Extended Data Figure 2: Optogenetic manipulation of the AC–CA projection.

a, Experimental design: RV-ChR2-eYFP (or eYFP alone) was injected into the dorsal hippocampus and light was delivered above the cell bodies in the AC. Five days after injection, ChR2 and eYFP mice were fear conditioned in context A while no-shock controls were only exposed to context A (day 1). All mice were tested with light on and off sessions in context B (day 2), and then tested for contextual memory retrieval in context A (day 3). Optogenetic stimulation was with 473 nm light in a train of 20 Hz, 15 ms pulses, 30 s duration, with 8–10 mW laser power at fibre tip. b, Freezing (no head motion observed) during day 2 is plotted in 5 s time bins over 150 s in context B (left). ChR2/shock (FC): black; ChR2/no shock (NS): red; eYFP/shock (eYFP): blue. Individuals in the FC group (each animal a different colour) are shown (middle). Summary (right): percentage time freezing (mean ± s.d.) 20 s before light on (darker shade) versus 20 s after light on (lighter shade); FC: 60.9 ± 7.4% light on versus 6.5 ± 4.4% light off, n = 8; NS: 2.7 ± 0.65% light on versus 3.4 ± 0.95% light off, n = 6; eYFP: 2.9 ± 0.75% light on versus 3.6 ± 1% light off, n = 6; P < 0.001, two-way ANOVA with repeated measures. c, Preservation of contextual fear memory (percentage time freezing) on day 3 in the original context (mean ± s.d., P < 0.001, unpaired t-test). d, Preservation of contextual memory in medial septum injected mice (Fig. 1f); percentage time freezing on day 3 in the original context (mean ± s.d., P < 0.001, comparisons shown, unpaired t-test). e, Preservation of contextual memory in hippocampus injected mice (Fig. 1h); percentage time freezing on day 3 in the original context (mean ± s.d., n = 8 mice, P < 0.001, paired t-test). f, The successful loss-of-function experiments targeting hippocampus-dependent memory formation mediated by cells giving rise to the AC–CA projection (reported in Fig. 2) were designed to allow the most robust inhibition of this circuit element. An alternative design (attempting to target the projection field despite the broad and long septotemporal extent of the hippocampal formation) was also explored as shown here but was not effective, as expected; we injected AAV5-eNpHR3.0-eYFP (or AAV5-eYFP in a parallel cohort) bilaterally into the AC, and targeted light stimulation bilaterally to axon terminals in the hippocampus. Eight weeks after injection, all mice were fear conditioned to context (day 1), and tested for context retrieval during light on/off sessions (day 2), and again for context retrieval in light-off only (day 3). Optogenetic inhibition was with constant illumination of 589 nm light, 30 s duration, with 8–10 mW laser power at fibre tip. g, We observed a trend towards reduction in freezing due to optical inhibition of the AC–CA projection during memory retrieval. Percentage time freezing in context A during day 2 before light (darker bar on left) versus after light (lighter bar at right). eNpHR3.0: 73.5 ± 8.5% light off versus 55.5 ± 11.4% light on, n = 10; eYFP: 74 ± 7.4% light off versus 74 ± 11.3% light on, n = 10; percentage time freezing in context A with light off (dark bars) during day 3 is shown after dotted line. eNpHR3.0: 67.5 ± 7.2%, n = 10; eYFP: 66.5 ± 9.1%, n = 10 (P = 0.067, two-way ANOVA). As expected, point illumination may be less effective for inhibiting broad axon terminal field volumes. h, Extension of findings from effective loss-of-function experiments (Fig. 2) targeting hippocampus-dependent memory formation mediated by cells giving rise to the AC–CA projection: significant effect on speed of onset of memory expression. Experimental design: CAV-Cre was injected into the dorsal hippocampus, DIO-eNpHR3.0 (or DIO-eYFP) was injected into the AC, and light was delivered above cell bodies in the AC. All mice were fear conditioned in context A (day 1), tested for latency to contextual retrieval with light-on only (day 2), and then for latency to context retrieval in light-off only (day 3). i, Day 2: 66.1 ± 18.1 s for eNpHR3.0 (n = 12) versus 43.8 ± 11.1 s for eYFP (n = 8) during light on; day 3: 53.8 ± 13.7 s for eNpHR3.0 versus 48.8 ± 7.7 s for eYFP during light off; P < 0.05 two-way ANOVA with repeated measures. *P < 0.05, **P < 0.01, ***P < 0.001.