Electrophysiology
HEK293T cells, seeded in 35 mm diameter plastic dishes at low confluency
(1x104 cells) and transfected with
pcDNA3.1-Myc-mGlu5-Venus (0.25 µg) and pCAGGS-TRPC6-ires2-mbtdtomato
(0.5 µg) plasmids, were targeted by fluorescence microscopy.
Measurements were performed in the whole cell configuration, in voltage
or current clamp mode, with pipettes of 2-4 MΩ resistance when filled
with the appropriate intracellular medium.
At 10 to 12 days in vitro, hippocampal neurons were used for endogenous
NMDA current measurements. Patch clamp recordings were performed in the
whole-cell configuration with pipettes of 3-5 MΩ resistance when filled
with the appropriate intracellular medium. NMDA stimulations were
separated by 2 min to avoid receptor desensitization. All recordings
were performed at room temperature for less than 1 h per dish. Drugs
were applied using a gravity perfusion system allowing complete exchange
of the cellular environment in less than 30ms. Currents were recorded
using an Axopatch 200B amplifier (Axon Instruments), with noise removal
at 50-60 Hz (Hum Bug Noise, Quest Scientific) and digitized at 3 kHz
before being stored in Clampex software (version 8.1). The data were
then analyzed using the Clampfit 11.2 software from Axon instruments
(Molecular Devices).