Combined use of the canine adenovirus-2 and DREADD-technology to activate specific neural pathways in vivo

Abstract

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM3D(Gq)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM3D(Gq)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM3D(Gq)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM3D(Gq)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.

Figure 1. hM₃D(G_q)-mCherry is expressed on VTA to Acb neurons.

Depicted in the upper row are single section images of (A) hM₃D(G_q)-mCherry expression, (B) Tyrosine hydroxylase (TH) expression and (C) combined hM₃D(G_q)-mCherry and TH expression. hM₃D(G_q)-mCherry expression is restricted to the ventral tegmental area (VTA), as no mCherry expression could be observed in the substantia nigra pars compacta (SNpc) or surrounding areas. Depicted in the lower are single section confocal images of (D) hM₃D(G_q)-mCherry expression, (E) TH expression and (F) combined expression of hM₃D(G_q)-mCherry and TH. Two populations of hM₃D(G_q)-mCherry expressing VTA to nucleus accumbens (Acb) projection neurons can be distinguished, one population that does not express TH (vertical white arrows) and one population that does express TH (horizontal yellow arrows).

Figure 2. CNO increases PR-performance and locomotor activity.

(A) Injections of different doses of clozapine-N-oxide (CNO) led to transient increases in the number of active lever presses. (B) The number of inactive lever presses was dose-dependently affected by injections of CNO. The amount of lever presses are depicted as bars representing group means+SEM. *indicates a significant difference in lever presses between CNO and saline treatments of p<0.05. (C) Injections of CNO also had a transient and dose-dependent effect on locomotor activity. Data in C are presented as the group means of home cage activity ± SEM, binned into one hour periods. Shaded in light gray are circles and triangles that represent the effect of saline injections the day before and after CNO injection respectively. *indicates a significant difference between CNO and saline treatments in locomotor activity 3–7 hours after injections of p<0.05.

Figure 3. The effect of CNO on PR-performance is dose-dependently suppressed by a DRD1-antagonist.

(A) In seven animals cannulae were correctly placed in the nucleus accumbens (Acb). The outlined numbers represent the location of the ending of the guide cannulae, for each animal (1–8) individually. Abbreviations: AcbC = nucleus accumbens core, AcbSh = nucleus accumbens shell, CPu = caudate putamen. (B) Representative image (animal 5) of cannula placement in the Acb. The horizontal arrow indicates the ending of the cannula (C) Intra-Acb infusions of the D1-antagonist SCH22390 dose-dependently suppressed CNO-induced increases in the number of active lever presses. Data are depicted with bars representing group means+SEM, with * indicating a significant difference of i.p. CNO vs. i.p. saline of p<0.05 and & indicating a significant difference of intra-Acb SCH23390 vs. intra-Acb saline of p<0.05.