Extended Data Fig. 7. Comparison of synaptic strength in local inhibitory engram inputs to mPFC engram versus nonengram pyramidal neurons.

(a) Top: AAV-DIO-ChR2-eYFP was injected into the mPFC/PL in Fos-iCreER^T2 × ROSA-LSL-tdTomato mice. mPFC engram neurons expressed ChR2-eYFP and tdT. Photostimulation activated axons of local GABAergic engram neurons and induced inhibitory postsynaptic responses recorded in engram (E−E synapses) and nonengram pyramidal neurons (E−NE synapses). (b) Left: 28 days after CFC, the mice were tested for the recall of remote contextual fear memories, and electrophysiological experiments were performed. Right: quantification of freezing behavior during memory recall (11 mice). Data are presented as the mean ± SEM. (c) Representative traces of quantal IPSCs (qIPSCs) induced by the photostimulation of local GABAergic engram inputs and recorded in a tdT− nonengram pyramidal neuron (E−NE synapses) and a tdT+ engram pyramidal neuron (E−E synapses) in the mPFC. Photostimulation (blue triangles) activated local GABAergic inputs of ChR2+ engram neurons and induced IPSCs in postsynaptic pyramidal neurons. qIPSCs were recorded at 0 mV in voltage-clamp mode. Presynaptic GABA release was desynchronized in Ca²⁺-free and 4 mM Sr²⁺-containing extracellular solution. In each neuron, peak amplitudes of well-separated qIPSCs recorded 0.5–1.5 s after photostimulation (gray areas) were calculated and averaged. The average qIPSC (red) was overlaid onto individual qIPSC traces (gray). TTX (1 μM) and 4-AP (1 mM) were added in the extracellular solution to block polysynaptic IPSCs. NBQX (10 μM) was also added to block excitatory glutamatergic transmission. Scale bar, 10 μm (inset). (d) Comparison of the peak amplitude of evoked qIPSCs in GABAergic engram inputs to 24 pairs of nonengram neurons (E−NE synapses) versus engram neurons (E−E synapses). Paired t-test. Data are presented as the mean ± 95% confidence interval.

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