Compound 21, a two-edged sword with both DREADD-selective and off-target outcomes in rats

Abstract

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) represent a technical revolution in integrative neuroscience. However, the first used ligands exhibited dose-dependent selectivity for their molecular target, leading to potential unspecific effects. Compound 21 (C21) was recently proposed as an alternative, but in vivo characterization of its properties is not sufficient yet. Here, we evaluated its potency to selectively modulate the activity of nigral dopaminergic (DA) neurons through the canonical DREADD receptor hM4Di using TH-Cre rats. In males, 1 mg.kg-1 of C21 strongly increased nigral neurons activity in control animals, indicative of a significant off-target effect. Reducing the dose to 0.5 mg.kg-1 circumvented this unspecific effect, while activated the inhibitory DREADDs and selectively reduced nigral neurons firing. In females, 0.5 mg.kg-1 of C21 induced a transient and residual off-target effect that may mitigated the inhibitory DREADDs-mediated effect. This study raises up the necessity to test selectivity and efficacy of chosen ligands for each new experimental condition.

Fig 1. Representative example of the waveform average of recordings.

The total duration of the triphasic spike is indicated in blue. The duration, from spike initiation to the maximal negative phase of action potential, is indicated in green. Data are presented as the waveform average +/- SEM.

Fig 2. hM4Di-mCherry and mCherry expression in mesencephalic DA neurons of TH-Cre rats.

(A) On the left, schema of the three levels of mesencephalon used for quantified viral expression with three areas: lateral SNc (lSNc), medial SNc (mSNc) and Ventral Tegmental Area (VTA). The black rectangle indicates the level at which representative images, on the right, were taken to illustrate TH immunostaining and hM4Di-mCherry or mCherry expression. (B-C) Percent of transgenes expression in the lSNc, mSNC and VTA for males (B) (hM4Di, orange, n = 18 hemispheres, 13 animals; mCherry, black, n = 23 hemispheres, 16 animals) or females (C) (hM4Di, red, n = 7 hemispheres, 7 animals; mCherry, grey, n = 8 hemispheres, 6 animals). A similar pattern of expression was observed between males and females with a gradient of expression from lSNc to the VTA (two-way ANOVAs: Fs > 18.83, p < 0.001, partial η2 > 0.49), in both transgene conditions (two-way ANOVAs: Fs < 2.45, p > 0.16, partial η2 < 0.06). For each area, the given percent of expression correspond to the mean expression of the three levels of mesencephalon. Scale bar: 200 μm. Data were expressed as the mean number of quantified hemispheres for which recordings were included +/- SEM.

Fig 3. Dose dependent effect of C21 on multi-unit activity of SNc neurons expressing hM4Di-mCherry or mCherry.

(A) Schema of the bilateral electrodes implantations. (B) Representative data obtained during recording of neuronal subpopulation within the SNc from male mCherry rat treated with 1 mg.kg^-1 of C21. (C) Representative data obtained during recording of neuronal subpopulation within the SNc from male hM4Di rat treated with 0.5 mg.kg^-1 of C21. (D-F) On the left, effect of C21 along time on SNc neuronal multi-unit activity, during vehicle (VEH, a 20-minutes interval) and C21 periods (30-minutes intervals), normalized to 10-minutes baseline recording. In the middle and on the right, mean neuronal multi-unit activity, during the VEH period and between the 90 and 180 minutes post-C21 injection intervals, normalized to baseline. (D) Effect of C21 in mCherry male rats treated with 1 (white, n = 6 recordings, 4 animals) or 0.5 (black, n = 9 recordings, 7 animals) mg.kg^-1. (E) Effect of C21 in mCherry (black, n = 8 recordings, 7 animals) or hM4Di (orange, n = 10 recordings, 8 animals) male rats treated with 0.5 mg. kg^-1. (F) Effect of C21 in mCherry (grey, n = 8 recordings, 6 animals) or hM4Di (red, n = 7 recordings, 7 animals) female rats treated with 0.5 mg.kg^-1. Data were expressed as the mean number of recording sides +/- SEM. BL: baseline, VEH: vehicle. *P < 0.05, **P < 0.01, ***P < 0.001.