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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. vHipp-nAcc Synaptic Co-cultures
<ol>
	<li>Sacrifice mice (postnatal day 0 - 3, from wild-type (WT) or &#945;7 nAChRs transgenic mouse line) with CO2. Decapitate each pup and save the tail in a 1.5 ml centrifuge tube for post facto genotyping. Perform the following steps in a sterilized hood.</li>
	<li>Remove the top of the skull with scissors and forceps to expose the whole brain. Starting at the juncture where the cerebral cortices part from each other caudally, obtain a coronal section that contains the posterior cortices, including the ventral hippocampal (vHipp), under a dissecting microscope.</li>
	<li>Dissect vHipp tissue strips containing the cornu ammonis 1 (CA1)-subiculum region out according to a developing mouse brain atlas (Figure 1A). Transfer to a 35 mm culture dish containing cold culture media (4 &#176;C), and cut into small thin slices (around 100 &#181;m &#215; 100 &#181;m microslices).</li>
	<li>Pre-incubate 12 mm poly-D-lysine/laminin-coated glass coverslips inside 24 well tissue culture plates with culture media (~ 500 &#956;l) for at least 30 min in the incubator at 37 &#176;C. Remove media before plating microslices.</li>
	<li>Plate with a fire-polished Pasteur pipette at the center of the coverslip with a minimal amount of media (~ 50 &#956;l, containing ~ 20 pieces of microslices) to facilitate attachment of the explants. Plate vHipp microslices originating from a single animal (either the +/+ or the -/- genotype of the transgenic &#945;7 line) on each coverslip.</li>
	<li>After the explants settle onto the coverslip, gently add additional media (~ 100 &#956;l) by the wall so that the level of the media is high enough to completely cover the explants on the periphery but not those at the center of the coverslip.</li>
	<li>After an O/N incubation in 37 &#176;C, 5% CO2 incubator, disperse nucleus accumbens (nAcc) neurons from embryonic days 18 to postnatal day 1 WT mice and add to the explants.
	<ol>
		<li>Dissect out nAcc tissues according to a developing mouse brain atlas (Figure 1B).</li>
		<li>Cut into small pieces (around 500 &#181;m &#215; 500 &#181;m microslices) and transfer to a 15 ml tube.</li>
		<li>Treat with 0.25% trypsin (2 ml) for 15 min at 37 &#176;C. Wash tissue chunks three times with cold washing media (5 ml, 4 &#176;C) followed by one wash in cold culture media (5 ml, 4 &#176;C). Allow suspension to rest for 5 min in each wash step, and then pour off supernatant carefully.</li>
		<li>Dissociate cells by gentle trituration in 2 ml of culture media with a lightly fire-polished Pasteur pipette.</li>
		<li>Transfer cell suspension to another 15 ml tube and centrifuge at 2,000 x rpm for 5 min. Remove the supernatant and resuspend the cells in culture media at 1 ml per 6 pups. Disperse cells by gently pipetting in media several times with a fire-polished Pasteur pipette.</li>
		<li>Add 0.25 ml of dispersed nAcc cells to each coverslip with vHipp explants.</li>
		<li>Maintain the cultures in a humidified 37 &#176;C, 5% CO2 incubator. Place culture plates on dampened sterile gauze pads to mechanically stabilize and maintain humidity, facilitating synapse formation.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Culture Media (50 ml)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>GIBCO</td>
			<td>10888022</td>
			<td>48 ml</td>
		</tr>
		<tr>
			<td>B-27 Supplements</td>
			<td>GIBCO</td>
			<td>0080085-SA</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO</td>
			<td>10908-010</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>Brain-derived neurotrophic factor (BDNF)</td>
			<td>GIBCO</td>
			<td>15140-122</td>
			<td>20 ng/ml</td>
		</tr>
		<tr>
			<td>washing media (HBSS, 100 ml)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium, no phenol red</td>
			<td>GIBCO</td>
			<td>14175-095</td>
			<td>99 ml</td>
		</tr>
		<tr>
			<td>HEPES ( 1M)</td>
			<td>GIBCO</td>
			<td>15630-130</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>HEPES buffered saline (HBS) pH=7.3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>HBS Cocktail for live imaging pH=7.3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>135 mM</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>CaCl2,</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G0350500</td>
			<td>10 mM</td>
		</tr>
	</tbody>
</table>

co-culturing of ventral hippocampal and nucleus accumbens

Source: Zhong, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52730">Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons.</a> J. Vis. Exp. (2015)
This video demonstrates the process of preparing ventral hippocampal and nucleus accumbens tissues from mouse brains for co-culture. Neurons extend projections to form synaptic connections between the ventral hippocampal and nucleus accumbens tissues. This method allows studying neuronal interactions between these two brain regions, offering insights into cognition, behavior, and neurological disorders.

<img alt="Figure 1" src="/files/ftp_upload/22381/22381_Figure1.jpg" /> 
Figure 1. Synaptic co-culture of vHipp with nAcc allows examination of pre-synaptic localization of nAChRs. (A-C) Schematic cartoon of genotype-specific in vitro circuits (C) prepared by separate plating of ventral hippocampus/subiculum (A). &#160;slices from an individual WT or &#945;7 -/- mouse and dispersed neurons from WT nucleus accumbens (B). Aq, aqueduct (Sylvius); DG, dentate gyrus; MM, medial mammillary nucleus; PMCo, posteromedial cortical amygdaloid nucleus; rf, rhinal fissure, fmi, forceps minor of the corpus callosum; LV, lateral ventricle; Tu, olfactory tubercle; VP, ventral pallidum. (D) vHipp microslices extend vGluT1 positive (red) axonal projections that contact GAD65 positive (green) nAcc neurons (white arrows are examples of those contact sites). Scale bar: 10 &#181;m. (E-G) Representative micrographs of WT vHipp axons (staining with vGluT1, green) are shown for &#945;4*nAChR (E), &#945;5*nAChR (F), and surface &#945;7*nAChR (nicotinic acetylcholine receptors) (G) staining in red clusters (white arrows). Scale bar: 5 &#956;m. (H) There are no surface &#945;7*nAChR clusters on dispersed GABA(gamma-aminobutyric acid)ergic neurons (GAD65 positive) from nAcc alone. (I) Red &#34;clusters&#34; of surface &#945;7*nAChR (white arrows) can be seen in those dispersed neurons co-cultured with vHipp microslices. Scale bar: 5 &#956;m.

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a postnatal mouse brain coronal section containing a ventral hippocampal region, an area within the brain&#39;s lower region.
Cut the ventral hippocampal tissue and transfer it to a suitable medium.
Slice the tissue into smaller pieces and let them attach to a matrix-coated coverslip in a multiwell plate.
The neurons from the tissue begin extending projections.
Next, take a mouse embryo-derived nucleus accumbens, a small tissue deep inside the forebrain.
Cut the nucleus accumbens tissue into small pieces. Transfer them to a tube.
Add digestion enzymes to dissociate the extracellular matrix proteins. Wash to reduce enzyme levels.
Pipette the tissue pieces up and down to generate single cells.
Centrifuge the cells. Remove the supernatant and resuspend the cells.
Add this suspension to the coverslip with the ventral hippocampal tissues and incubate.
The nerve endings from the ventral hippocampal tissue piece connect with the neuronal cells of the nucleus accumbens, forming a communication junction called a synapse.

co-culturing-of-ventral-hippocampal-and-nucleus-accumbens

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

55 Views. Fonte: Zhong, C., et al. Imaging dal vivo della segnalazione del calcio indotta dalla nicotina e dal rilascio di neurotrasmettitori lungo gli assoni dell'ippocampo ventrale. J. Vis. Exp. (2015) Questo video dimostra il processo di preparazione dei tessuti ventrali dell'ippocampo e del nucleo accumbens da cervelli di topo per la co-coltura. I neuroni estendono le proiezioni per formare connessioni sinaptiche tra i tessuti ventrali, ippocampali e nucleus accumbens. Questo metodo consente di studi...

Video: Co-coltura dell'ippocampo ventrale e del nucleo accumbens - Concept

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.

Cultura primario di topo neuroni dopaminergici

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

Ricerca

JoVE Journal

Neuroscienze

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

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

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.

Uso di neuroni ippocampali coltivati primari per studiare l'assemblaggio dei segmenti iniziali di Axon

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Coltura primaria a bassa densità facile e riproducibile utilizzando stock congelati di neuroni ippocampali embrionali

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Trascrizione

Co-culturing of Ventral Hippocampal and Nucleus Accumbens