TITLE: Primary Ventral Midbrain Culture
AUTHORS: Douglas Miller, Min Lin, Joseph Lebowitz, Danielle Sambo, Kaustuv Saha, Habibeh Khoshbouei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Primary ventral midbrain cultures provide a reproducible medium to explore neuronal dynamics that are experimentally difficult to perform </span><span style = "font-style:italic;">in vivo</span><span>. The ventral midbrain contains two important dopaminergic-rich regions: substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). These regions have been shown to be critical in regulation of goal-directed behavior and reward prediction error. Furthermore, dysfunction of the SNc and VTA has been identified as targets in Parkinson's disease and addiction, respectively. Therefore, primary ventral midbrain culture provides a model to explore the relative impact of dopaminergic modulation from molecular to neural network scales.</span></div></div>

[STEPS]
?. [Washing Coverslips]
Boil cloverslips in 1M HCL for 5 minutes, allow to cool.Wash 3X with ddH2O.Wash for 30 min in 100% ethanol. Wash 3X with ddH2O. Cover flask with aluminum foil and autoclave.Wash for 30 min in 100% ethanol.Wash 3X with ddH2O. Cover flask with aluminum foil and autoclave.
?. [Coating Coverslips]
Distribute 2 coverlips into each 35 x 10 mm petri dish with flamed forceps. Dispense approximately 175 µl of 0.1 mg/ml Poly-D-Lysine on each coverslip and incubate for 1 hour. Wash 3X with ddH2O.Allow to air dry at least 30 minutes. Dilute laminin in MEM (5 µg/ml final concentration) and coat coverslips with 100 µl of solution.Cover petri dishes to prevent evaporation of the solution. Dispense approximately 175 µl of 0.1 mg/ml Poly-D-Lysine on each coverslip and incubate for 1 hour.Wash 3X with ddH2O.Allow to air dry at least 30 minutes. Dilute laminin in MEM (5 µg/ml final concentration) and coat coverslips with 100 µl of solution.Cover petri dishes to prevent evaporation of the solution. Allow to air dry at least 30 minutes.Dilute laminin in MEM (5 µg/ml final concentration) and coat coverslips with 100 µl of solution.Cover petri dishes to prevent evaporation of the solution.
?. [Dissociation Media Preparation]
Prepared fresh day of dissection.Mix the following solutions in order, otherwise papain will not dissolve15.5 ml Cysteine water400 units papain 4 ml DISCO20 µl kynurenic acidAdjust pH to 7.4 with 5N HCl and sterile filter into Nunc vial
?. [Dissection]
All orientation instructions assumes user is right-handed.Place 10 cm petri dishes on ice and fill with Gey's Balanced Salt Solution (GBSS).Remove post-natal day 0 pups from mother in container with bedding and place on heating pad (set to low) until ready to use.Prepare a trough in ice and line with aluminum foil.Place pups on ice.Once movement has slowed and pups stop moving, rapidly decapitate and transfer to 10 cm petri dish.Cut skin to expose skull and remove the skull by cutting around the base with 3 mm Vanna Scissors.Using a spatula, gently push the brain from the skull from the front into GBSS.Place brain in center of stage, ventral surface upwards and anterior pointing left. Using 5A style forceps angled downward, pierce the brain through the rostral hypothalamus and pin to stage.Make first cut caudal to the midbrain flexture.Make second cut rostral to midbrain flexture, including caudal hypothalamus.Take resulting slice and orient ventral surface to the right.Hold slice in place by inserting forceps into slice at the acqueduct.Remove the hypothalamus by cutting the ventral portion.Isolate ventral midbrain by cutting approximately halfway between previous cut and acqueduct.Remove left and right meninges. Dissect ventral midbrain into 1 mm3 sections and transfer to dissociation media using sterile transfer pipette. Incubate in dissociation media for 20 minutes.
?. [Neuronal Media]
Neuronal Media (Prepare fresh)25 ml Neurobasal Plus0.5 ml B-27 Plus250 µl L-glutamineFor day one of plating add:40 ul GDNF (1 ng/ml)20 ul kynurenic acidFor day one of plating add:40 ul GDNF (1 ng/ml)20 ul kynurenic acidWarm to 37ºC.
?. [Maintenance]
Every 4 days following plating, remove 1 ml of old media and replace with 1 ml of fresh Neuronal Media. Media can be changed more often, if necessary.
DIV 9-12 is the ideal window for general neuronal health and activity.
?. [Pre-surgical preparation]
Clean 10 cm glass petri dish with 70% ethanol and allow to dryUsing VetBond, glue a single Sylgard stage in the middle of the dish and allow to cureWarm water bath to 37ºC.Thaw Glia Media in water bath.Transfer Dissociation media to water bath and oxygenate with 95% O2 5% CO2. Flow rate approximately 1 bubble per second.
?. [Plating]
Following incubation, transfer tissue to biosafety cabinet. Add 5 ml of Glia Media to a 15 ml tube and transfer tissue, ensuring to transfer as little dissociation media as possible.Wash tissue 2-3x with 10 ml Glia Media, allowing tissue to settle between solution removal.After final wash, resuspend tissue in 5 ml Glia Media.Starting with large bore pipette, triturate tissue until it no longer decreases in size (10-20X) before switching to medium then small bore pipettes. Final appearance will be opalescent, indicating cells are in suspension. Pipette slowly to minimize the entrance of air bubbles and significant shear force. Centrifuge cell suspension for 5 minutes at 1550 RPM at room temperature.Remove supernatant and resuspend in 1 ml of Glia Media and pass through a 70 µm cell strainer.Aspirate 10 µl of cell suspension (ensure it does not settle) and mix thoroughly with 10 µl Trypan Blue Solution and count number of live cells / µl.Adjust cell concentration to 80,000 cells per 100 µl with Glia Media.Pipette 100 µl of suspension directly into the laminin coating solution on each coverslip, slowly and in a clockwise motion to ensure homogenous plating.Allow cells to settle for 2 hours in incubator set to 37ºC with 5% CO2.  Gently flood dishes with 2 ml of Neuronal Media supplemented with GDNF and kynurenic acid.Transduce neurons with AAV5-hSyn-GCaMP6f (number of genome copies per cell = 30,000).Allow cells to settle for 2 hours in incubator set to 37ºC with 5% CO2. Gently flood dishes with 2 ml of Neuronal Media supplemented with GDNF and kynurenic acid.Transduce neurons with AAV5-hSyn-GCaMP6f (number of genome copies per cell = 30,000).