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. 2016 Aug:107:294-304.
doi: 10.1016/j.neuropharm.2016.03.039. Epub 2016 Mar 26.

Habenula cholinergic neurons regulate anxiety during nicotine withdrawal via nicotinic acetylcholine receptors

Xueyan Pang  1 , Liwang Liu  2 , Jennifer Ngolab  1 , Rubing Zhao-Shea  2 , J Michael McIntosh  3 , Paul D Gardner  1 , Andrew R Tapper  4
Affiliations

Habenula cholinergic neurons regulate anxiety during nicotine withdrawal via nicotinic acetylcholine receptors

Xueyan Pang et al. Neuropharmacology. 2016 Aug.

Abstract

Cholinergic neurons in the medial habenula (MHb) modulate anxiety during nicotine withdrawal although the molecular neuroadaptation(s) within the MHb that induce affective behaviors during nicotine cessation is largely unknown. MHb cholinergic neurons are unique in that they robustly express neuronal nicotinic acetylcholine receptors (nAChRs), although their behavioral role as autoreceptors in these neurons has not been described. To test the hypothesis that nAChR signaling in MHb cholinergic neurons could modulate anxiety, we expressed novel "gain of function" nAChR subunits selectively in MHb cholinergic neurons of adult mice. Mice expressing these mutant nAChRs exhibited increased anxiety-like behavior that was alleviated by blockade with a nAChR antagonist. To test the hypothesis that anxiety induced by nicotine withdrawal may be mediated by increased MHb nicotinic receptor signaling, we infused nAChR subtype selective antagonists into the MHb of nicotine naïve and withdrawn mice. While antagonists had little effect on nicotine naïve mice, blocking α4β2 or α6β2, but not α3β4 nAChRs in the MHb alleviated anxiety in mice undergoing nicotine withdrawal. Consistent with behavioral results, there was increased functional expression of nAChRs containing the α6 subunit in MHb neurons that also expressed the α4 subunit. Together, these data indicate that MHb cholinergic neurons regulate nicotine withdrawal-induced anxiety via increased signaling through nicotinic receptors containing the α6 subunit and point toward nAChRs in MHb cholinergic neurons as molecular targets for smoking cessation therapeutics.

Keywords: Acetylcholine; Anxiety; Habenula; Nicotine; Withdrawal.

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Figures

Figure 1
Figure 1
α4* nAChRs are necessary and sufficient for nicotine activation of MHb cholinergic neurons. A. Representative images of coronal sections from the MHb of C57BL/6J wild type mice, α4 knockout mice or L9′A mutant mice injected with saline or nicotine. Immunolabeling was performed to detect c-Fos expression (green, left columns) and ChAT expression (red, middle columns). Merged images are represented in the right column. The MHb is highlighted by the white-dotted line. B. Quantification of the number of c-Fos immunopositive neurons within the MHb after each drug treatment (10–15 slices analyzed per mice, n = 3 mice/treatment). Note that L9′A mice were not tested at the 3 mg/kg dose (N.A. = Not Analyzed). *** p < 0.001, Student’s t test.
Figure 2
Figure 2
Selective expression of agonist-hypersensitive α4* nAChRs in MHb cholinergic neurons. A. Schematic diagram of the double-floxed Cre-dependent AAV vector expressing L9′S α4-YFP nAChR subunit cDNA under the control of the Ef-1α promoter. B. Left, Representative images of coronal sections illustrating the expression of L9′S-YFP (green signal) in the MHb (20x). Right, 60x photomicrograph from the section indicated in the square outline of the 20x image. YFP signal (green, top panel), cholinergic signal (red, middle panel) and merged signals (yellow, bottom panel) are shown. C. Representative whole-cell voltage-clamp recording from eGFP-positive (left, control) or L9′S-YFP-positive neurons (middle) in response to 0.3 mM ACh in acute MHb slices from ChAT-Cre mice. Average currents recorded from eGFP- and L9′S-YFP-positive neurons are shown on the right. D. Locomotor activity (ambulation) did not differ between Control-infected and AAV2 L9′S-infected ChAT-Cre mice. Each data point represents summed 5 min total ambulation. E. Time spent in the open arms of the EPM in Control- and AAV2 L9′S-infected ChAT-Cre mice (left). Total EPM arm entries of each group (right). F. Cumulative number of buried marbles at different time points in the MBT from Control-infected and AAV2 L9′S-infected ChAT-Cre mice. Inset, averaged marbles buried at t=20 min. Data are expressed as average values ± S.E.M. *p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Increased anxiety-like behavior is mediated by increased nAChR activity in mice expressing Leu9′Ser α4-YFP nAChRs in MHb cholinergic neurons. A. Time spent in the open arms of the EPM in Control- and AAV2 L9′S-infected ChAT-Cre mice after challenge with saline (Sal) or low dose mecamylamine (Mec, 0.3 mg/kg, i.p.). B. Total EPM arm entries of each group from panel A. C. Cumulative number of buried marbles at different time points in the MBT from control-infected ChAT-Cre mice challenged with Sal or Mec. Inset, averaged marbles buried at t=20 min. D. Cumulative number of buried marbles at different time points in the MBT from AAV2-L9′S-infected ChAT-Cre mice challenged with Sal or Mec. Inset, averaged marbles buried at t=20 min. E. Time spent in the open arms of the EPM in Control- and AAV2 L9′S-infected ChAT-cre mice after infusion of vehicle (Veh) or low dose mecamylamine (Mec, 0.5 µg). F. Total EPM arm entries of each group from panel E. G. Cumulative number of buried marbles at different time points in the MBT from control-infected ChAT-cre mice infused with Veh or Mec. H. Cumulative number of buried marbles at different time points in the MBT from AAV2-L9′S-infected ChAT-cre mice infused with Veh or Mec. ^^p<0.01 compared to between group control. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to within group vehicle.Data are expressed as average values ± S.E.M.
Figure 4
Figure 4
Nicotine regulation of MHb α4/α6β2* nAChRs mediates increased anxiety during nicotine withdrawal. A. Time spent in the open arms of the EPM in control and nicotine withdrawn mice after vehicle (Veh), α-conotoxin MII[E11A] (α-Ctx[E11A]), DHβE, or SR16584 infusion into the MHb. B. Total arm entries in the EPM from the groups in panel A. C. Total number of marbles buried in the MBT (t = 20 min) in control and nicotine withdrawn mice after Veh, a-Ctx[E11A], DHβE, or SR16584 infusion into the MHb. Data are expressed as average values ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, Two-way ANOVA, Bonferroni post-hoc. D. Representative whole-cell response to 10 µM nicotine under control conditions and in the presence of 100 nM α-Ctx[E11A], 1 µM DHβE, or 20 mM SR16584 in MHb slices from control-treated mice (left recordings) and chronic nicotine-treated mice (right recordings). B. Averaged peak whole-cell current responses to 10 mM nicotine from the indicated groups as recorded in panel D from control mice (top) or chronic nicotine-treated mice (bottom). Data are expressed as average values ± S.E.M. **p < 0.01, ***p < 0.001 compared to Veh within groups.
Figure 5
Figure 5
Expression of α4* nAChRs in ventral medial MHb. A. Representative whole-cell traces from the medial ventral portion of the MHb in WT and α4 KO mice (top, (recorded region is highlighted in inset). Averaged peak current in response to 1 mM nicotine in WT and α4 KO mice (bottom). B. A representative DNA agarose gel is shown illustrating a typical result from a cholinergic neuron from the ventral medial MHb. α4 and α6 nAChR subunit transcript was detected in 18 out of 24 neurons from C57Bl/6J mice (as recorded in Fig. 3). ** p < 0.01, unpaired t-test.
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