Extended Data Fig. 6: Enhanced synaptic efficacy by optogenetic stimulation of CA1 pyramidal neurons and Anandamide regulates long-term depression (LTD) in the dorsolateral striatum.
(a) Drawing showing viral vectors (GFAP-GCaPM6f and Syn-CrimsonR-Tdtom) injected into the CA1 of the hippocampus. (b) Fluorescence image showing ChrimsonR expression in neurons (left upper image), GCaMP6f in astrocytes (left bottom image) and merge (right image) in the CA1 of the hippocampus. (c) Neuronal response to optogenetic stimulation of CA1 pyramidal neuron expressing the opsin crimsonR (red upper trace) and empty virus (black middle trace) during an opto-stimulation pulse (square red bottom trace; (λ = 580 nM, 5 s). Scale bars 10 mV, 100 ms. (d) Left. schematic representation of the experimental design in which a patched neuron is activated during an opto-stimulation pulse (λex = 580 nM, 5 s). Right top. EPSCs evoked by minimal stimulation (0.1–10 mA) showing EPSC amplitudes and failures of synaptic transmission (20 consecutive stimuli) before (basal) and after opto-stimulation (opto-stim). (Scale bars: 10 pA, 20 ms). Bottom. Synaptic efficacy of EPSC during baseline (black bar) and after opto-stimulation (red bar; n = 6/3, p < 0.01). (e) Schematic representation of the experimental design showing AEA puff. (f) Left. Pseudocolor images showing fluorescence intensities in CA1 astrocyte in basal condition and during AEA puff. Right. Representative traces showing astrocytes responding to AEA puff. Scale bars 0.4 ΔF/F, 20 s. (g) Relative astrocyte calcium event probability before and after AEA puff in astrocytes expressing GCaMP6f in control condition (n = 7/2, p < 0.05), in the presence of AM-251 (n = 7/2, p = 0.2) and in Gfap-Cnr1−/− (n = 7/2, p = 0.8). The increase in the calcium probability was abolished in the presence of AM-251 (n = 7, p < 0.05) and in Gfap-Cnr1−/− mice (n = 7, p < 0.05). Adult animals ( ≥ 6 weeks; males and females) were used; One-way ANOVA, post hoc Holm–Sidak. *p < 0.05, **p < 0.01, **p < 0.001; Student’s paired t-test. #p < 0.01, ##p < 0.01, ###p < 0.001; one-way ANOVA with post hoc Holm–Sidak, nonsignificant (p > 0.05). Data are mean ± s.e.m. (h) Time course of the LTD induced by HFS in control condition (green circles), under DAGL inhibitor, THL (10 µM; orange circles) and in the presence of NAPE-PLD inhibitor, LE I-401 (10 µM; purple circles) (i) Relative changes from control basal values of EPSC amplitude before and after HFS in control conditions (n = 7/5, p < 0.001; green circles), under DAGL inhibitor, THL (10 µM; n = 5/3, p < 0.001; orange circles), and in the presence of NAPE-PLD inhibitor, LE I-401 (10 µM; n = 5/3, p < 0.01; purple circles). The LTD was preserved in the presence of THL (n = 5, p = 0.6) and it turned in LTP in the presence of LEI-401 (n = 5, p < 0.001); Adult animals ( ≥ 6 weeks; males and females) were used; one-way ANOVA, post hoc Holm–Sidak. *p < 0.05, **p < 0.01, **p < 0.001; Student’s paired t-test. #p < 0.01, ##p < 0.01, ###p < 0.001; one-way ANOVA with post hoc Holm– Sidak, nonsignificant (p > 0.05). Data are mean ± s.e.m.
