For cell-attached recording of synaptically evoked spikes, neurons were patched in voltage-clamp mode with K+ internal solution. To avoid biasing the cell’s Vm (and therefore its excitability), holding potential (Vhold) was adjusted so that Ihold ≈0 pA. Capacitative action currents were recorded in the intact patch. After cell-attached recording, we broke in and measured spiking patterns in current-clamp mode to classify the cell physiologically as FS or RSNP. To measure L4-evoked synaptic conductances, we made recordings in voltage clamp using Epacadostat normal Ringer’s with
50 μM APV (Tocris Bioscience), and Cs gluconate internal with 5 mM BAPTA (Sigma-Aldrich). Mean Rseries and Rinput were 10.8 ± 0.4 MΩ and 401 ± 22 MΩ. As before, excitatory-response threshold was the stimulus intensity necessary to evoke a discernible EPSC in a L2/3 pyramidal cell without failures. An average L4-evoked PSC (6–10 repetitions, 10 s interval) was measured at 1.2 × excitatory-response threshold at −90, −68, −40, and 0mV holding potentials. L4-evoked excitatory and inhibitory
synaptic conductance was calculated using published methods (Wehr and Zador, 2003). First, total synaptic conductance (Gsyn) and the synaptic reversal potential (Erev) were calculated at each time point by linear fit to the equation: Isyn(t)=Gsyn(t)×(Vhold−Erev(t)).Isyn(t)=Gsyn(t)×(Vhold−Erev(t)). Gi and Ge were then calculated based on the measured reversal potentials already for excitation
(Ee = 0mV) and inhibition (Ei = −68mV): Gi(t)=Gsyn(t)×(Ee−Erev(t))(Ee−Ei) www.selleckchem.com/ATM.html Ge(t)=Gsyn(t)−Gi(t).Ge(t)=Gsyn(t)−Gi(t). Ee and Ei were directly measured in separate voltage-clamp experiments from pharmacologically isolated EPSCs (in 50 μM D-APV and 100 μM picrotoxin) and IPSCs (in 50 μM D-APV and 10 μM DNQX). This calculation assumes an isopotential neuron. Peak conductance was averaged in a 2 ms window. Latency was calculated relative to L4-stimulus onset, unless otherwise stated. For experiments measuring the threshold Ge required to evoke 50% spike probability in FS cells (Figure 5), L4-evoked EPSCs were recorded near the reversal potential for inhibition. Ge was calculated at this single-holding potential, based on the driving force for excitation: Ge = I/(Vhold − Ee). Immediately after break in, Vrest was measured, and Rinput and membrane time constant were measured from the Vm response to a 500 ms negative-current injection. The current-firing rate relationship was measured by injecting 500 ms depolarizing current in steps from rheobase (minimum current to elicit at least one spike) to rheobase + 200 pA, in steps of 20 pA. Spike threshold was determined at rheobase + 40 pA injected current as the prespike Vm at which dV/dt > 10mV/ms. L2/3 pyramidal cells were identified by soma shape under differential interference contrast optics.