PMC

CBD induces autophagy in C. elegans and neuronal cells. A Adult transgenic animals expressing rgef-1p::gfp::lgg-1, imaged at day 1 and day 5 of adulthood at 20 ℃. GFP::LGG-1 punctae were counted in control and CBD animals on day 1 and day 5 of adulthood in nerve-ring neurons (day 1: control, n = 21; CBD, n = 23; day 5: control, n = 20; CBD, n = 21 animals). B–D Adult transgenic animals expressing lgg-1p::gfp::lgg-1, imaged at day 1 and day 5 of adulthood at 20 ℃. GFP::LGG-1 punctae were counted in control and CBD animals on day 1 and day 5 of adulthood in body-wall muscle (day 1: control, n = 21; CBD, n = 23; day 5: control, n = 20; CBD, n = 23), intestinal cells (day 1: control, n = 34; CBD, n = 21; day 5: control, n = 22; CBD, n = 22 animals), and terminal pharyngeal bulb (day 1: control, n = 21; CBD, n = 21; day 5: control, n = 20; CBD, n = 20 animals). Values are mean ± SEM. *Compared with control group; #Compared with control group in day 1. **p ≤ 0.01, *** p ≤ 0.001, ### p ≤ 0.001 by unpaired t test. TB, terminal bulb. Scale bar = 25 µm. E Western blot analysis results showing LC3-II:LC3-I ratio, and SQSTM1 and ATG5 levels normalized to actin in neuroblastoma SH-SY5Y cells at the indicated time of CBD treatment. F The Western blot analysis results of LC3-II:LC3-I ratio and SQSTM1 and ATG5 levels normalized to actin in SH-SY5Y cells treated with or without CBD (n = 3 biological replicates). *Compared with control group. *p ≤ 0.05, **p ≤ 0.01 by unpaired t test. G Immunofluorescence double-labeled staining for co-localization of LC3 with LAMP1 treated with or without CBD for 48 h in primary hippocampal neurons (green: LAMP1, red: LC3). Cell nucleus was stained with DAPI (blue). Rapamycin was used as a positive control. Scale bar = 25 µm. H Quantification of intensity of LC3 positive area as observed in the immunofluorescence images. n > 10 cells per group. I Quantification of co-localization of LC3 and LAMP1 observed in the immunofluorescence images. n > 10 cells per group. Values are mean ± SEM. *p ≤ 0.05, *** p ≤ 0.001 by unpaired t test

Effect of CBD on action potential shape compared with bupivacaine. A, The first action potential evoked by a 1 s 80 pA current injection in control and 2 μm CBD (same cell as in ). B, The first action potential evoked by a 1 s 40 pA current injection in control and 2 μm bupivacaine. C, Collected results for the effect of 2 μm CBD (n = 10 cells from two animals) or 2 μm bupivacaine (n = 6 cells from two animals) on the peak of the first action potential elicited by 40 pA current injections. Control versus 2 μm CBD, p < 0.0001; control versus 2 μm bupivacaine, p = 0.07. D, Collected results for the effect of 2 μm CBD (n = 10 cells from two animals) or 2 μm bupivacaine (n = 6 cells from two animals) on the width of the first action potential (measured at half action potential height). Control versus 2 μm CBD, p = 0.0002; control versus 2 μm bupivacaine, p = 0.27. E, Collected results for the effect of 2 μm CBD (n = 10 cells from two animals) or 2 μm bupivacaine (n = 6 cells from two animals) on the membrane potential of the trough after the first action potential. Control versus 2 μm CBD, p < 0.0001; control to 2 μm bupivacaine, p = 0.009.

Effects of 300 nm and 1 μm CBD on TTX-R sodium channels. A, Inhibition of TTX-R sodium current by 300 nm CBD (red; n = 6 cells from three animals) and by 1 μm CBD (blue; n = 6 cells from two animals). Currents were evoked by a 15 ms step to +10 mV after a 2 s depolarization at −40 mV, applied once every 30 s. Black symbols: data for cells with the same protocol with extended recording in control solution (n = 6 cells from four animals). p < 0.05 after 60 s between 300 nm CBD and control; p < 0.05 after 0 s between 1 μm CBD and control. B, Effect of 300 nm CBD on the time course of the recovery of availability of TTX-R sodium channels following a 2 s prepulse to −40 mV. Cells were preincubated with 300 nm CBD or control (DMSO-containing) solution for 1–2 h. Representative recovery time course in control (black) and 300 nm CBD (red). Right, Collected results for fraction available after 4 ms recovery at −70 mV, normalized to test pulse before conditioning pulse. Control: relative current = 0.68 ± 0.04; n = 12 cells from three animals; 300 nm CBD: relative current = 0.48 ± 0.05; n = 11 cells from three animals; p = 0.009. C, Availability using the protocol of B to measure voltage dependence of slow inactivation, with 5 s conditioning pulses followed by a 10 ms return to −100 mV to allow recovery from fast inactivation followed by the test pulse. Control: V_h = −38.3 ± 1.2 mV, n = 12 from three animals; 300 nm CBD: V_h = −43.6 ± 1.9 mV, n = 11 cells from three animals; p = 0.026. D, Prediction of the model in for channel fraction in R (i.e., available channels) with the protocol in C in control (solid black line) and with 300 nm CBD (solid red line). Dashed red line shows channel availability in CBD normalized to 1 at −100 mV. Blue line shows occupancy of the slow inactivated state in control; in the presence of 300 nm CBD, blue dashed line shows occupancy of CBD-bound slow inactivated state, and green dashed line shows occupancy of CBD-free slow inactivated state.

a Change in body temperature 2 h after either vehicle, THC (30 mg/kg i.p.), or CBD (30 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice showed the predicted decrease in body temperature in both groups, whereas CBD showed no change (n = 4 per group). b Change in locomotor activity after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls (n = 3–4 per group). c Change in latency to pain response 2 h after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or 30 min after morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice in both groups showed a similar increase in analgesic response, while CBD animals showed no change. Drug-naïve morphine injected animals showed analgesia, while there was no change in mice that have consumed morphine for 7 days (n = 4–8 per group). d Change in paw withdrawal threshold 2 h after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or 30 min after morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice in both groups showed a similar increase in paw withdrawal threshold, while CBD animals again showed no change. Drug-naïve morphine injected animals showed an increase in paw withdrawal threshold, while there was no change in mice that have consumed morphine for 7 days (n = 3–4 per group). *p < 0.05, **p < 0.01, data represent the mean ± SEM.