Hippocampal acetylcholine modulates stress-related behaviors independent of specific cholinergic inputs

Abstract

Acetylcholine (ACh) levels are elevated in actively depressed subjects. Conversely, antagonism of either nicotinic or muscarinic ACh receptors can have antidepressant effects in humans and decrease stress-relevant behaviors in rodents. Consistent with a role for ACh in mediating maladaptive responses to stress, brain ACh levels increase in response to stressful challenges, whereas systemically blocking acetylcholinesterase (AChE, the primary ACh degradative enzyme) elicits depression-like symptoms in human subjects, and selectively blocking AChE in the hippocampus increases relevant behaviors in rodents. We used an ACh sensor to characterize stress-evoked ACh release, then used chemogenetic, optogenetic and pharmacological approaches to determine whether cholinergic inputs from the medial septum/diagonal bands of Broca (MSDBB) or ChAT-positive neurons intrinsic to the hippocampus mediate stress-relevant behaviors in mice. Chemogenetic inhibition or activation of MSDBB cholinergic neurons did not result in significant behavioral effects, while inhibition attenuated the behavioral effects of physostigmine. In contrast, optogenetic stimulation of septohippocampal terminals or selective chemogenetic activation of ChAT-positive inputs to hippocampus increased stress-related behaviors. Finally, stimulation of sparse ChAT-positive hippocampal neurons increased stress-related behaviors in one ChAT-Cre line, which were attenuated by local infusion of cholinergic antagonists. These studies suggest that ACh signaling results in maladaptive behavioral responses to stress if the balance of signaling is shifted toward increased hippocampal engagement.

Figure 1:

ACh levels increase in hippocampus in response to stress. A genetically-encoded fluorescent ACh sensor (AAV1-GRAB_ACh3.0) was infused into the dorsal and ventral hippocampus to record ACh-induced fluorescent transients (A) in mice implanted with an optic fiber (insets). Animals experienced two foot shocks, 0.4 mA and 0.7 mA, for 1 sec. Traces represent the average fluorescence 50 sec before and after shock onset in ventral or dorsal hippocampus. Shaded areas represent bootstrapped 95% confidence intervals; n = 9 mice per group. To determine the behavioral consequences of increasing ACh levels in hippocampus, BAC-ChAT-Cre and ChAT-KI-Cre male mice were infused with AAV2-DREADD-carrying virus into the medial septum with some diffusion in the diagonal band (B) allowing modulation of the activity of MSDBB cholinergic neurons with CNO (10 mg/kg, i.p.) (C). Time spent in the light side of the light-dark box (BAC-ChAT-Cre (D), ChAT-KI-Cre (H)). Time spent immobile in the tail suspension test (BAC-ChAT-Cre (E), ChAT-KI-Cre (I) or forced swim test (BAC-ChAT-Cre (F), ChAT-KI-Cre (J)). Ratio of interaction (time with CD1/time without CD1) following 8 bouts of social defeat (BAC-ChAT-Cre (G), ChaT-KI-Cre (K)). Results are expressed as Mean +/− SEM. *: p < 0.05; **: p < 0.01. BAC-Chat-Cre: Control, n = 10; Gq, n = 9; Gi, n = 12. ChAT-KI-Cre: Control, n = 11; Gq, n = 13; Gi, n = 11. Red dots represent data from animals with mistargeted placements (provided for reference and not included in calculation of means or SEM (see example in supp. Fig)).

Figure 2:

Effects of optogenetic stimulation of hippocampal terminal fields on anxiety-related behaviors following ChR2 infusion in the medial septum of C57BL/6J mice. AAV2-ChR2 was infused into the MSDBB (A), and an optic fiber was placed above the dorsal hippocampus (B) allowing stimulation of the septo-hippocampal pathway (C). Time spent in the light side of the light-dark box (D). Time spent immobile in the tail suspension test (E). Time to first feeding episode in the novelty-suppressed feeding test (F). Results are expressed as Mean +/− SEM. *: p < 0.05. Control males, n = 9; ChR2 males, n = 9. Control females, n = 7; ChR2 females, n = 6.

Figure 3:

Behavioral effects of retrograde infusion of a Cre-dependent DREADD construct into the hippocampus of ChAT-Cre mouse lines. BAC-ChAT-Cre and ChAT-KI-Cre male mice were infused with a Gq-DREADD-carrying retrograde virus into the hippocampus (A) allowing activation of hippocampal cholinergic projections (with CNO 10 mg/kg) (B). Time spent in the light side of the light-dark box (BAC-ChAT-Cre (C), ChAT-KI-Cre (G)). Time spent immobile in the tail suspension test (BAC-ChAT-Cre (D), ChAT-KI-Cre (H)) or forced swim test (BAC-ChAT-Cre (E), ChAT-KI-Cre (I)). Ratio of interaction (time with CD1/time without CD1) following 8 social defeats (BAC-ChAT-Cre (F), ChaT-KI-Cre (J)). Results are expressed as Mean +/− SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001. BAC-Chat-Cre: Control, n = 9; Gq, n = 9. ChAT-KI-Cre: Control, n = 9; Gq, n = 10.

Figure 4:

Stimulation of hippocampal ChAT-positive interneurons increases stress-relevant behavioral responses. BAC-ChAT-Cre and ChAT-KI-Cre male mice were infused with AAV2 carrying Cre-dependent Gq- or Gi-DREADD in the hippocampus (A), allowing modulation of cholinergic neurons intrinsic to the hippocampus with CNO (10 mg/kg) (B). Time spent in the light side of the light-dark box (BAC-ChAT-Cre (C), ChAT-KI-Cre (G)). Time spent immobile in the tail suspension test (BAC-ChAT-Cre (D), ChAT-KI-Cre (H)) or forced swim test (BAC-ChAT-Cre (E), ChAT-KI-Cre (I)). Ratio of interaction (time with CD1/time without CD1) following 8 social defeats (BAC-ChAT-Cre (F), ChaT-KI-Cre (J)). Results are expressed as Mean +/− SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001. BAC-Chat-Cre: Control, n = 9; Gq, n = 14; Gi, n = 9. ChAT-KI-Cre: Control, n = 9; Gq, n = 11; Gi, n = 11.

Figure 5:

Cholinergic receptor antagonists infused into the hippocampus decrease the effect of DREADD-mediated stimulation of hippocampal ChAT-Cre-expressing neurons. The effects of local infusion of cholinergic antagonists into the hippocampus were evaluated in BAC-ChAT-Cre male mice during Cre-dependent Gq-DREADD stimulation of the hippocampus with CNO (10 mg/kg), at baseline (+CSF), with mecamylamine (1 ng) or scopolamine (0.5 ng). Mice were infused with AAV2-Gq-DREADD into the hippocampus through a canula. After 3 weeks recovery, to allow for expression of the DREADD construct, animals were infused with either mecamylamine or scopolamine through the same guide canula 15 min before testing (A), allowing for activation of ChAT-positive neurons of the hippocampus by the DREADD construct and infusion of cholinergic antagonists into the same hippocampal area (B). Time spent in the light side of the light-dark box (A). Time spent immobile in the tail suspension (C) or forced swim test (D). Results are expressed as Mean +/− SEM. *: p < 0.05; **: p < 0.01. Control, n = 9; Gq+CSF, n = 10; Gq+MEC, n = 9; Gq+SCOP, n = 11. Red dots represent data from mistargeted animals (provided for reference and not included in calculation of means or SEM).