5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, and 1 mM MgCl2 (pH 7.4). Slices were then kept at room temperature until they were transferred to a recording chamber and continuously superfused with oxygenated standard ACSF. All recordings were made at 33°C ± 1°C from slices that had been maintained
at this temperature for at least 20 min (in light of the fact that gap junctional coupling is temperature sensitive [Payton et al., 1969]). Patch pipettes were pulled from borosilicate glass on a PC-10 puller (Narishige) to a resistance of 5 MΩ for somatic recordings. The internal solution contained 130 mM KMeSO4, 7 mM KCl, 0.1 mM EGTA, 2 mM Na2ATP, 2 mM MgATP, and 0.3 mM Na2GTP (pH 7.2) (0.5% biocytin was included in some recordings). For voltage-clamp experiments, we used the same internal,
except that KMeSO4 Dabrafenib solubility dmso was replaced with 130 mM CsMeSO3. Olivary neurons in the principal and medial accessory olives were identified using infrared-DIC optics and somatic patch-clamp recordings were find more made under direct visual control (Stuart et al., 1993 and Mathy et al., 2009), with most recordings being made from the principal olive. We selected somata within 50 μm of each other to maximize the probability of obtaining coupled cells (Devor and Yarom, 2002). Cells were rejected if their resting potential was depolarized to −40 mV. Coupling was assessed in current-clamp mode by injecting negative current pulses alternately (from −400 to −800 pA, every 4 to 6 s) in both cells. Bridge balance was monitored and corrected online and offline. For stimulation of synaptic inputs, a bipolar tungsten electrode (stimulus duration 50–100 μs) was placed in the white matter at the border of the olivary subnucleus from which the recording was obtained. During induction in current clamp, continuous depolarizing current was used to maintain a baseline membrane potential of 44 ± 1 mV,
Cytidine deaminase ensuring that cells fired with the physiological burst pattern seen in vivo and enhancing relief of magnesium block of NMDA receptors in experiments where spiking was not triggered by depolarizing current steps. Each slice was only used for one paired recording. To assess the strength of chemical synapses, we injected a 300 ms hyperpolarizing pulse (1 nA) in both cells and triggered an EPSP when the voltage had reached steady state. Biocytin processing was performed using standard techniques (Horikawa and Armstrong, 1988), with overnight paraformaldehyde (0.5%) fixation followed by conjugation with streptavidin-Alexa Fluor 488 (Invitrogen). Filled neurons were imaged using a 40× oil-immersion objective (NA 1.3) on a spinning-disk confocal microscope (Perkin-Elmer). Data were low-pass filtered at 3–10 kHz and acquired at 20–100 kHz using an ITC-18 board (Instrutech) in conjunction with AxoGraph (AxoGraph Scientific) software. Analysis was carried out using custom-written software for MATLAB (MathWorks) and Igor Pro (Wavemetrics).