Co-culturing of Ventral Hippocampal and Nucleus Accumbens

Protocol

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

1. Sacrifice mice (postnatal day 0 - 3, from wild-type (WT) or α7 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.
2. 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.
3. 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 °C), and cut into small thin slices (around 100 µm × 100 µm microslices).
4. Pre-incubate 12 mm poly-D-lysine/laminin-coated glass coverslips inside 24 well tissue culture plates with culture media (~ 500 μl) for at least 30 min in the incubator at 37 °C. Remove media before plating microslices.
5. Plate with a fire-polished Pasteur pipette at the center of the coverslip with a minimal amount of media (~ 50 μl, 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 α7 line) on each coverslip.
6. After the explants settle onto the coverslip, gently add additional media (~ 100 μl) 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.
7. After an O/N incubation in 37 °C, 5% CO₂ incubator, disperse nucleus accumbens (nAcc) neurons from embryonic days 18 to postnatal day 1 WT mice and add to the explants.
   1. Dissect out nAcc tissues according to a developing mouse brain atlas (Figure 1B).
   2. Cut into small pieces (around 500 µm × 500 µm microslices) and transfer to a 15 ml tube.
   3. Treat with 0.25% trypsin (2 ml) for 15 min at 37 °C. Wash tissue chunks three times with cold washing media (5 ml, 4 °C) followed by one wash in cold culture media (5 ml, 4 °C). Allow suspension to rest for 5 min in each wash step, and then pour off supernatant carefully.
   4. Dissociate cells by gentle trituration in 2 ml of culture media with a lightly fire-polished Pasteur pipette.
   5. 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.
   6. Add 0.25 ml of dispersed nAcc cells to each coverslip with vHipp explants.
   7. Maintain the cultures in a humidified 37 °C, 5% CO₂ incubator. Place culture plates on dampened sterile gauze pads to mechanically stabilize and maintain humidity, facilitating synapse formation.

Results

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).  slices from an individual WT or α7 -/- mouse and dispersed neurons from WT nucleus acc...

Materials

Name	Company	Catalog Number	Comments
Culture Media (50 ml)
Neurobasal	GIBCO	10888022	48 ml
B-27 Supplements	GIBCO	0080085-SA	1 ml
Penicillin-Streptomycin	GIBCO	10908-010	0.5 ml
GlutaMAX Supplement	GIBCO	35050-061	0.5 ml
Brain-derived neurotrophic factor (BDNF)	GIBCO	15140-122	20 ng/ml
washing media (HBSS, 100 ml)
HBSS, no calcium, no magnesium, no phenol red	GIBCO	14175-095	99 ml
HEPES ( 1M)	GIBCO	15630-130	1 ml
HEPES buffered saline (HBS) pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
MgCl2	Sigma	M8266	1 mM
CaCl2,	Sigma	C1016	2 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM
HBS Cocktail for live imaging pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
MgCl2	Sigma	M8266	1 mM
CaCl2,	Sigma	C1016	2 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM