KO mice show decreased amplitude of monosynaptic EPSPs in mPFC pyramidal neurons evoked by optogenetic stimulation of vHP terminals. A, vHP microinjection of AAV2/CamKII-hChR2(H134R)-EYFP results in selective EYFP fluorescence (green) expression in transduced vHP region. Low-magnification photomicrograph shows a representative example of the vHP targeted area. B, Top, Low-power photomicrograph of a representative brain slice (300-μm-thick) used for patch-clamp recordings in mPFC after streptavidin-Cy3 staining for visualization of the neurobiotin-filled recorded neuron. Dense innervation of vHP terminals loaded with ChR2-EYGP (green channel) can be observed together with the neurobiotin-filled pyramidal neuron recorded in layer 3 of mPFC (red channel, boxed area). Bottom, Higher-magnification image of the neuron shown at top. Maximum intensity projection of series of Z stack of confocal images shows a filled cell with typical pyramidal neuron morphology, including apical dendrite and dendritic spines (inset). C, Representative example of a short latency (blue light-evoked) EPSP in an mPFC pyramidal cell during whole-cell current-clamp recording. Gray represents individual responses. Green represents average trace from 30 stimuli. Bottom, Response jitter is consistent with monosynaptic responses in both experimental groups. Each dot represents individual jitter of a recorded neuron from control (Flox: pink dots, n = 8; red dot, mean ± SEM) or mutant mice (KO: gray dots, n = 9; black dot, mean ± SEM). No significant differences between genotypes were observed (Student's t test, p = 0.51). D, EPSPs evoked by optogenetic stimulation of vHP terminals in mPFC is reduced in mutant mice (KO) compared with age-matched controls (Flox) during GABAergic blockade. EPSPs were recorded in whole-cell current-clamp configuration with 50 μm picrotoxin in the bath. *p = 0.023 (Student's t test). Error bar indicates mean ± SEM from 10 cells for flox and 8 for mutant. Each dot represents one recorded neuron (18 neurons from 14 slices from 11 animals).
