Osmolarity

The soma and axonal bleb of identical pyramidal neurons in layers IV-V of cerebral cortex were simultaneously recorded (MultiClapm-700B, Axon Instrument Inc. USA) under a fluorescent and DIC microscope (Nikon FN-E600; [14 (link)]. Electrical signals were inputted into pClamp-10 with 50 kHz sampling rate. In whole-cell recording, action potentials were induced by the signals recorded intracellularly in vivo. The judgment for recording two sites from an identical neuron is based on the synchronous presence of direct and corresponding electrical signals. Transient capacitance was compensated. Output bandwidth was 3 kHz. Pipette solution contains (mM) 150 K-gluconate, 5 NaCl, 0.4 EGTA, 4 Mg-ATP, 0.5 Tris- GTP, 4 Na-phosphocreatine and 10 HEPES (pH 7.4 adjusted by 2M KOH). The osmolarity of pipette solution was 295-305 mOsmol. The pipette resistance was 10-15 MΩ.
Neuronal intrinsic properties include spike thresholds (Vts) and refractory periods (RP). Vts were measured by depolarization pulses. RPs were measured by injecting two pulses (5% above threshold) into neurons after each spike under current-clamp, in which inter-pulse intervals were adjusted [12 (link),13 ,25 (link),54 (link),55 (link)]. The duration of pulses was 50 ms, the minimal time period of in vivo signals (Figure 1C)
Latencies between axonal spikes and somatic ones, used to judge spike initiation, were measured based on the following thoughts. Elements in an electrical circuit of cell membrane includes voltage- gated conductance (Rv) for the generation of active signals, such as action potentials and synaptic signals, as well as passive membrane properties (input resistance, Rin; membrane capacitance, Cm; inset in Figure 3A). We ruled out the effects of Rin and Cm on the analyses of temporal signals via subtracting the responses (gray lines in 3A) evoked by depolarization and hyperpolarization in the same intensities, such that spike potentials (black line in Figure 3A) were mediated by voltage-gated channels. The derivative of somatic and axonal spike potentials vs. time (dv/dt) was calculated. The site of spike initiation was defined as a time point with a minimal dv/dt but larger than zero (Figure 3B), which accurately represents the locus of spike initiation in the comparison with the peak, 50% rising phase or initial phase (onset point) of spikes [20 (link)-24 ,50 (link),56 (link)-58 (link)]. Latencies between somatic spikes and axonal ones were the time difference of their initiation (ΔT = Tsoma-Taxon).

Single slices were transferred to a recording chamber that was constantly perfused (∼3 mL/min) with oxygenated aCSF at 35 °C. The CA1 neurons were visualized under a Zeiss upright microscope (40 X water-immersion objective) and an enhanced differential interference contrast (DIC) video microscope system. Recording pipettes with the resistance of 3–5 MΩ were pulled from borosilicate glass capillaries (1.5 mm outer diameter) using a P97 electrode puller (Sutter Instruments, Novato, CA). Access resistance and input capacitance were electronically compensated by ∼60–70% and monitored throughout the experiment to confirm the stability of the recording.
Fast spontaneous inhibitory postsynaptic currents (sIPSCs) mediated by the GABA_A receptor were recorded from hippocampal CA1 pyramidal neurons held at −70 mV in the presence of 2-amino-5-phosphonovaleric acid (APV)(50 μM), 6-cyano-7-nitroquinoxaline-2,3-dione (DNQX) (20 μM), and CGP55845A (1 μM). The internal solution contained the following (in mM): Cs-gluconate 130, CsCl 10, EGTA 0.2, Mg ATP 4, Tri-GTP 0.3, HEPES 10, and QX-314, 4 The pH was adjusted to 7.4 with CsOH, and the osmolarity was 290 mOsm. In all instances, the recordings of spontaneous GABAergic IPSCs usually began at least 5 min after a whole-cell configuration was established with a stable baseline. Spontaneous IPSCs were completely blocked with bath-applied bicuculline methiodide (BMI, 20 μM), confirming that they are mediated by GABA_A receptors. Tonic currents were isolated after bath application of the GABA_A receptor antagonist picrotoxin (100 μM).
Data were recorded with a MultiClamp 700B amplifier, filtered at 10 kHz, and digitized at 20 kHz through a Digidata 1440 interface controlled by pClamp10.7 software (Molecular Devices, CA). Both the frequencies and amplitudes of CA1 sIPSCs were analyzed using Clampfit 10.7 software, and the threshold for detecting sIPSCs was used and followed by visual inspection to ensure the accuracy of detection.

Cells were plated on glass coverslips at very low density to obtain isolated single cells, incubated overnight in culture medium and used between 40 and 50 hs after plating. Coverslips were attached with Vaseline to the perforated bottom of 4–5 mL plastic chambers (35 mm cell culture dish caps) and placed on the stage of an upright microscope (BX50WI, Olympus) for patch-clamp work. Two discontinuous single-electrode voltage-clamp (DSEVC) amplifiers (SEC-05LX NPI, Germany) were used to simultaneously clamp the voltage and record membrane currents from two single cells in whole-cell voltage clamp (WCVC) mode, or from a single cell with one electrode in WCVC and the second in cell-attached voltage clamp (CAVC) mode. In some experiments, WC configuration was achieved but voltage clamp was not performed: instead, the resting membrane potential (Vm) was documented using the amplifiers’ bridge mode (BR) function. Cells were bathed in external solution (in mM: NaCl 140, KCl 4.7, CaCl₂ 1.8, MgCl₂ 1.2, Glucose 10, EGTA 0.1, HEPES 10) adjusted to pH = 7.2 and osmolarity 319–330 mOsm. Patch pipettes were filled with internal (in mM: KCl 138, MgCl₂ 3, TEACl 9, CaCl₂ 0.5, EGTA 9, Glucose 5, Na₂ ATP 5, HEPES 9; adjusted to pH = 7.4 and osmolarity 315–319 mOsm) or external solution.
Pulsing protocols were as follows: “steady HP”, continuously voltage clamping at any holding potential (HP); “−70 to +80 mV”, from an HP = −70 mV, 600 ms, +80 mV square pulses applied every 10–30 s; “±100 mV ramps”, from and HP = −50 mV, voltage ramps from −100 to +100 mV (slope ∼32 mV per sec) applied every 10–30 s; “step or IV”, from HP = −70 mV, 600 ms square pulses from −80 to +80 mV in 20 mV steps, every 5.5 s; “±80 paired pulses”, from HP = 0 mV, 10 s pulses to −80 and +80 mV, preceded by 100 ms prepulses at ±10 mV, every 30 s; “long pulses”, HP at any given value, usually −80, +80 mV or 0 mV, held for variable periods >1 s. Recordings were acquired at 13 kHz and low pass filtered at 1 kHz. Channel recordings were further filtered (100–200 Hz) and decimated (10–50) for ease of display. Macroscopic Im was analyzed with Clampfit10 (Molecular Probes) and Excel (Microsoft). For single channel event amplitude, long traces were surveyed with the Histogram function of Clampfit10 to locate sections with discrete transitions, then transferred those sections to Excel to produce refined all-points histograms with bin sizes of 0.25–0.5 pS; traces and histograms were plotted with SigmaPlot (SPSS Inc.). When suitable, results are reported as average ±SEM.