Dec 21, 2023

Public workspaceLive Imaging of Primary Mouse Neuron Cultures

DOI
dx.doi.org/10.17504/protocols.io.e6nvwdjydlmk/v1
  • Robert Edwards1,
  • Shweta Jain1
  • 1University of California San Francisco
Kelsey Barcomb
Open access
DOI: dx.doi.org/10.17504/protocols.io.e6nvwdjydlmk/v1
Protocol Citation: Robert Edwards, Shweta Jain 2023. Live Imaging of Primary Mouse Neuron Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdjydlmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86286
Keywords: ASAPCRN, Cell Culture, Neuron, Mouse, Live Imaging, Microscopy, Vesicle Release
Funders Acknowledgement:
ASAP-CRN
Grant ID: 020529
Abstract
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.
Protocol materials
ReagentB27 supplement without retinoic acid (50x)Gibco, ThermoFisherCatalog #17504044
In 3 steps
ReagentNeurobasal™ MediumGibco - Thermo FisherCatalog #21103049
In 2 steps
ReagentGlutamax (100x)Gibco - Thermo FischerCatalog #35050-061
In 2 steps
ReagentBasal Medium Eagle (BME)Thermo FisherCatalog #21010046
Step 20
ReagentGDNF Recombinant Human ProteinThermo FisherCatalog #PHC7045
Step 20
ReagentHyClone Fetal Bovine SerumFisher ScientificCatalog #SH3039603
In 2 steps
ReagentD-( )-Glucose solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8769
Step 8
Preparation of Hippocampal Cultures
Preparation of Hippocampal Cultures
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 supplementReagentB27 supplement without retinoic acid (50x)Gibco, ThermoFisherCatalog #17504044
  • 5% Fetal Bovine Serum (FBS)ReagentHyClone Fetal Bovine SerumFisher ScientificCatalog #SH3039603
  • 21 mM glucose ReagentD-(+)-Glucose solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #G8769

Store cultures in incubator at Temperature37 °C

After day 1 in vitro (DIV1), replace 3/4 of the medium with Neurobasal medium ReagentNeurobasal™ MediumGibco - Thermo FisherCatalog #21103049 containing:
  • 1X B27 supplementReagentB27 supplement without retinoic acid (50x)Gibco, ThermoFisherCatalog #17504044
  • GlutaMAX ReagentGlutamax (100x)Gibco - Thermo FischerCatalog #35050-061

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)
Preparation of Dopamine Neuron Cultures
Preparation of Dopamine Neuron Cultures
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 MediumReagentNeurobasal™ MediumGibco - Thermo FisherCatalog #21103049 , 30 % Basal Eagle Medium ReagentBasal Medium Eagle (BME)Thermo FisherCatalog #21010046 , 10% FBS ReagentHyClone Fetal Bovine SerumFisher ScientificCatalog #SH3039603 containing:
  • 1X B27 supplementReagentB27 supplement without retinoic acid (50x)Gibco, ThermoFisherCatalog #17504044
  • 2 mM GlutaMAX ReagentGlutamax (100x)Gibco - Thermo FischerCatalog #35050-061
  • 10 ng/mL glial cell line-derived neurotrophic factor (GDNF)ReagentGDNF Recombinant Human ProteinThermo FisherCatalog #PHC7045
  • 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
Live Imaging
Live Imaging
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 TemperatureRoom 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