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 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.
Preparation Preceding the Culture
Note: ** Glial cells must be plated well in advance so that they have time to proliferate and cover the bottom of the dishes. For mice with atypical or experimental genetic background, make sure to match glial and neuronal cultures in terms of genotype.
Note: ** 1-7 days before dissection, prepare fresh neuronal medium and replace the glial medium in the dishes with 2ml.
Note: ** On the day before dissection, the following items need to be left under UV light overnight:
Day of Culture
Note: * BE SURE NOT TO TOUCH ANYTHING THAT WAS LEFT OUT FOR UV OVERNIGHT!
Maintaining a sterile environment under the hood is very important.
1) Set Up:
Note: ** Set aside all frozen ingredients to make papain solution.
2) Dissection
3) Trituration:
4) Plating Cells
5) Mitotic Inhibition: THE NEXT DAY
MEDIA/SOLUTIONS
Neuronal Medium
(for 200mL)
**Best if conditioned on glia from flasks overnight before use for washes/trituration
Ingredient | Amount | Notes |
BSA 5% | 0.5 g | Fraction V |
MEM liquid | 94.0 ml | Sigma |
DMEM liquid | 80.0 ml | Sigma |
F-12 liquid | 20.0 ml | Sigma |
Glucose 45% liquid | 1.50 ml | Sigma solution |
Glutamine 200mM | 0.5 ml | Aliquotted Sigma solution |
Diporzio Conc. | 2.0 ml | Sigma solution |
Liquid Catalase | 0.1 ml | |
Kynurenic acid 0.5M | 200μl | In 1N NaOH |
HCL 5N | 50 μl |
Kynurenic Acid
(FW = 189.2)
0.5M = 94.6mg/ml
Make 8ml stock: 756.8mgKA/8ml 1N NaOH and pipette into STERILE aliquots of 200μl
DiPorzio Media
A) DiPorzio Conc. Stocks:
NEED | Combine | Alliquot | ||||||
Additive | Solvent | Tube | Amount | ml | ml/tube | Conc. | Amount | # Aliq. |
Insulin | 20mM HCL (1) | plastic | 250mg bottle | 10 | 1 | 25mg/ml | 25mg | 10 |
Transferrin | Hank’s | 500mg bottle | 5 | 1 | 100mg/ml | 100mg | 5 | |
SOD | Hank’s | 70mg bottle | 14 | 1 | 5mg/ml | 5mg | 14 | |
Putrescine | Hank’s | 50mg | 3 | 0.12 | 20mg/ml | 2.4mg | 21 | |
Na2SeO3 | Hank’s | 0.104mg | 10 | 0.5 | 10μg/ml | 5.2μg | 20 | |
T3 | 10mM NaOH | 2mg | 10 | 0.1 | 0.20mg/ml | 0mg | 100 | |
Progesterone | 100% EtOH | Glass | 12.5mg | 10 | 0.05 | 1.25mg/ml | Use pipet | |
Cortisol | 100% EtOH | Glass | 20mg | 10 | 0.02 | 2.00mg/ ml | Use pipet |
B) DiPorzio Media Stock:
Additive | Amount (ml) | Amount (ml) X2 | Final Conc. μg/ml | Final Conc. Molarity |
Progesterone | 0.05 | 0.1 | 0.06 | 200nM |
Cortisol | 0.02 | 0.04 | 0.04 | 125nM |
Hank’s BSS | 6.21 | 12.42 | ||
Insulin | 1 | 2 | 25 | |
Na2SeO3 | 0.5 | 1 | 0.01 | 30nM |
T3 | 0.1 | 0.2 | 0.02 | 30nM |
SOD | 1 | 2 | 5 | |
Putrescine | 0.12 | 0.24 | 2.4 | 15nM |
Transferrin | 1 | 2 | 100 |
Dissociation Media
A. Papain (for 1 vial):
**Prepare day of plating right before dissection.
Ingredient | Amount | (Notes) Final Conc. |
Cysteine Water | 7.8mL | 1mM cysteine |
Papain | Varies (*190μl for the bottle of 44.0mg/ml) |
20 units/ml (400 units total for 20ml worth of sol’n) |
H&B conc. | 2mL | |
Carbogen | 95% O2 + 5% CO2 | |
HCl 5N | 10μl | |
0.5% Phenol red | 20μl | 0.001% |
Kynurenate 0.5M | 10μl | (In 1N NaOH) 0.5mM |
B. H&B Concentrate (100ml of 5X):
Ingredients | M.W. | Powder/50ml H2O | Conc. (M) | Combine (ml) | Final Conc. (mM) |
NaCl | 58.44 | 11.699 g | 4 | 14.5 | 116 |
KCl | 74.56 | 3.728 g | 1 | 2.7 | 5.4 |
NaHCO3 | 84.01 | 4.2 g | 1 | 13 | 26 |
NaH2PO4*H2O | 137.99 | 6.90 g | 1 | 1 | 2 |
MgSO4 | 120.38 | 6.019 g | 1 | 0.5 | 1 |
EDTA (ED2- SS) |
292 | Sigma 5% (make 5g/100ml stock) |
0.134 | 0.3722 | 0.5 |
Glucose | 180 | Sigma 45% | 2.5 | 5 | 25 |
TC H2O | 62.93 |
C. Cysteine Water (157.5ml of 1X):
Ingredients | M.W. | Components Powder (mg) |
H2O (ml) | Stock Conc.(mM) | Combine (ml) | Final Conc. (mM) |
CaCl2 | 147.2 | 736 | 10 | 500 | 0.6 | 1.9mM |
Cysteine | 121.7 | (1.5) | 24 | 20mM (0.02M) | 10 | 1.27mM (0.88) |
TC water | 146.9 |
GDNF Preparation
FDU Preparation
FDU-sol’n 1000X Stock:
Ingredient | Amount | (Notes) Final Conc. |
Uridine | 247 mg | 16.5 mg/ml |
5-FDU (5-fluorodeoxyuridine) | 100 mg (bottle) | 6.7 mg/ml |
TC Water | 15 ml |
Dilute for Use:
The methods described here allow for fine resolution of morphological and neurochemical features of central dopamine neurons that are otherwise not available in a systemic or in vivo approach.
The work was supported by DK065872 (ENP), a Smith Family Foundation Award of Excellence in Biomedical Research (ENP), F31 DA023760 (BMG, ENP) and P30 NS047243 (Tufts Center for Neuroscience Research).