Supplementary Figure 3: oPSD enhanced NMDAR-mediated EPSPs (EPSP_NMDA) and Ca²⁺ influx.

AAV-C1V1-eYFP (a-e), AAV-Chrimson-tdTomato and AAV-GCaMP6s (f, g) were infused into the DMS. (a) Sample confocal image of an Alexa Fluor 594-filled C1V1-eYFP-expressing neuron showing the placement of stimulating (150 µm from the soma) and recording electrodes. (b) Representative traces depicting optogenetic-mediated EPSP in the distal dendrites in response to single synaptic stimulation. After blockade of the AMPAR-mediated EPSP (EPSP_AMPA) with NBQX (10 µM), simultaneous presynaptic stimulation and oPSD elicited a depolarization (C1V1-mediated response [V_c1v1] + EPSP_NMDA) that was reduced by bath application of APV (V_c1v1). The optogenetic-mediated EPSP_NMDA was calculated by digital subtraction of V_c1v1 from V_c1v1 + EPSP_NMDA. (c) Comparison of the amplitudes of the electrically and optogenetically induced responses in the absence (E+O) and presence (E+O+APV) of APV. t₍₉₎ = 4.93, ***P = 0.00082; n = 10 neurons, 3 rats. (d) Top, sample EPSP traces in response to eHFS in the presence (color) and absence (black) of APV. Bottom, traces were generated by digital subtraction of the EPSP with APV from that without APV. (e) Paired eHFS+oPSD induced a greater area under EPSP_NMDA than eHFS alone. t₍₇₎ = -4.50, **P = 0.0028; n = 8 neurons, 4 rats per group. (f) Representative traces of Ca²⁺ transients in distal dendrites in response to eHFS (left) and eHFS+oPSD (right) with and without APV application. (g) eHFS+oPSD induced greater NMDAR-mediated Ca²⁺ transients than that of eHFS. t₍₁₁₎ = 4.30, **P = 0.0012; n = 12 neurons, 3 rats per group. Two-sided paired t test for c, e, g. Data are presented as mean ± s.e.m.