LPHN3 regulates the strength of synaptic input from L2/3 to L5. (A- C) Images of an acute coronal slice from a P14 mouse electroporated at E16 to express ChR2-YFP in L2/3 PNs. (B, C) A L5 PN is filled with Alexa 595 during a whole cell recording with a pipette nearby to record the axonal field potential (AfP). (D) Schematic of the optogenetic electrophysiology experiment. A whole cell recording from a L5 PN is used to record EPSCs while the AfP is recorded nearby. Flashes of blue light over the recorded cell (blue circle) are used to stimulated ChR2-expressing axon of L2/3 neurons in L5. (E) Example average EPSC (top: Vhold = +40 mV) and AfP (bottom) before (black) and after (red) 1 μM TTX. (F) Example recordings with small (green), medium (blue), and large (red) AfPs from different Control, shLPHN3, and Rescue slices. (G) shLPHN3 reduced mean EPSC amplitude versus Control and Rescue (one-way ANOVA, F(2,68) = 3.34, P = 0.04: Control 81.72 ± 11.42 pA, n = 25; shLPHN3 49.5 ± 5.8 pA, n = 23; Rescue 83.48 ± 12.42 pA, n = 23). (H) AfP amplitude did not differ (one-way ANOVA, F(2,68) = 1.06, P = 0.35: Control, 4.44 ± 0.27 pA, n = 25; shLPHN3, 3.89 ± 0.39 pA, n = 23; Rescue, 4.68 ± 0.48 pA, n = 23). (I) Plot of AfP vs. EPSC amplitude for Control (black), shLPHN3 (red), and Rescue (gray) inputs, fitted with a line through the origin. The slopes of the fitted lines differed between conditions (extra sum-of-squares F test, F(2,68) = 4.82, P = 0.01). The slope of the shLPHN3 condition (11.14 pA/μV, 95% confidence interval 7.82-14.46) was determined to be significantly different from Control (18.91, 14.69-23.12) and Rescue (17.97, 14.53-21.42) since the 95% confidence intervals were non-overlapping.