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

Abstract

Although Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) represent a technical revolution in integrative neuroscience, the first ligands used were not as selective as expected. 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 aspecific 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.

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“…In hM4Di rats, a 30%-decrease in aDLS DA level was observed between 90 and 135 minutes after C21 injection (Fig. 2E), which is consistent with the temporal inhibition of SNc neurons previously reported ( 25 ). Importantly, this strategy did not influence NAc DA levels (Fig.…”

“…Chemogenetic manipulation of SNc DA neurons was achieved through stereotaxic bilateral infusion of a AAV5-hSyn-DIO-hM4D(Gi)-mCherry (10 12 particles/mL, plasmid #44362, Addgene, Watertown, MA, USA) or AAV5-hSyn-DIO-mCherry (10 12 particles/mL, plasmid #50459, Addgene) in the SNc of TH::Cre rats (20,21). As previously described and validated in (20), 1 μL was infused at a rate of 0.2 μL/min on each hemisphere and coordinates for SNc injections were set at: -4.3 mm (AP), ±2.4 mm (ML), -7.9 mm (DV) relative to bregma (22).…”

“…27 TH::Cre rats were infused with a virus coding for the inhibitory DREADD (designer receptor exclusively activated by designer drug(19)) hM4Di-mCherry or mCherry alone in the substantia nigra pars compacta (SNc). Microdialysis experiments were performed at least two weeks after the surgeries to allow the proper recovery of the animals and a stable transgene expression (20). At the end of the experiment, rats were euthanized, and brains were processed for histological validation of the probe placement and transgenes expression.…”

“…Sections were then stained using Cresyl violet staining and visualized under a light microscope (Nikon Eclipse 80i, TRIBVN, Châtillon, France) coupled to the ICS FrameWork computerized image analysis system (Calopix 2.9.2 software, TRIBVN, Châtillon, France). Finally, to quantified transgenes expression, floating coronal sections (30 µm) of three levels of the mesencephalon were selected according to (1).…”

“…2, A-C). hM4Di was activated by the synthetic ligand Compound 21 (C21) ( 24 ), at a dose that we previously reported as potent and selective to inhibit SNc neurons in TH-Cre rats ( 25 ). We first confirmed with in vivo microdialysis that this approach efficiently decreases aDLS DA levels (Fig.…”