(A) Experimental design: we recorded from fast-spiking interneurons labeled in a PV-Cre:: Ai14 in mPFC (top). Images of a recorded neuron in DIC and showing tdTomato expression (bottom). (B) Example FSI responses to hyperpolarizing (−200 pA) and depolarizing (50 pA above rheobase) current steps at baseline and after application (Post, 10 min after drug wash in) of 5HT (30 µM, blue), 5HT + synaptic blockers (10 uM CNQX, 100 uM DL-AP5, 10 uM gabazine, green), 5HT + synaptic blockers+5HT2A antagonist (1 uM MDL-100907, purple), or time-locked control aCSF (gray). (C–D) Subtracted change in membrane potential (C) and percent change in input resistance (D) after pharmacological manipulations listed above. (E) Experimental design: Cre-dependent ChR2 was injected into the dorsal raphe of SERT-Cre mice. Top and bottom rows: Images of ChR2 expression and 5HT immunohistochemistry in dorsal raphe injection side (top) and mPFC recording site (bottom). Confocal images of ChR2 (green), 5HT immunohistochemistry (red), and merged. Yellow sections indicate overlap. Arrows point to examples of overlap. (F) Top: FSIs in mPFC were injected with light depolarizing current to elicit spiking and ChR2 expressing terminals were activated with blue light (10 Hz, 10 s) to release endogenous 5HT (top). Bottom: Peristimulus time histograms of FSI firing rate during current step with ChR2-activated release of 5HT (G) These experiments were repeated after washing in a 5HT2A antagonist (1 uM MDL100907). (H) Experimental design: ChR2 was injected into one hemisphere of mPFC and FSIs were patched on the opposite hemisphere. (I) Example traces of FSI responses at baseline (black) and after 5HT (blue) in response to activation of synaptic inputs from ChR2-expressing terminals with either a train of blue light pulses (5 Hz, 2 mW, top) or single light flashes (0.5–1 mW, bottom). (J) Number of spikes fired in response to each light flash in the stimulus train depicted before and after application of 5HT. (K–L) Change in amplitude (K) and decay time constant (tau, (L) of synaptic responses before (black) and after 5HT (blue). *p<0.05, **p<0.01.