Whole-cell recordings
Slices were visualized using the Olympus BX51WI microscope equipped with Olympus 5x and 60x water immersion lens and the Andor Neo sCMOS camera (Oxford Instruments, Abingdon, Oxfordshire, UK). In most cases, neurons were patched randomly within layers 2/3 of RSG with the exception of experiments in which PV neurons were targeted for patching based on their expression of either an eYFP tag (PV-IRES-Cre x Ai32 cross) or a tdTomato tag (PV-IRES-CRE x Ai14 cross). All recordings were done under current clamp conditions using the Multiclamp 700B and Digidata 1400 (Molecular Devices). Neurons were adjusted for series resistances and held at a resting potential of -65 mV (unless otherwise stated) using a constant holding current injection. In order to characterize the different neuron types, intrinsic and firing properties of recorded neurons were calculated using the Clampfit and Matlab software packages.
The following intrinsic neuronal properties were calculated: resting membrane potential, spike threshold, spike amplitude, spike width, input resistance (Rin), membrane time constant (\(\tau\)m), capacitance (Cin), afterhyperpolarization (AHP) amplitude, AHP latency, spike frequency adaptation ratio, and rheobase. Resting membrane potential was recorded within 2 minutes of break-in. Cells with depolarized break-in potentials (> -55 mV) were not included in this study. Spike threshold, amplitude, width, AHP amplitude, and AHP latency were calculated by average all spikes in the first sweep of a 600 ms current step protocol that elicited a firing rate of at least 5 Hz. Spike threshold is calculated from the peak of the third derivative of membrane potential (Cruikshank et al., 2012). Spike amplitude was measured as the voltage change from the spike threshold to the peak of the action potential. Spike width was calculated as the full-width at half-max of the spike amplitude. AHP amplitude was calculated as the voltage change from spike threshold to the peak negativity of the AHP, and AHP latency as the time from peak of the spike to peak negativity of the AHP. Input resistance (Rin), membrane time constant (\(\tau\)m), and input capacitance (Cin) were calculated from a series of small negative current steps ranging from -5 pA to -30 pA, creating a deflection in membrane potential of -2 to -4 mV. Rin was calculated using Ohm’s law, as the mean voltage change divided by mean current amplitude.\(\tau\)m was calculated by fitting a single exponential to the average of the initial 60 ms voltage response, ignoring the first 20 ms. Cin was then calculated from those two parameters using the formula \(\tau\)m = Rin×Cin. Spike frequency adaptation ratio was calculated from the first sweep of the 600ms current step protocol that elicited a firing rate of at least 10Hz (6 spikes per 600ms) using the equation ISIlast / ISIfirst. Rheobase was calculated from 1 sec current pulses increasing in steps of 1-5 pA as the minimum current required to elicit at least one action potential.
A two-tailed Wilcoxon rank sum was used to compute the statistical significance between the intrinsic properties of various neuronal subtypes. To establish the statistical significance between the probability of E→I and I→E connections, a bootstrap resampling (1000 bootstrap samples) method was used to generate a distribution of connectivity probabilities (Sudhakar et al., 2017). Statistical significance was then computed using two-tailed t-test with a confidence interval of 95%.