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%.