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. 2020 Dec;237(12):3729-3739.
doi: 10.1007/s00213-020-05650-5. Epub 2020 Aug 28.

Positive allosteric modulation of the cannabinoid type-1 receptor (CB1R) in periaqueductal gray (PAG) antagonizes anti-nociceptive and cellular effects of a mu-opioid receptor agonist in morphine-withdrawn rats

Udita Datta  1   2 , Leslie K Kelley  1 , Jason W Middleton  3   4   5 , Nicholas W Gilpin  6   7   8   9
Affiliations

Positive allosteric modulation of the cannabinoid type-1 receptor (CB1R) in periaqueductal gray (PAG) antagonizes anti-nociceptive and cellular effects of a mu-opioid receptor agonist in morphine-withdrawn rats

Udita Datta et al. Psychopharmacology (Berl). 2020 Dec.

Abstract

Opioid drugs are a first-line treatment for severe acute pain and other chronic pain conditions, but long-term opioid drug use produces opioid-induced hyperalgesia (OIH). Co-administration of cannabinoids with opioid receptor agonists produce anti-nociceptive synergy, but cannabinoid receptor agonists may also produce undesirable side effects. Therefore, positive allosteric modulators (PAM) of cannabinoid type-1 receptors (CB1R) may provide an option reducing pain and/or enhancing the anti-hyperalgesic effects of opioids without the side effects, tolerance, and dependence observed with the use of ligands that target the orthosteric binding sites. This study tested GAT211, a PAM of cannabinoid type-1 receptors (CB1R), for its ability to enhance the anti-hyperalgesic effects of the mu-opioid receptor (MOR) agonist DAMGO in rats treated chronically with morphine (or saline) and tested during withdrawal. We tested the effects of intra-periaqueductal gray (PAG) injections of (1) DAMGO, (2) GAT211, or (3) DAMGO + GAT211 on thermal nociception in chronic morphine-treated rats that were hyperalgesic and also in saline-treated control rats. We used slice electrophysiology to test the effects of DAMGO/GAT211 bath application on synaptic transmission in the vlPAG. Intra-PAG DAMGO infusions dose-dependently reversed chronic morphine-induced hyperalgesia, but intra-PAG GAT211 did not alter nociception at the doses we tested. When co-administered into the PAG, GAT211 antagonized the anti-nociceptive effects of DAMGO in morphine-withdrawn rats. DAMGO suppressed synaptic inhibition in the vlPAG of brain slices taken from saline- and morphine-treated rats, and GAT211 attenuated DAMGO-induced suppression of synaptic inhibition in vlPAG neurons via actions at CB1R. These findings show that positive allosteric modulation of CB1R antagonizes the behavioral and cellular effects of a MOR agonist in the PAG of rats.

Keywords: Dose-response; Hyperalgesia; Morphine; Opiates; Opioids; PAG; Pain; Positive allosteric modulators.

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Figures

Figure 1.
Figure 1.. Intra-PAG infusions of the MOR agonist DAMGO produce dose-dependent and time-dependent anti-hyperalgesia in rats treated with chronic morphine and this effect is attenuated by co-infusion of the CB1R PAM GAT211 into the PAG.
(A) Timecourse of chronic in vivo morphine treatment and intra-PAG infusions. (B to G) show mean ± SEM hindpaw withdrawal latencies in response to a noxious thermal stimulus using the Hargreaves test. Hindpaw withdrawal latency for each animal at each dose/timepoint is the average of two trials on the right hindpaw and two trials on the left hindpaw. (B) Rats treated with 20 mg/kg morphine per day for 6 days (two s.c. injections of 10 mg/kg/day at 9:00 a.m. & 4:00 p.m.) exhibited thermal hyperalgesia, as exhibited by shorter hindpaw withdrawal latencies, relative to the same rats treated with morphine for 1 day and also relative to a separate group of rats treated with saline for 6 days, **p<0.001; (C) dose-response effects of DAMGO infused into the PAG on hindpaw withdrawal latencies in morphine-treated (n=6) and saline-treated (n=5) rats; *p<0.001 DAMGO dose effect relative to the zero dose condition in morphine-treated rats; (D) dose-response effects of GAT211 infused into the PAG on hindpaw withdrawal latencies in morphine-treated (n=6) and saline-treated (n=6) rats; *p<0.05 main effect of morphine; (E) timecourse effects of DAMGO (0.03 ug) infused into the PAG on hindpaw withdrawal latencies in morphine-treated (n=3) and saline-treated (n=4) rats; red asterisk: p<0.02 DAMGO dose effect relative to zero minutes time point in morphine-treated rats, black asterisk: p<0.001 DAMGO dose effect relative to zero minutes time point in saline-treated rats; (F) timecourse effects of DAMGO (0.03 ug) and GAT211 (10 ug) co-infused into the PAG on hindpaw withdrawal latencies in saline-treated rats (n=4); black asterisk: p<0.01 DAMGO effect relative to zero minutes time point, red asterisk: p<0.01 DAMGO+GAT211 effect relative to zero minutes time point; (G) timecourse effects of DAMGO (0.03 ug) and GAT211 (10 ug) co-infused into the PAG on hindpaw withdrawal latencies in morphine-treated rats (n=6); black asterisk: p<0.01 DAMGO effect relative to zero minutes time point, red asterisk: p<0.01 DAMGO+GAT211 effect relative to zero minutes time point.
Figure 2 –
Figure 2 –. Chronic morphine hyperpolarizes PAG cells and reduces the number of spontaneously active cells in the PAG of hyperalgesic rats.
(A) Rats treated with 20 mg/kg morphine (n=15) per day for 6 days (two s.c. injections of 10 mg/kg/day at 9:00 a.m. & 4:00 p.m.) exhibited thermal hyperalgesia, as exhibited by shorter mean ± SEM hindpaw withdrawal latencies, relative to saline-treated control rats (n=16), *p<0.001; (B) Whole cell recordings in vlPAG neurons revealed some neurons that were spontaneously active (example trace above) and some neurons that had stable subthreshold resting membrane potentials (example recording below). (C) Resting membrane potential (RMP) from saline-treated control rats (n=22) was higher than morphine-treated rats (n=25), *p<0.05. (D) The proportion of spontaneously active neurons was higher for saline-treated rats relative to morphine-treated rats, **p<0.01. (E) Postsynaptic current was recorded at −50 mV and spontaneous inhibitory postsynaptic currents (sIPSCs) were detected. sIPSC frequency (F) and amplitude (G) were not different between neurons from saline-treated rats and morphine-treated rats (n=47 neurons from saline-treated rats and n=30 neurons from morphine-treated rats).
Figure 3 –
Figure 3 –. Positive allosteric modulation of CB1Rs by GAT211 functionally antagonizes MOR function.
(A) Electrically evoked inhibitory postsynaptic currents (eIPSCs) were recorded periodically every 20 sec for 40 mins. Baseline eIPSCs were recorded for 10 mins after which GAT211 was applied to the bath and 15 mins after this DAMGO was applied. (B) Example traces show that DAMGO significantly attenuated eIPSC amplitude. (C) There was a main effect of GAT211 concentration on the suppression of eIPSC amplitude by DAMGO (p < 0.05), although posthoc comparisons with multiple comparison correction showed no significant differences. (D) GAT211 (1 μM) attenuates DAMGO-induced suppression of synaptic inhibition, an effect that is not observed in the presence of the CB1R antagonist rimonabant (RIM), *indicates p<0.05 difference between the two bracketed conditions.
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