This research was supported by grants CONACYT 252756 and 253631; UNAM-DGAPA-PAPIIT IN202818 and IN203518; INPER 2018-1-163, and NIH P51OD11132. We thank Deisy Gasca, Carlos Lozano, Mart&#237;n Garc&#237;a, Alejandra Castilla, Nidia Hernandez, Jessica Norris and Susana Castro for their excellent technical assistance.

The study was approved by the Research Ethics Committee of the Instituto de Neurobiolog&#237;a, Universidad Nacional Aut&#243;noma de M&#233;xico, Mexico and Instituto Nacional de Perinatologia (2018-1-163). The reproduction, care and humane endpoints of the animals were established following the Official Mexican Standard (NOM-062-Z00-1999) based on the &#8220;Ley General de Salud en Materia de Investigaci&#243;n para la Salud&#8221; (General Health Law for Health Research) of the Mexican Secretaria of Health.
1. Solutions and stocks preparation
<ol>
	<li>Prepare an N2 culture medium with 485 mL of Dulbecco&#39;s Modified Eagle Medium-F12 (DMEM-F12), 5 mL of N2 supplement (100x), 5 mL of glutamine supplement (100x) and 5 mL of antibiotic-antimycotic (100x).</li>
	<li>Prepare a B27 culture medium with 480 mL of Neurobasal medium, 10 mL of B27 supplement (50x), 5 mL of glutamine supplement, and 5 mL of antibiotic-antimycotic (100x).</li>
	<li>Reconstitute collagenase powder in 1x PBS (Phosphate-buffered saline) to obtain aliquots with an activity of 100 units/&#181;L (1000x) and store at -20 &#176;C. Notice, collagenase activity depends on the lot number of the companies.</li>
	<li>Prepare dispase stock aliquots by dissolving 5 mg of dispase powder in 1x PBS (50 mg/mL). Store at -20 &#176;C.</li>
	<li>Prepare an enzymatic solution with 100 mL of DMEM-F12 medium, 50 &#181;L of stock collagenase (100 units/&#181;L) to have a final concentration of 50 U/mL and 333 &#181;L of stock dispase (50 mg/mL) to have a final concentration of 0.33 mg/mL.</li>
	<li>To prepare a washing solution, to 1,000 mL of 1x PBS, add 0.4766 g of HEPES (final concentration 2 mM), 3.6 g of D-glucose (final concentration 20 mM) and 2.1 g of NaHCO3 (final concentration 25 mM).</li>
	<li>Prepare poly-L-ornithine stock aliquots (1 mg/mL) using sterile water and store at -20 &#176;C.</li>
	<li>Prepare a working solution of poly-L-ornithine. Dilute a stock aliquot (1mg/mL) in 49 mL of sterile water for a final concentration of 20 &#181;g/mL.</li>
	<li>Prepare a working solution of laminin. Dilute 25 &#181;L of laminin (1 mg/mL original stock) in 5 mL of sterile water for a final concentration of 5 &#181;g/mL. 
	NOTE: After preparation, filter the culture media, working and stock solutions to avoid contamination. Use a syringe or bottle-top vacuum filters (polyethersulfone membrane with a 0.2 &#181;m pore size). The culture media and work solutions can be stored for up to 30 days at 4 &#176;C, while the stocks can be stored for up to four months at -20 &#176;C.</li>
</ol>
2. Preparation before starting the microdissection
<ol>
	<li>Sterilize surgical instruments by autoclaving or with a hot glass bead dry sterilizer.</li>
	<li>Clean the microdissection surface area under strict aseptic and antiseptic conditions (e.g., with ozonized water). 
	NOTE: The timing of microdissection of both neurogenic niches from each vole brain is approximately 30 min. Working with 1-4 animals for the entire procedure is recommended.</li>
</ol>
3. Extraction of the whole brain
<ol>
	<li>Anesthetize the adult vole (12-16 weeks) with an overdose of pentobarbital (6.3 mg/animal) through intraperitoneal injection. Verify the depth of anesthesia by the absence of pedal reflex in response to a firm toe pinch.</li>
	<li>Once the vole is entirely anesthetized, induce euthanasia by decapitation and recover the head.</li>
	<li>Dissect the skin from the skull with scissors, making a caudal-rostral incision (15 mm long) to expose the skull.</li>
	<li>Cut the occipital and interparietal bones and trace an incision into the skull along the sagittal and parietal sutures.</li>
	<li>Make a hole in the skull at the junction of frontal and parietal bones using scissors, being very careful not to damage the brain tissue.</li>
	<li>To expose the brain, remove the remaining cranium fragments that cover both brain hemispheres with sharp-pointed tweezers.</li>
	<li>Use a stainless-steel spatula to lift the entire brain from the cranial base.</li>
	<li>Collect the brain into a centrifuge tube (50 mL) with 20 mL of cold wash solution.</li>
	<li>Wash the brain twice with the cold wash solution.</li>
</ol>
4. Microdissection of the neural tissue
<ol>
	<li>Place a Petri dish on a surface surrounded by ice.</li>
	<li>Deposit the brain on the dish and add 20 mL of cold wash solution.</li>
	<li>With a scalpel, in the coronal plane, divide the brain into two blocks of tissue (rostral and caudal). As a neuroanatomical reference, perform the coronal cut at Bregma level in the anterior-posterior axis11 (Figure 1A, solid line).</li>
	<li>From the rostral block, extract the VZ tissue (Figure 1B), while from the caudal block remove the DG (Figure 1C).</li>
	<li>Dissect the VZ under a stereo microscope.
	<ol>
		<li>With a Dumont forceps, hold one of the hemispheres; then, insert, at the height of the ventricle, the fine tips of a second Dumont forceps under the tissue that lines the caudate-putamen (Figure 2A).</li>
		<li>Open the forceps along the dorsoventral axis to separate the tissue.</li>
		<li>Collect the VZ tissue per individual in a centrifuge tube with 2 mL of cold wash solution. Do not pool the tissue of more than two animals.</li>
		<li>Repeat the microdissection in the other hemisphere.</li>
		<li>Store the tube containing the bilateral VZ tissue on ice and continue dissecting the DG.</li>
	</ol>
	</li>
	<li>Dissect the DG from the caudal block under a stereo microscope.
	<ol>
		<li>With a scalpel, make a coronal cut into the block to obtain two slices, in which the hippocampal formation is observed. As a landmark, the cut is made at -2 mm Bregma coordinates in the anterior-posterior axis according to the mouse brain atlas11 (Figure 1A, dotted line and Figure 1C).</li>
		<li>With a Dumont forceps, hold one of the slices, and with fine-point Dumont forceps make a horizontal cut between DG and CA1 and then perform a vertical incision between the DG and CA3 to separate the DG (Figure 2B).</li>
		<li>Repeat the dissection in the first slice of the other hemisphere.</li>
		<li>Repeat the dissection in both hemispheres in the second slice.</li>
		<li>Collect the four DG pieces of each vole in a centrifuge tube. Do not pool the DG tissue of more than two animals. 
		NOTE: If dissection of more than one animal is required, store the centrifuge tubes with the VZ or DG tissue on ice while continuing to dissect the rest of the brains. Remove all blood vessels that cover the brain tissue while dissecting. If the vessels are not discarded, the culture could be mixed with an excess of erythrocytes and disturb neurosphere formation.</li>
	</ol>
	</li>
</ol>
5. Isolation of neural cells
<ol>
	<li>Place the centrifuge tubes inside the biosafety cabinet and wait about 10 min for the tissue fragments to precipitate by gravity.</li>
	<li>Remove the wash solution and add 1 mL of the warm enzymatic solution to each tube.</li>
	<li>Incubate the tubes at 37 &#176;C for 10 min.</li>
	<li>Disintegrate the tissue fragments; pipette up and down with a 1 mL tip. Do not pipette more than 30x.</li>
	<li>Carry out a second incubation of 10 min at 37 &#176;C.</li>
	<li>At the end of the second incubation, pipette to break up the tissues. Do not pipette more than 30x. 
	NOTE: After pipetting, the tissue fragments should be completely disintegrated; if they are not disintegrated, incubate for another 10 min at 37 &#176;C and re-pipette. The digestion period should not exceed 30 min.</li>
	<li>Add 9 mL of N2 medium per tube to dilute the enzymatic treatment.</li>
	<li>Centrifuge the tubes at 200 x g for 4 min at room temperature.</li>
	<li>Discard the supernatant and wash with 10 mL of N2 medium.</li>
	<li>Centrifuge under the same conditions as step 5.8.</li>
	<li>Remove the supernatant from each tube and resuspend the cell pellets of the VZ and DG in 2 mL and 1 mL of the B27 medium, respectively.</li>
	<li>To remove any non-disintegrated tissue, filter each cellular suspension using a cell strainer (size 40 &#181;m).</li>
</ol>
6. Neurospheres formation
<ol>
	<li>Culture the cells passed through the strainer into an ultra-low attachment, 24-well plate. Use two wells for the VZ and one well for the DG (1 mL of B27 medium/well).</li>
	<li>Add 20 ng/mL of FGF2 and 20 ng/mL of EGF to each well (final concentration 1x).</li>
	<li>Incubate at 37 &#176;C, 5% CO2 and high humidity (90-95%). Do not disturb for 48 h (day 1 and day 2 of culture, D1-D2).</li>
	<li>On the third day (D3), remove half of the culture medium and replace it with fresh B27 medium (500 &#181;L per well) supplemented with double concentration (2x) of growth factors.</li>
	<li>Repeat every third day, change the culture medium (half of it) and replace it with a fresh B27 medium supplemented with double concentration (2x) of growth factors.</li>
	<li>On days when it is not necessary to change the culture medium, add growth factors to a final concentration of 1x.</li>
	<li>Ensure that the neurospheres are formed around D8-D10.</li>
	<li>At the D10, change the complete culture medium to remove all debris.
	<ol>
		<li>Collect the medium and neurospheres individually of each well in centrifuge tubes.</li>
		<li>Incubate for 10 min at room temperature. This procedure allows neurospheres precipitation by gravity.</li>
		<li>Remove the supernatant and resuspend in 1 mL of fresh B27 medium supplemented with growth factors.</li>
		<li>Place the neurospheres back into the same ultra-low attachment plate and incubate at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>From D10 to D15, continue changing half of the medium and adding growth factors.</li>
</ol>
7. Passage of the neurospheres
<ol>
	<li>At D15 of the primary culture, collect the neurospheres into centrifuge tubes using 1 mL pipette. Cut the pipette tip to increase the size of the opening to avoid damage to the neurospheres.</li>
	<li>Incubate for 10 min at room temperature. Neurospheres precipitate by gravity.</li>
	<li>Remove the medium and add 1 mL of the cell detachment medium per tube.</li>
	<li>Incubate the tubes for 7 min at 37 &#176;C.</li>
	<li>Pipette up and down with a 1 mL tip to dismantle the neurospheres.</li>
	<li>Dilute the cell detachment medium with 3 mL of B27 medium per tube.</li>
	<li>Centrifuge the cell suspension for 5 min at 200 x g.</li>
	<li>Discard the supernatant and resuspend each cell pellet with a fresh B27 medium supplemented with growth factors.
	<ol>
		<li>Resuspend the VZ-derived cells in 4 mL of medium and the DG-derived cells in 2 mL of medium.</li>
	</ol>
	</li>
	<li>Culture the cells (passage 1) in a new ultra-low attachment plate by doubling the number of wells that were used in the primary culture (4 and 2 wells for VZ and DG, respectively).</li>
	<li>Change half of the medium every third day and add growth factors daily.</li>
	<li>After 10 days (D10) in passage 1, change to adherent conditions in the next passage.</li>
</ol>
8. The passage in adherent conditions
<ol>
	<li>Before carrying out the passage 2, prepare coated plates with poly-L-ornithine and laminin.
	<ol>
		<li>In 24-well plates, add 500 &#181;L of 1x poly-L-ornithine (20 &#181;g/mL) per well. Incubate at 37 &#176;C overnight.</li>
		<li>Remove the poly-L-ornithine and wash 4x with 1x PBS (500 &#181;L/well).</li>
		<li>Add 200 &#181;L (minimum volume to cover the surface of a single well) of 1x laminin (5 &#181;g/mL) per well and incubate for 2-3 h at 37 &#176;C before cultivating the cells.</li>
	</ol>
	</li>
	<li>Collect the neurospheres with 1 mL pipette with cut tips into a centrifuge tube.</li>
	<li>Incubate for 10 min at room temperature to precipitate the neurospheres by gravity.</li>
	<li>Discard the supernatant and resuspend the neurospheres in fresh B27 medium without growth factors.</li>
	<li>Aspirate the laminin from the coated plate and deposit the neurospheres into the wells using 1 mL pipettes with cut tips. 
	NOTE: Prevent coated wells from drying out between laminin removal and plating neurospheres.</li>
	<li>Divide the culture into two conditions:
	<ol>
		<li>Maintain differentiated neurospheres for 6 days (D6). Change the medium every third day and add growth factors daily.</li>
		<li>Observe differentiation of the neurosphere-derived cells by 12 days (D12). Change the medium every third day without growth factors. 
		NOTE: At the end of D6 for undifferentiated or D12 for differentiation conditions, the cells can be used for conventional immunohistochemistry, cell sorting analysis, 5-Ethynyl-2&#180;-deoxyuridine (EdU) staining, RNA extraction, among others.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

A stage to obtain a neural stem cell culture is the digestion period with the enzymatic solution, which should not exceed more than 30 min because it might decrease cell viability. The neurospheres should emerge at 8-10 days after initial culture; if they do not emerge by day 12, discard the culture and repeat the experiment, reducing the digestion period. Another issue is the blood vessels that cover the brain tissue. They should be completely removed during the dissection because the excess of erythrocytes can interfer...

The prairie vole (Microtus ochrogaster), a member of the Cricetidae family, is a small mammal whose life strategy develops as a socially monogamous and highly sociable species. Both males and females establish an enduring pair bond after mating or long periods of cohabitation characterized by sharing the nest, defending their territory, and displaying biparental care for their progeny1,2,3,4. Thus, the prairie vole is a valuable model for understanding the neurobiological basis of socio-sexual behavior and impairments in social cognition5.
Adult neurogenesis is one of the most paramount processes of neural plasticity that leads to behavioral changes. For example, our research group reported in male voles that social cohabitation with mating increased cell proliferation in the subventricular zone (VZ) and subgranular zone in the dentate gyrus (DG) of the hippocampus, suggesting that adult neurogenesis can play a role in the formation of pair bonding induced by mating in prairie voles (unpublished data). On the other hand, although the brain regions where new neurons are generated and integrated are well known, the molecular and cellular mechanisms involved in these processes remain undetermined due to technical drawbacks in the whole brain model6. For instance, the signaling pathways controlling gene expression and other cellular activities have a relatively short activation period (detection of phosphoproteome)7. One alternative model is isolated and cultured adult neural stem cells or progenitor cells to elucidate molecular components involved in adult neurogenesis.
The first approach to maintain in vitro neural precursors from adult mammal (mouse) brain was the assay of neurospheres, which are cellular aggregates growing under non-adherent conditions which preserve their multipotent potential to generate neurons, as well as astrocytes8,9,10. During their development, there is a selection process where only the precursors will respond to mitogens such as the Epidermal Growth Factor (EGF) and Fibroblast Growth Factor 2 (FGF2) to proliferate and generate neurospheres8,9,10.
To our knowledge, no protocol is reported in the literature to obtain adult neural progenitors from prairie voles. Here, we established the culture conditions to isolate neuronal progenitors from neurogenic niches and their in vitro maintenance through the neurosphere formation assay. Thus, experiments can be designed to identify the molecular and cellular mechanisms involved in proliferation, migration, differentiation and survival of the neural stem cells and progenitors, processes that are still unknown in the prairie vole. Moreover, elucidating in vitro differences in the properties of the cells derived from the VZ and DG could provide information about the role of neurogenic niches in neural plasticity associated with changes in socio-sexual behavior and cognitive behaviors, and deficits in social interactions (autism spectrum disorder and schizophrenia), which could also be sex-dependent.

<table>
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td>Antibody ID</td>
		</tr>
		<tr>
			<td>Anti-Nestin</td>
			<td>GeneTex</td>
			<td>GTX30671</td>
			<td>RRID:AB_625325</td>
		</tr>
		<tr>
			<td>Anti-Doublecortin</td>
			<td>MERCK</td>
			<td>AB2253</td>
			<td>RRID:AB_1586992</td>
		</tr>
		<tr>
			<td>Anti-Ki67</td>
			<td>Abcam</td>
			<td>ab66155</td>
			<td>RRID:AB_1140752</td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>GeneTex</td>
			<td>GTX50810</td>
			<td>RRID:AB_11170769</td>
		</tr>
		<tr>
			<td>Anti-GFAP</td>
			<td>SIGMA</td>
			<td>G3893</td>
			<td>RRID:AB_477010</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11029</td>
			<td>RRID:AB_2534088</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit Alexa Fluor 568</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11036</td>
			<td>RRID:AB_10563566</td>
		</tr>
		<tr>
			<td>Goat Anti-Guinea Pig Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11073</td>
			<td>RRID:AB_2534117</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>15240062</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>17504044</td>
			<td>50X</td>
		</tr>
		<tr>
			<td>Collagenase, Type IV</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>17104019</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>17105041</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>DMEM/F12, HEPES</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>11330032</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>any brand</td>
			<td></td>
			<td>Powder, Cell Culture Grade</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>35050061</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>any brand</td>
			<td></td>
			<td>Powder, Cell Culture Grade</td>
		</tr>
		<tr>
			<td>Mouse Laminin</td>
			<td>Corning</td>
			<td>354232</td>
			<td>1 mg/mL</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>17502048</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>NAHCO3</td>
			<td>any brand</td>
			<td></td>
			<td>Powder, Suitable for Cell Culture</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>10010023</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher Scientific/Gibco</td>
			<td>A1110501</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3473</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well Clear TC-treated Multiple Well Plates</td>
			<td>Corning/Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Corning/Falcon</td>
			<td>352340</td>
			<td>Blue</td>
		</tr>
		<tr>
			<td>Bottle Top Vacuum Filter, 0.22 &#181;m pore</td>
			<td>Corning</td>
			<td>431118</td>
			<td>PES membrane, 45 mm diameter neck</td>
		</tr>
		<tr>
			<td>Non-Pyrogenic Sterile Centrifuge Tube</td>
			<td>any brand</td>
			<td></td>
			<td>with conical bottom</td>
		</tr>
		<tr>
			<td>Non-Pyrogenic sterile tips of 1,000 &#181;l, 200 &#181;l and 10 &#181;l.</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton gauzes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile microcentrifuge tubes of 1.5 mL</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile serological pipettes of 5, 10 and 25 mL</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filters, 0.22 &#181;m pore</td>
			<td>Merk Millipore</td>
			<td>SLGPR33RB</td>
			<td>Polyethersulfone (PES) membrane, 33 mm diameter</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment and surgical instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized Pipet Filler/Dispenser</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless-steel Spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

culture of neurospheres derived from the neurogenic niches in adult prairie voles

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the differentiation and proliferation potential of neural stem cells, as well as to generate cell lines than can be assayed over time. Also, neurospheres can create a niche (in vitro) that allows the modeling of the dynamic changing environment, such as varying growth factors, hormones, neurotransmitters, among others. Microtus ochrogaster (prairie vole) is a unique model for understanding the neurobiological basis of socio-sexual behaviors and social cognition. However, the cellular mechanisms involved in these behaviors are not well known. The protocol aims to obtain neural progenitor cells from the neurogenic niches of the adult prairie vole, which are cultured under non-adherent conditions, to generate neurospheres. The size and number of neurospheres depend on the region (subventricular zone or dentate gyrus) and sex of the prairie vole. This method is a remarkable tool to study sex-dependent differences in neurogenic niches in vitro and the neuroplasticity changes associated with social behaviors such as pair bonding and biparental care. Also, cognitive conditions that entail deficits in social interactions (autism spectrum disorders and schizophrenia) could be examined.

Neurospheres were formed from neural stem cells isolated from the VZ and DG of both female and male adult prairie voles. About 8-10 days after starting the culture, cells should have formed the neurospheres. Note that the plate may contain debris in the primary culture (Figure 3A). However, in passage 1 the culture should only consist of neurospheres (Figure 3B).
A higher number of neurospheres were obtained from the female VZ as comp...

Watch this Scientific Journal Video about Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles at JoVE.com

Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

Neurospheres are primary cell aggregates that comprise neural stem cells and progenitor cells. These 3D structures are an excellent tool to determine the ...

culture-neurospheres-derived-from-neurogenic-niches-adult-prairie

Research

JoVE Journal

Neuroscience

3.9K Views.  Universidad Nacional Autónoma de México. We established the conditions to culture neural progenitor cells from the subventricular zone and dentate gyrus of the adult brain of prairie voles, as a complementary in vitro study, to analyze the sex-dependent differences between neurogenic niches that could be part of functional plastic changes associated with social behaviors.

Video: Culture of Neurospheres Derived from the Neurogenic Niches in Adult Prairie Voles

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Organotypic Culture of Full-thickness Adult Porcine Retina

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.

Here we describe a cost-effective technique for organotypic culture of adult porcine retina for seven days. Briefly, a sterile filter paper was used to lift the neural retina off from the RPE and place photoreceptor side up on an insert raised by a custom-made stand.

There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

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 ...

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...