(A) Low-magnification view of sagittal section from Chat i-Cre; Rosa26 lsl-ChR2-EYFP mouse forebrain. ChR2-EYFP (green) is expressed in the nucleus basalis (NB), globus pallidus externus (GP), striatum and cortex (Ctx). (B) Higher-magnification view of ChR2-EYFP expression combined with ChAT immunostaining (magenta) in frontal cortex (top) and nucleus basalis (bottom). Arrowheads indicate cells immunopositive for ChAT. (C) Example 2-photon stack from a layer 1 interneuron following whole-cell recording and dialysis with Alexa Fluor 594. (D) High-magnification view of ChR2-EYFP+ somata and fibers in layers 1 and 2/3 of frontal cortex. NeuN immunostain (magenta) highlights the distribution of neuronal somata across layers. Arrowhead indicates an example layer 1 interneuron surrounded by ChR2-EYFP fibers. (E) Example PSCs from voltage-clamp recordings of three different layer 1 interneurons in response to blue light stimulation (blue bar) of ChR2+ cholinergic fibers. Neurons were voltage-clamped at −70 mV (left) to isolate EPSCs or at 0 mV (middle and right) to isolate IPSCs. PSCs recorded in the presence of glutamate receptor antagonists CPP and NBQX are shown in black and after bath application of nAChR antagonists (MEC, MLA, and DHβE) in red. (F) Example light-evoked IPSCs from a layer 1 interneuron in a Chat i-Cre; Rosa26 lsl-ChR2-EYFP mouse voltage-clamped at 0 mV in the presence of CPP and NBQX (baseline) and following subsequent bath application of (from left to right) nAChR antagonists, TTX, 4AP, and SR95531. (G) Summary graph of IPSC peaks normalized to baseline (n = 5 cells from 4 mice). Asterisk, condition vs baseline p < 0.05, Mann–Whitney test). (H) Onset latencies for monosynaptic nEPSCs (n = 41 cells), monosynaptic IPSCs (n = 9 cells), and polysynaptic IPSCs (n = 19 cells from 9 mice). Mean (±sem) are shown in green. Asterisk, p < 0.05, Mann–Whitney test.