5 mM

NaGTP, 0 2 mM EGTA, and 2 5 mM glutamate Drugs were

5 mM

NaGTP, 0.2 mM EGTA, and 2.5 mM glutamate. Drugs were applied to slices through the perfusion system unless otherwise noted. In the case of selective block of GABAergic input within glomeruli, gabazine (SR-95531; 100 μM) was puff applied to the indicated location through a patch pipette (7–10 MΩ resistance) with pressure supplied by a Picospritzer (Parker Instrumentation) set to 500 ms puff duration and 10 psi. AMPA receptor-mediated EPSCs were recorded by holding cells at −70mV, whereas mixed AMPA and NMDA receptor-mediated EPSCs were recorded at +40mV. In select experiments, we added the GABAA receptor blockers picrotoxin (PTX, 0.1 mM, Tocris) or gabazine (SR-95531, 10 μM, Tocris) in the ACSF to prevent ON-01910 purchase inhibitory responses. GABAA receptor-mediated IPSCs were recorded at 0mV, and for some cells both EPSCs and IPSCs were recorded at −40mV. ChR2 was activated in the entire optical

field of view using a custom-built illuminator (Albeanu et al., 2008). A super-bright light-emitting diode (LED) array (CBT-120B, Luminus Devices) was coupled to the rear lamp-housing of an Olympus BX51 upright microscope, with an intensity of 5–10 mW/mm2 in the sample plane. Stimulation sometimes elicited brief electric artifacts (from the LED power source) that were easily distinguished from synaptic Rucaparib nmr currents and were not affected by blockers. We relied on published characterization of juxtaglomerular cells to identify ETCs, PGCs, and SACs (Hayar et al., 2004; Gire and Schoppa, 2009; Shao et al., 2009). ETCs were identified in a few recordings based on their bursts of spikes in cell-attached recordings. They were mainly identified

based on their location in the border between glomeruli and EPL, their lower input resistance (194 ± 32 MΩ in Hayar et al., 2004) and the absence of spontaneous bursts of synaptic input (Figure S3; compare Figure 1 of Hayar et al., 2004). Conversely, Resminostat SACs and PGCs almost always have bursting spontaneous synaptic activity (Hayar et al., 2004). In addition, PGCs have much higher input resistance (1,054 ± 106 MΩ, in Hayar et al., 2004). There remains some uncertainty about lower input resistance SACs, but these will comprise a small fraction of our total sample. Responses were recorded with an Axopatch 200B amplifier (Molecular Devices), filtered at 2 kHz, and digitized at 20 kHz (Axon Digi1440A) using PClamp acquisition software (Molecular Devices). The recorded data were analyzed using Clampfit (Version 10.1.0.10, Molecular Devises). We used the peak amplitudes of synaptic currents recorded at −70mV to characterize AMPA EPSCs, and the amplitude at 50 ms to estimate the contribution of NMDA to EPSCs (AMPA currents are negligible at this time point) from the currents recorded at +40mV. Latencies were measured as time between light onset and the onset of synaptic currents, detected as a systematic deviation of more than 3 SDs from baseline noise.

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