The present study provides the first evidence of behavioral and slice electrophysiological efficacy of the DREADD ligand, DCZ, in rats. Our data show that a relatively low dose of DCZ (0.1 mg/kg) administered systemcially effectively supports hM3Dq and hM4Di DREADD-mediated behavior and electrophysiological effects without apparent confounding, non-specific alterations in locomotor behavior or associatve conditioning. Systemic administration of DCZ to selectively induced robust cFos expression in hM3Dq-expressing neurons without producing any detectable cFos in the uninfected control hemisphere. In an slice electrophysiological preparation, DCZ effectively inhibited neuronal firing in hM4Di-expressing neurons. Moreover, DCZ produced greater inhibition of action potential firing at a lower concentration, compared to CNO. Systemic DCZ-mediated hM4Di inhibition reduced ethanol intake in ethanol dependent and non-dependent rats expressing hM4Di in the central amygdala. Lastly, systemic DCZ did not alter gross locomotor activity or produce a conditioned place preference/aversion in control animals that lacked DREADD expression, providing evidence that DCZ alone does not induce noticeable off-target behavioral alterations. Taken together, our findings suggest that DCZ is an effective ligand for use with hM3Dq and hM4Di DREADDs in rats and supports prior evidence that DCZ is a superior DREADD ligand that does not appear to produce off-target behavioral or physiological outcomes.
The goal of the hM3Dq cFos experiment was to assess whether low dose DCZ could produce a robust and selective activation of cells expressing the excitatory DREADD, hM3Dq in rats. We chose cFos immunolabeling as the readout because induction of cFos protein is a widely used index of neuronal activity with a temporally defined expression window14,15. DCZ-mediated activation of hM3Dq resulted in robust induction of cFos expression in hM3Dq-expressing cells and surrounding non-infected cells in the viral injected hemisphere, but did not induce any detectable cFos expression in the opposite non-injected control hemisphere. Because identical imaging settings were used for both hemispheres, it is possible that DCZ induced very low levels of cFos in the control hemisphere that we did not detect. However, given the robust staining in the virus hemisphere, any sparse cFos expression in other brain regions is likely negligible. Overall, results from this set of experiments reveal that systemically adminstered DCZ robustly and selectively activates hM3Dq-expressing neurons in vivo.
Prior studies that assessed hM4Di-mediated inhibition with CNO in slice electrophysiology applications typically monitored changes in membrane potential, action potential firing, rheobase, etc., and reported near-maximal inhibitory responses with 5 or 10 uM CNO16–19. The highest CNO concentration used in the present study was 1 uM, which produced ~40% inhibition of firing. This result appears appropriate relative to studies using 5 or 10-fold higher CNO concentrations that appear to achieve near-maximal inhibition. However, the same concentration of DCZ (1 uM) produced ~75% percent inhibition, providing further support that DCZ offers better potency for hM4Di-mediation inhibition, relative to CNO.
For the hM4Di DCZ behavioral experiments, we chose to examine ethanol dependence-induced escalation of drinking as a number of previous studies have consistently demonstrated that the central amygdala is necessary for escalated intake in ethanol dependent animals20–22. To our knowledge, this is the first report to examine the effect of DREADD hM4Di inhibition of the central amygdala on dependence-induced escalation of drinking. While we observed that both DCZ and CNO-mediated hM4Di inhibition of the central amygdala reduced ethanol intake to a similar extent, DCZ was effective at a 100-fold lower dose. This finding is consistent with the slice electrophysiological observations of concentration differences and supports the contention that DCZ is a superior DREADD ligand13. In addition to a reduction in ethanol intake in dependent rats in response to systemic administration of either DCZ or CNO, we also observed a significant reduction in ethanol consumption in the non-dependent rats that had not undergone CIE exposure. A prior study reported a similar reduction in ethanol intake in non-dependent rats using a chemogenetic Daun02 inactivation approach to inhibit central amygdala neuronal ensembles23. Therefore, these observations are consistent with the suggestion that while global inhibition of the CeA may reduce ethanol intake regardless of dependence status, inhibition of specific cell-types and circuits in the CeA may selectively reduce ethanol intake in dependent rats20.
The present study also assessed whether systemic DCZ used in the prior experiments produced changes in gross locomotor behavior or was intrinsically rewarding/aversive in control rats lacking DREADD expression. Locomotor activity was utilized as it is a high-throughput assay that captures obvious changes in motor function. Furthermore, prior reports have observed changes in gross locomotor activity following administration of other DREADD ligands12,24. However, it should be noted that while we did not observe any gross changes in locomotor activity following administration of DCZ, we cannot rule out potential changes to fine locomotor movements that would be captured by more nuanced analyses.
To assess whether DCZ is intrinsically rewarding or aversive we used a place conditioning assay to determine whether repeated injections of DCZ paired with a distinct context could produce a place preference/aversion25,26. The results of these experiments revealed that DCZ did not support a conditioned place preference or aversion of the DCZ-paired compartment. Thus, systemic administration of the same low dose of DCZ that was shown to be an effective agonist of DREADD receptors in the cFos induction experiments, slice electrophysiological analysis of neuronal firing, and ethanol drinking, did not exhibit intrinsic rewarding or aversive properties. These control experiments provide encouraging evidence that DCZ alone does not produce obvious non-specific behavioral effects.
In summary, our findings demonstrate that systemic DCZ is effective for hM3Dq and hM4Di DREADD activation studies in rats. While the seminal publication on DCZ use was carried out in mice13, it is important to establish its effectiveness in rats to account for potential species differences in the response to DCZ and not simply rely of extrapolation of observations between species. For example, rats and mice have different sensitivities to the interoceptive effects of CNO and exhibit different metabolite profiles following clozapine and CNO administration11. Compared to other available DREADD ligands, DCZ is a promising tool with advantages including increased potency and lack of apparent off-target effects at concentrations that we demonstrate are effective in-vivo for DREADD activation. Moreover, DCZ provides a substantial cost savings benefit due to the low dose requirement relative to CNO and other DREADD ligands. Our observations in rats are consistent with the original findings in primates and mice13, that DCZ is a clear upgrade from past DREADD ligands and should provide researchers with an improved tool for DREADD activation.