez Biologicals</td><td>P60101-0&amp;nbsp;</td><td></td></tr><tr><td>Poly-L-ornithine, 0.01% solution</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Anti-Map2 (Microtubule associated protein-2A and -2B) antibody</td><td>Millipore</td><td>MAB3418</td><td></td></tr><tr><td>Anti-Synapsin-1 antibody</td><td>Millipore</td><td>AB1543P</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21206</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-rabbit IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21207</td><td></td></tr><tr><td>Alexa Fluor 488 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11015</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-sheep IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-11016</td><td></td></tr><tr><td>Alexa Fluor 594 donkey anti-mouse IgG&amp;nbsp;&amp;nbsp; antibody</td><td>Molecular Probes</td><td>A-21203</td><td></td></tr><tr><td>Trypan blue solution (0.4% (wt/vol))</td><td>Biowhittaker</td><td>17-942E</td><td></td></tr><tr><td>Stereo Microscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Inverted phase contrast microscope</td><td>Carl Zeiss</td><td>Axiovert 40 C</td><td></td></tr><tr><td>Dumont Forceps</td><td>Fine Scientific Tools</td><td>May-45</td><td></td></tr><tr><td>Cover Slip Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11251-33</td><td></td></tr><tr><td>Two Dumont #45 Forceps &amp;ndash; Dumoxel</td><td>Fine Scientific Tools</td><td>11245-30</td><td></td></tr><tr><td>Blade Holder/Breaker Flat Grip - 11cm</td><td>Fine Scientific Tools</td><td>10052-11</td><td></td></tr><tr><td>Student Iris Scissors - Straight 11.5 cm</td><td>Fine Scientific Tools</td><td>91460-11</td><td></td></tr><tr><td>Fiber optic halogen illuminator</td><td>Nikon</td><td>MKII</td><td></td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipettes</td><td>Fisherbrand</td><td>13-678-20C</td><td></td></tr><tr><td>Hemocytometer</td><td>Proscitech</td><td>SVZ2NIOU</td><td></td></tr><tr><td>0.2 &amp;micro;m sterile filter units</td><td>Nalgene</td><td>NL-CE-156-4020</td><td></td></tr><tr><td>100x20 mm Petri dishes</td><td>BD Biosciences</td><td>351005</td><td></td></tr><tr><td>Round Cover Slip #1 Thickness German Glass 12 mm</td><td>Bellco Glass</td><td>1943-10012&amp;nbsp;</td><td></td></tr></tbody></table>

isolation, culture and long-term maintenance of primary mesencephalic dopaminergic neurons from embryonic rodent brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

Immunohistochemistry against Tyrosine hydroxylase (Th) shows that between 0.5-1% of the cells in culture are dopaminergic. Neuronal projections appear within 2 hr after plating and by the first day, axons and dendrites are distinguishable (Figure 2), using Tyrosine Hydroxylase (TH) and Microtubule-Associated Protein 2 (Map2) antibodies (Figure 3). The neurons survive for more than six weeks and show extensive outgrowth. The neuron-glia ratio in the cultures is directly related to the concentration of serum in the medium, as shown previously. While eliminating FBS from the cultures may yield relatively pure neuronal cultures, we and others have found that addition of serum increases survivability of dopaminergic neurons in culture 9.
<img alt="Figure 1" src="/files/ftp_upload/52475/52475fig1.jpg" /> 
Figure 1.&#160;Procedure for isolation of embryonic ventral midbrain. The schematic view of E12.5 mouse brain, midbrain boundaries (dashed lines), and the approximate position of the region of interest, ventral midbrain, within the red rectangle (A) and the tools for dissection of the ventral midbrain, a bioscissor, foreceps, and a blade holder (B) are shown. The embryos are taken out from amniotic sac by rupturing it (C). The region of interest, ventral midbrain, is shown by the rectangle (D). Midbrain is isolated by cutting at the isthmus (left of the cut piece) and the midbrain-forebrain boundary (right; E). The mediodorsal part of the midbrain is cut (F) and it is flattened, to resemble a butterfly shape (G,H). Then, meninges are dissociated from brain (H) and ventral midbrain (rectangle) is isolated from the dorsal part by cutting off half of the wings, using a shaving blade (I). Ventral midbrain (J) is transferred to a 15 ml conical tube and kept on ice, until dissociation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52475/52475fig2.jpg" /> 
Figure 2.&#160;Representative brightfield images of ventral midbrain cultures. Images were taken on DIV1 (left), DIV2 (center), and DIV15 (right). Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52475/52475fig3.jpg" /> 
Figure 3.&#160;Long term survival, outgrowth of processes and synaptogenesis in two week old ventral midbrain cultures. Midbrain dopaminergic neurons, identified by tyrosine hydroxylase antibody grow extensive axons and dendrites, identified by Map2 antibody, in the ventral midbrain cultures two weeks after dissociation from E12.5 embryos. Dendrites can be distinguished by overlap of Th and Map2-positive processes, where only Th-positive fibers represent axons. Antibodies were used at a dilution of 1:200. Scale bar: 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52475/52475fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains at JoVE.com

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

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Nanyang Technological University

Research

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Neuroscience

13.9K Views.  Imperial College London. The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Video: Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Biology

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 ...

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

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

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.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

 Primary Microglia and Neuron Co-Culture Protocol for CNS Interaction Studies

Generating and Co-culturing Murine Primary Microglia and Cortical Neurons

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting&#160;reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Author Spotlight: In Vitro Co-Culture Model for Studying Microglia-Neuronal Interactions in Disease Conditions

This protocol describes a microglia-neuronal co-culture established from primary neuronal cells isolated from mouse embryos at embryonic days 15-16 and primary microglia generated from the brains of neonatal mice at post-natal days 1-2.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS ...

Culturing and Maintaining Dopaminergic Neurons from Mouse Embryonic Brain Tissue

Source: Weinert, M. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52475">Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains.</a> J. Vis. Exp. (2015)
This video outlines a method for cultivating dopaminergic neurons from mouse embryonic brain tissue. The process includes treating the midbrain with a proteolytic enzyme, detaching the cells, and culturing them on polymer-coated coverslips in a nutrient-rich medium with antibiotics for their growth and long-term viability.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

Source: Landeira, B. S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56063">Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System.</a> J. Vis. Exp. (2017)
This video demonstrates a protocol for isolating and culturing primary cerebral cortex cells from the dorsolateral telencephalon of mouse embryos.

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

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