Coat coverslips with Poly-L-lysine

Collect mouse pups at postnatal day 0

For Jain et al., 2023, cultures were prepared from WT, β3A KO, β3B KO, and mocha mice

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

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

Wash hippocampi three times with HBSS containing 10 mM HEPES and 20 mM glucose

Triturate hippocampi to get a single cell suspension

Using a hemocytometer, count the density of cells in the suspension

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

Store cultures in incubator at 37°C

After day 1 in vitro (DIV1), replace 3/4 of the medium with Neurobasal medium containing:

• 1X B27 supplement
• GlutaMAX

Infect cells at DIV4-5 using lentiviral vectors

For Jain et al., 2023, viruses encoding VGLUT2-pH, VMAT2-pH, or VGAT-pH were used

At DIV 6-7, inhibit glial proliferation by treating cultures with 4 µM cytosine arabinoside (Ara-C)

Coat coverslips with poly-L-lysine and laminin; plate astrocyte monolayer onto coverslips

Collect mouse pups at postnatal day 0

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

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

Wash digested tissue three times with HBSS containing 10 mM HEPES and 20 mM glucose

Triturate tissue to get a single cell suspension

Using a hemocytometer, count the density of cells in the suspension

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

At DIV 2-4, infect with lentivirus

For Jain et al., 2023, cells were infected with lentivirus encoding VMAT2-pH or VGLUT2-pH

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

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

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

Make Tyrode's +JHC1-64 by adding the fluorescent DAT ligand JHC1-64 at 30 nM to Tyrode's buffer

Incubate dopamine neuron culture in Tyrode's +JHC1-64 for 5 minutes at Room temperature

Rinse neurons in Tyrode's buffer (no JHC 1-64)

Add fresh Tyrode's buffer to culture dishes, mount coverslips in a perfusion and stimulation chamber of a TE300 inverted epifluorescence microscope

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

Place platinum-iridium stimulating electrodes near the dopamine neuron being observed

Acquire images, typically at 1 Hz except for Fluo-5F imagine where images are acquired at 10 Hz

While acquiring images, evoke action potentials using 1 ms bipolar current pulses through the stimulating electrode at 5-10 V/cm