Subthreshold Kir and Ih currents modulate excitability of layer 1 VIP interneurons in the medial prefrontal cortex

Abstract Cortical layer 1 (L1) is a key site for integrating top-down and bottom-up information and is populated by inhibitory interneurons, including vasoactive intestinal peptide (VIP)-expressing cells. These interneurons regulate information flow across the cortical column by disinhibiting pyramidal neurons, yet the subthreshold ionic mechanisms that shape their excitability in the medial prefrontal cortex (mPFC) remain poorly understood. Here, we characterize the electrophysiological properties of L1b VIP interneurons in mouse mPFC, focusing on the role of the hyperpolarization-activated cation current (I h ) and inward-rectifying potassium current (Kir). Using whole-cell recordings in acute slices, we find that L1b VIP interneurons exhibit constitutively active I h and Kir conductances. Inhibition of I h with ZD-7288 hyperpolarized the resting membrane potential (RMP), increased input resistance (R in ), and prolonged action potential (AP) duration without altering rheobase or firing frequency, while increasing EPSP-spike coupling probability. Blocking Kir with BaCl 2 depolarized the RMP, increased R in and membrane time constant, reduced rheobase, increased firing frequency, and similarly enhanced EPSP-spike probability and AP duration. Voltage-clamp experiments confirmed the presence of ZD-7288-sensitive I h and BaCl 2 -sensitive Kir currents of small amplitude but operating on a high-resistance membrane, consistent with a strong impact on excitability. In contrast, neither I h nor Kir inhibition affected the amplitude or frequency of spontaneous or miniature EPSCs, indicating that these currents do not measurably alter basal excitatory synaptic transmission. Immunofluorescence revealed weak somatic HCN1 and no detectable HCN2 expression in L1b VIP interneurons. Notably, Kir inhibition unmasked an I h -dependent voltage sag during hyperpolarizing steps, suggesting that constitutive Kir activity normally masks I h recruitment at subthreshold potentials. Together, these results indicate that I h and Kir are active near RMP in L1b VIP interneurons and jointly regulate their passive properties, intrinsic excitability, and EPSP-spike coupling, therefore shaping how L1 VIP cells filter incoming signals and influence information flow within the prefrontal cortical column.