(A) Left, schematic of a mouse coronal brain section showing the injection site of an opsin-expressing viral vector (AAV-CAG-ChR2-mCherry) in the CA3 region. Right, schematic showing the opto-activation of Schaffer collateral inputs combined with juxtacellular recording from single CA1 pyramidal neurons. (B) Confocal images showing a light-responsive CA1 pyramidal neuron (recording shown in C, D) labeled in vivo. Right panel, high-magnification view on the neuron (white, Neurobiotin, Nb) relative to the labeled Schaffer collaterals (red, ChR2-mCherry) and Calb1 staining (green). Scale bars = 100 µm (left), 20 µm (right). (C) High-pass filtered juxtacellular trace (bottom) for the neuron shown in B. Note the partial spiking responses to the light pulses (3 Hz, ~1.3 mW). Top, high magnification showing long-latency spike responses upon pulses of blue light (5 ms, indicated in blue). Scale bar = 2 mV, 2 s. (D) Raster plot (top) and peristimulus time histogram (bottom) showing the spike latency to the light stimuli for the CA1 pyramidal neuron shown in B. The average latency is indicated. (E) Histogram of average spike latencies of CA1 neurons (n = 11) showing spiking responses to Schaffer collateral input activation. The average latency is indicated. (F) Left, schematic of a mouse coronal brain section showing the injection site of recombinase-dependent opsin-expressing viral vectors (e.g., AAV-EF1a-flex-ChR2-eYFP, AAV-hSyn1-flex-ChR2-mCherry, or AAV-hSyn1-flex-oChIEF-TdTomato) in the dentate gyrus of Calb1cre mice. Right top, schematic showing the opto-activation of mossy fiber inputs combined with juxtacellular recording from single CA3 pyramidal neurons. Right bottom, epifluorescence image showing eYFP signal (pseudocolored to red for display purposes) following the injection of AAV-CAG-flex-eYFP. Note the labeling of mossy fibers (red) along the transverse CA3 axis. DG, dentate gyrus; SL, stratum lucidum; SO, stratum oriens; Pyr, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum moleculare; ML, molecular layer of dentate gyrus. Scale bar = 200 µm. (G) Epifluorescence image showing a nonresponsive (Cell 1) and a responsive (Cell 2) CA3a pyramidal neuron, recorded (as in F) along the same electrode penetration and labeled in vivo. Left panel, high magnification on the somata of the two neurons (green, Nb) relative to the labeled mossy terminals (red, oChIEF-TdTomato). Scale bars = 100 µm (left inset), 200 µm (right). (H) Representative recordings from the nonresponsive (Cell 1) and responsive (Cell 2) CA3a pyramidal neurons shown in G. The recording form Cell 1 (top, gray) shows the absence of spiking, but increasing amplitude of the negative local field potential (LPF) deflection during the stimulus train (25 Hz, 5-ms pulse duration) − consistent with the expected facilitation of neurotransmitter release at mossy terminals. Scale bars = 2 mV, 100 ms. The recording from Cell 2 (middle and bottom, black) shows partial spiking responses to low and high-frequency light pulses (3 and 40 Hz, 5 ms). Note the increase of the local field potential (LFP) amplitude (indicated with an arrow) during the stimulus trains. Scale bars = 2 mV, 500 ms (middle trace); 2 mV, 50 ms (bottom trace). (I) Raster plot (top) and peristimulus time histogram (bottom) showing the spike latency to the light stimuli for Cell 2 shown in G,H. 40 Hz light stimulus trains were excluded because of a high probability of spiking failure (see details in Materials and Methods). The average latency is indicated. (J) Boxplots showing comparison of latencies between directly activated CA1 cells (n = 10), indirectly activated CA3 (via mossy fiber photostimulation, n = 7), and CA1 cells (via Schaffer collateral photostimulation, n = 11). Significant p values after multiple group comparison are indicated (Kruskal–Wallis, p = 0.00006). Red lines indicate medians. Outliers are shown as crosses.