GCaMP variants were made in a modified SIV-based lentiviral construct, pGP-syn-GCaMP-nls-mCherry-WPRE, derived from pCL20cSLFR MSCV-GFP51 (link). The prolentiviral vector included a 476-bp human synapsin promoter, GCaMP, a nuclear localization sequence fused to mCherry, and the woodchuck hepatitis post-transcriptional regulatory element. Site-directed mutagenesis was conducted by PCR and mutated regions were incorporated into the lentiviral constructs by gene assembly52 (link).
Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 μg/mL transferrin, B-27 supplement (1X, Invitrogen), 500 μM L-glutamine, 50 units/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum in MEM).
Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G)51 (link),53 (link) into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal cultures were infected at 3 days in vitro. Each well of a 24-well plate was incubated overnight with 0.5 mL of lentivirus in conditioned growth medium. The growth medium was supplemented with 4 μM AraC to inhibit glial proliferation. In some experiments, OGB1-AM was loaded into cells by incubating neurons in 1 mL of 2 μM OGB1-AM (Invitrogen) for 30 min and rinsing 3 times with imaging buffer (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 2 mM CaCl2, 1 mM MgCl2).
Neurons were stimulated in imaging buffer containing a drug cocktail to inhibit synaptic receptors (10 μM CNQX, 10 μM (R)-CPP, 10 μM gabazine, 1 mM (S)-MCPG, Tocris). Under these conditions, intracellular calcium increases are presumably caused by the opening of voltage sensitive calcium channels.
Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10× (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 μm × 800 μm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F0 response over the standard deviation of the signal during a one second period prior to stimulation.
Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor54 (link) confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system was controlled by custom scripts written in MetaMorph software (version 7.7.5, Molecular Devices) and Ephus software55 (link) (ephus.org). Detailed neuronal culture screening methods will be described elsewhere (T.J.W., T.W.C., E.R.S., R.A.K., V.J., L.L.L., K.S., and D.S.K., manuscript in preparation).
Hippocampi were dissected and dissociated in papain. Cells were plated at a density of 225,000 viable cells/well in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips), pre-coated with Matrigel (BD Biosciences). Cells were cultured in growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 μg/mL transferrin, B-27 supplement (1X, Invitrogen), 500 μM L-glutamine, 50 units/mL penicillin, 50 mg/mL streptomycin, 5% fetal bovine serum in MEM).
Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G)51 (link),53 (link) into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal cultures were infected at 3 days in vitro. Each well of a 24-well plate was incubated overnight with 0.5 mL of lentivirus in conditioned growth medium. The growth medium was supplemented with 4 μM AraC to inhibit glial proliferation. In some experiments, OGB1-AM was loaded into cells by incubating neurons in 1 mL of 2 μM OGB1-AM (Invitrogen) for 30 min and rinsing 3 times with imaging buffer (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 2 mM CaCl2, 1 mM MgCl2).
Neurons were stimulated in imaging buffer containing a drug cocktail to inhibit synaptic receptors (10 μM CNQX, 10 μM (R)-CPP, 10 μM gabazine, 1 mM (S)-MCPG, Tocris). Under these conditions, intracellular calcium increases are presumably caused by the opening of voltage sensitive calcium channels.
Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10× (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 μm × 800 μm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F0 response over the standard deviation of the signal during a one second period prior to stimulation.
Control experiments varying stimulation voltage, frequency, and pulse width insured suprathreshold stimulation of neurons. Voltage imaging using the ArchWT-GFP archaerhodopsin-based voltage sensor54 (link) confirmed that individual pulses (1 ms, 40 V, 83 Hz) reliably triggered single APs. The imaging and stimulation system was controlled by custom scripts written in MetaMorph software (version 7.7.5, Molecular Devices) and Ephus software55 (link) (ephus.org). Detailed neuronal culture screening methods will be described elsewhere (T.J.W., T.W.C., E.R.S., R.A.K., V.J., L.L.L., K.S., and D.S.K., manuscript in preparation).
