Multiclamp 700a amplifier

We placed coverslips with cultured neurons in a perfusion chamber in which the extracellular media consisted of 150 mM NaCl, 4 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl₂, and 2 mM MgCl₂. We pulled glass pipettes with resistances of 3–5 MΩ, and filled the pipettes with intracellular solution consisting of 129 mM K-gluconate, 10 mM KCl, 10 mM HEPES, and 4 mM Na₂ATP. To characterize the Ca²⁺ response of sensors, we treated the cultured HEK293 cells with the same extracellular solution that included an additional 10 μM of ionomycin and 5 mM of Ca²⁺ to greatly increase the cytosolic Ca²⁺ concentration. We named the conditions before and after the ionomycin treatment as the Ca²⁺-resting condition and Ca²⁺-saturated condition, respectively.
We loose-patched cultured neurons using an Axon Digidata 1550 A digitizer (Axon Instruments), a Multiclamp 700 A amplifier (Axon Instruments), and pClamp (version 7.1) software at room temperature. We touched the pipettes on neurons and formed partial seals with resistances of 30–50 MΩ. We electrically stimulated neurons using 2 ms pulses at intervals of 500 ms and current amplitudes ranging from 300 to 1500 pA. We manually removed stimulus artifacts from the electrophysiology traces. Dual-view fluorescent imaging simultaneously recorded the intensity of patched neurons in the donor and acceptor channels at 10 Hz.

Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the hippocampal CA1 region by means of a glass microelectrode filled with 3 M NaCl (resistance 1–4 MΩ). The Schaffer collateral pathway was stimulated with concentric bipolar electrodes (Frederick Haer Co, Bowdoinham, ME, USA). After an optimal fEPSP wave was found, an input–output (I/O) curve was established with stimulating pulses (0.2 ms duration) at different intensities. Baseline fEPSP was recorded at 0.033 Hz with a stimulating strength adjusted to yield about 40% of the maximal response. After baseline responses had stably lasted for at least 30 min, LTP was induced by delivering theta burst stimuli (TBS; four trains of 10 bursts of four stimuli with 20 s, 200 ms, and 10 ms intervals between trains, bursts, and stimuli, respectively). The electrophysiological data was acquired with a multiclamp 700 A amplifier (Axon instruments, Molecular Devices, USA), filtered at 0.1–5 KHz, digitized at 10 KHz, and analyzed with Clampfit version 10.0 (Axon Instruments, USA). The analyzed data was further processed with Origin 5.1 (Microcal Software Northampton, MA).

Patch-clamp experiments in transiently transfected HEK-P1KO cells were performed in standard whole-cell and cell-attached mode using a Multi-clamp700A amplifier (Axon Instruments) and followed the procedures described in our previous report^{2 (link)}.