TITLE: Live Imaging of Primary Mouse Neuron Cultures
AUTHORS: Robert Edwards, Shweta Jain

[DESCRIPTION]
This protocol describes live imaging of primary neuron cultures. Included are methods for preparing hippocampal or dopamine neuronal cultures from neonatal mouse brain tissue. The imaging described involves labeling of dopamine neurons with a fluorescent DAT ligand and using virally encoded pHlourin sensors to measure vesicular release of neurotransmitter as the neurons are electrically stimulated. This technique was used in Jain et al., 2023 to compare vesicular release in neurons between various transgenic knockout mouse lines.

[STEPS]
SECTION: Preparation of Hippocampal Cultures
2. Collect mouse pups at postnatal day 0

SECTION: Preparation of Hippocampal Cultures
3. Decapitate and remove brain, dissect out hippocampi from each hemisphere into Hank's balanced salt solution (HBSS) containing 10 mM HEPES and 20 mM glucose


SECTION: Preparation of Hippocampal Cultures
4. Digest with papain (20 units/mL) in papain digestion buffer (HBSS with 20 mM CaCl2, 5 mM EDTA, 0.2 mg/mL L-Cysteine, 10 mM HEPES at pH 7.4) for 15 minutes
SECTION: Preparation of Hippocampal Cultures
5. Wash hippocampi three times with HBSS containing 10 mM HEPES and 20 mM glucose
SECTION: Preparation of Hippocampal Cultures
6. Triturate hippocampi to get a single cell suspension
SECTION: Preparation of Hippocampal Cultures
1. Coat coverslips with Poly-L-lysine
SECTION: Preparation of Hippocampal Cultures
7. Using a hemocytometer, count the density of cells in the suspension
SECTION: Preparation of Hippocampal Cultures
8. Plate cells onto the poly-L-lysine-coated coverslip at a density of 350 cells/mm2 in Minimal Essential Medium containing:
1X B27 supplement  
5% Fetal Bovine Serum (FBS)  
21 mM glucose   

SECTION: Preparation of Hippocampal Cultures
10. After day 1 in vitro (DIV1), replace 3/4 of the medium with Neurobasal medium    containing:
1X B27 supplement  
GlutaMAX   

SECTION: Preparation of Hippocampal Cultures
2.1. For Jain et al., 2023, cultures were prepared from WT, β3A KO, β3B KO, and mocha mice
SECTION: Preparation of Hippocampal Cultures
11. Infect cells at DIV4-5 using lentiviral vectors
SECTION: Preparation of Hippocampal Cultures
11.1. For Jain et al., 2023, viruses encoding VGLUT2-pH, VMAT2-pH, or VGAT-pH were used
SECTION: Preparation of Hippocampal Cultures
12. At DIV 6-7, inhibit glial proliferation by treating cultures with 4 µM cytosine arabinoside (Ara-C)
SECTION: Preparation of Hippocampal Cultures
9. Store cultures in incubator at 37 °C 

SECTION: Preparation of Dopamine Neuron Cultures
13. Coat coverslips with poly-L-lysine and laminin; plate astrocyte monolayer onto coverslips
SECTION: Preparation of Dopamine Neuron Cultures
14. Collect mouse pups at postnatal day 0
SECTION: Preparation of Dopamine Neuron Cultures
15. Decapitate and remove brain, dissect out midbrain (including ventral tegmental area and substantia nigra) from each hemisphere into Hank's balanced salt solution (HBSS) containing 10 mM HEPES and 20 mM glucose
SECTION: Preparation of Dopamine Neuron Cultures
16. Digest with papain (20 units/mL) in papain digestion buffer (HBSS with 20 mM CaCl2, 5 mM EDTA, 0.2 mg/mL L-Cysteine, 10 mM HEPES at pH 7.4) for 15 minutes
SECTION: Preparation of Dopamine Neuron Cultures
17. Wash digested tissue three times with HBSS containing 10 mM HEPES and 20 mM glucose
SECTION: Preparation of Dopamine Neuron Cultures
18. Triturate tissue to get a single cell suspension
SECTION: Preparation of Dopamine Neuron Cultures
19. Using a hemocytometer, count the density of cells in the suspension
SECTION: Preparation of Dopamine Neuron Cultures
20. Plate cells onto the pre-prepared coverslips at a density of 1000 cells/mm2 in medium consisting of 60% Neurobasal Medium  , 30 % Basal Eagle Medium   , 10% FBS    containing:
1X B27 supplement  
2 mM GlutaMAX    
10 ng/mL glial cell line-derived neurotrophic factor (GDNF)  
1X penicillin/streptomycin




SECTION: Preparation of Dopamine Neuron Cultures
21. At DIV 2-4, infect with lentivirus
SECTION: Preparation of Dopamine Neuron Cultures
21.1. For Jain et al., 2023, cells were infected with lentivirus encoding VMAT2-pH or VGLUT2-pH
SECTION: Live Imaging
22. Maintain cultures until ready for imaging. Live imaging of cultures is performed at DIV14-16 for hippocampal cultures and DIV13-15 for dopamine neuron cultures
SECTION: Live Imaging
24. For midbrain cultures, label dopamine neurons, incubate dopamine neuron cultures in Tyrode's buffer (119 mM NaCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2, 2.5 mM KCl, and 30 mM
SECTION: Live Imaging
24.1. Make Tyrode's +JHC1-64 by adding the fluorescent DAT ligand JHC1-64 at 30 nM to Tyrode's buffer
SECTION: Live Imaging
24.2. Incubate dopamine neuron culture in Tyrode's +JHC1-64 for 5 minutes at Room temperature 

SECTION: Live Imaging
24.3. Rinse neurons in Tyrode's buffer (no JHC 1-64)
SECTION: Live Imaging
23. Prepare Tyrode's buffer:
119 mM NaCl
25 mM HEPES 
2 mM CaCl2
2 mM MgCl2 
2.5 mM KCl 
30 mM glucose
Adjust pH to 7.4
SECTION: Live Imaging
25. Add fresh Tyrode's buffer to culture dishes, mount coverslips in a perfusion and stimulation chamber of a TE300 inverted epifluorescence microscope
SECTION: Live Imaging
26. Monitor fluorescence: 
For pHlourin, use 470/40 nm excitation and 525/70 nm emission bandpass filters
For JHC1-64, use 545/25 nm excitation and 605/70 nm emission bandpass filters

Use the red channel to identify dopamine neurons then switch to the pHlourin channel for experiments
SECTION: Live Imaging
28. Acquire images, typically at 1 Hz except for Fluo-5F imagine where images are acquired at 10 Hz
SECTION: Live Imaging
27. Place platinum-iridium stimulating electrodes near the dopamine neuron being observed 
SECTION: Live Imaging
29. While acquiring images, evoke action potentials using 1 ms bipolar current pulses through the stimulating electrode at 5-10 V/cm