Rc 49mfsh

Cultured neurons on coverslips were placed in a stimulation chamber (RC-49MFSH, Warner Instruments) and stimulated at an intensity of 10 V/cm using a stimulus isolation unit (SIU-102B, Warner Instruments). Stimulation duration and frequency was controlled by an isolated pulse stimulator (2100, A-M Systems). Neurons were imaged in standard extracellular saline containing (in mM): NaCl 150, KCl 3, Glucose 20, HEPES 10, CaCl₂ 2, MgCl₂ 3, pH adjusted to 7.35 with NaOH, 310 mOsm; unless indicated otherwise.

Live-cell fluorescent imaging of pHluorin-expressing neurons was carried out under the same conditions as iGluSnFR imaging. Briefly, images were acquired on an Olympus IX83 inverted microscope equipped with a cellTIRF 4Line excitation system using an Olympus 60×/1.49 Apo N objective and an Orca Flash4.0 CMOS camera (Hamamatsu Photonics). This microscope runs Metamorph software with Olympus 7.8.6.0 acquisition software from Molecular Devices. The imaging media was extracellular fluid with 2 mM CaCl₂. Single image planes were acquired with 500-ms exposure using a white organic light-emitting diode with standard green fluorescent protein filters. Images were collected once a second for 3 min. A stimulation train was started 9 s into imaging. The trains (200 stimuli in 10 s [20 Hz]) were triggered by a Grass SD9 stimulator through platinum parallel wires attached to a field stimulation chamber (Warner Instruments; RC-49MFSH). All biosensor imaging experiments were performed at 32 to 34 °C. The environment was controlled by a Tokai incubation controller and chamber.

Mature hippocampal neurons (20–22 DIV) were incubated with 1 µL Fluo-4 AM (Thermo Fisher, Life Technologies Corporation, Carlsbad, CA, USA) for 30 min. The neurons were transferred to a RC-49MFSH magnetic imaging/recording chamber with removable electrodes (Warner Instruments, Hamden, CT, USA) and covered with 900 μL of 1× Tyrodes buffer (119 mM NaCl, 2.5 mM KCl, 25 mM HEPES, 30 mM Glucose, 2 mM MgCl₂ and 2 mM CaCl₂, all from Sigma-Aldrich, St. Louis, MO, USA) at 32 °C. An inverted microscope (Observer. D1; Zeiss, Oberkochen, Germany) equipped with a 63×/1.2 N.A. objective, GFP/RFP single band exciters ET filter set (excitation 470/40, excitation 572/35, emission 590/22, dichroic 59022BS), and an EMCCD camera (Evolve 512 Delta; Photometrics, Tucson, AZ, USA) controlled by VisiView^® Software (Visitron Systems GmbH, Puchheim, Germany) was used to perform live imaging (33 Hz acquisition rate). For each experiment, the somatic calcium transients were evoked electrically by extracellular depolarization using 5 pulses (1 ms duration each) at 20 Hz at 32 °C. Somatic calcium transient was obtained in basal condition (control) for each neuron. After 2 min of resting, neurons were incubated with a purified IgG monoclonal anti-GM1 antibody (1 μg/mL; [66 (link),68 (link)]) dissolved in 1× Tyrodes buffer (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and stimulated again.