Ventral tegmental area cholinergic mechanisms mediate behavioral responses in the forced swim test

Abstract

Recent studies revealed a causal link between ventral tegmental area (VTA) phasic dopamine (DA) activity and pro-depressive and antidepressant-like behavioral responses in rodent models of depression. Cholinergic activity in the VTA has been demonstrated to regulate phasic DA activity, but the role of VTA cholinergic mechanisms in depression-related behavior is unclear. The goal of this study was to determine whether pharmacological manipulation of VTA cholinergic activity altered behavioral responding in the forced swim test (FST) in rats. Here, male Sprague-Dawley rats received systemic or VTA-specific administration of the acetylcholinesterase inhibitor, physostigmine (systemic; 0.06 or 0.125mg/kg, intra-cranial; 1 or 2μg/side), the muscarinic acetylcholine receptor (AChR) antagonist scopolamine (2.4 or 24μg/side), or the nicotinic AChR antagonist mecamylamine (3 or 30μg/side), prior to the FST test session. In control experiments, locomotor activity was also examined following systemic and intra-cranial administration of cholinergic drugs. Physostigmine administration, either systemically or directly into the VTA, significantly increased immobility time in FST, whereas physostigmine infusion into a dorsal control site did not alter immobility time. In contrast, VTA infusion of either scopolamine or mecamylamine decreased immobility time, consistent with an antidepressant-like effect. Finally, the VTA physostigmine-induced increase in immobility was blocked by co-administration with scopolamine, but unaltered by co-administration with mecamylamine. These data show that enhancing VTA cholinergic tone and blocking VTA AChRs has opposing effects in FST. Together, the findings provide evidence for a role of VTA cholinergic mechanisms in behavioral responses in FST.

Keywords: Acetylcholine; Forced swim test; Mecamylamine; Physostigmine; Scopolamine; Ventral tegmental area.

Fig. 1

Effects of systemic physostigmine on immobility time in the FST and on total locomotor activity. A. Experimental timeline for the FST, including a 15 min pretest 24 hr prior to the FST test session. B. Administration of physostigmine led to increased immobility time in FST (p < 0.05, main effect of drug; p < 0.05, Tukey post-hoc for 0.125 mg/kg physostigmine versus saline). C. Systemic physostigmine administration did not alter locomotor activity as measured by locomotor photobeam breaks (p > 0.05, two-way repeated measures ANOVA).

Fig. 2

Brain-region specific physostigmine infusion: effects on immobility time in the FST and total locomotor activity. A. Experimental timeline for the FST experiment. Intra-VTA drug infusion was performed immediately prior to the 10 min FST test session. B. Administration of physostigmine increased immobility time in FST (p < 0.05, main effect of drug; p < 0.05, Tukey post-hoc for 2 μg/side physostigmine versus saline). C. Intra-VTA drug infusion did not significantly affect locomotor activity, as measured by photobeam breaks (p > 0.05, two-way repeated measured ANOVA). D. Immobility time in the FST following physostigmine infusion into a site 2 mm dorsal to the VTA. Administration of 2 μg/side physostigmine did not alter immobility time in the FST (p > 0.05, independent samples t-test). E. Histological verification of intra-cranial cannula placements. Filled circles indicate representative placements for intra-VTA infusions and open circles indicate representative placements for dorsal control infusions.

Fig. 3

Intra-VTA scopolamine effects on the FST immobility time and total locomotor activity. A. Scopolamine infusion into the VTA led to decreased immobility in the FST (p < 0.001, main effect of drug; p < 0.05, Tukey post-hoc for 2.4 μg/side scopolamine versus saline; p < 0.05, Tukey post-hoc for 24 μg/side scopolamine versus saline). B. Intra-VTA infusion of 24 μg/side scopolamine did not alter locomotor activity, as measured by photobeam breaks (p > 0.05, two-way repeated measures ANOVA). C. Representative cannula placements for intra-VTA infusions in the scopolamine experiment.

Fig. 4

Intra-VTA mecamylamine effects on the FST immobility time and total locomotor activity. A. Mecamylamine infusion into the VTA leads to decreased immobility time in the FST (p < 0.05, main effect of drug; p < 0.05, Tukey post-hoc for 30 μg/side mecamylamine versus saline). B. Intra-VTA infusion of 30 μg/side mecamylamine did not alter locomotor activity as measured by locomotor beam breaks (p > 0.05, two-way repeated measures ANOVA). C. Representative VTA cannula placements for the mecamylamine infusion experiment.

Fig. 5

Effect of scopolamine plus physostigmine co-administration or mecamylamine plus physostigmine co-administration on immobility time in the FST. A. VTA scopolamine plus physostigmine experiment. Intra-VTA physostigmine administration increased immobility time in the FST (p < 0.05, Tukey post-hoc of 2 μg/side physostigmine versus saline), but co-administration with scopolamine blocked the physostigmine-induced increase in immobility (p < 0.05, Tukey post-hoc of 2 μg/side physostigmine plus 24 μg/side scopolamine versus scopolamine alone; p > 0.05, Tukey post-hoc of 2 μg/side physostigmine plus 24 μg/side scopolamine versus saline). B. VTA mecamylamine plus physostigmine experiment. Intra-VTA 2 μg/side physostigmine (in a separate cohort from that in panel A) increased immobility time in the FST (p < 0.05, Tukey post-hoc of 2 μg/side physostigmine versus saline). Intra-VTA 30 μg/side mecamylamine co-administration with 2 μg/side physostigmine did not alter the physostigmine-induced increase in immobility time (p > 0.05, Tukey post-hoc of 2 μg/side physostigmine plus 30 μg/side mecamylamine versus to 2 μg/side alone; p < 0.05, Tukey post-hoc of 2 μg/side physostigmine plus 30 μg/side mecamylamine versus saline). C. Systemic 0.125 mg/kg physostigmine plus intra-VTA mecamylamine or scopolamine experiment. Systemic administration of physostigmine led increased immobility time (p < 0.001, Tukey post-hoc of 2 μg/side physostigmine versus saline). Intra-VTA infusion of 30 μg/side mecamylamine co-administered with systemic 0.125 mg/kg physostigmine also increased immobility time compared to saline administered rats (p < 0.05, Tukey post-hoc), with no difference between physostigmine alone versus physostigmine plus mecamylamine (p > 0.05, Tukey post-hoc). Intra-VTA infusion of 24 μg/side scopolamine co-administered with systemic 0.125 mg/kg physostigmine increased immobility time compared to control rats (p < 0.05, Tukey post-hoc), with no difference between physostigmine alone versus physostigmine plus scopolamine (p > 0.05, Tukey post-hoc). D. Histological verification of cannula placements. Dark filled circles indicate representative placements for the scopolamine plus physostigmine experiments. Light filled circles indicate representative placements for the mecamylamine plus physostigmine experiments.