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, 7 (1), 900

Down-regulation of Cholinergic Signaling in the Habenula Induces Anhedonia-Like Behavior

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Down-regulation of Cholinergic Signaling in the Habenula Induces Anhedonia-Like Behavior

Seungrie Han et al. Sci Rep.

Erratum in

Abstract

Dysfunction of cholinergic signaling in the brain has long been believed to be associated with depressive disorders. However, the functional impact of habenular cholinergic signaling on the specified depressive behaviors is not well understood. Here, we demonstrated that the expression levels of cholinergic signaling genes (CHAT, VACHT, CHT, CHRNA3, CHRNB3 and CHRNB4) were down-regulated in a chronic restraint stress (CRS) rat model of depression, in which rats display depression-like behaviors such as anhedonia and mood despair. Moreover, knockdown of CHAT in the rat habenula was sufficient to evoke anhedonia-like behavior. The anhedonia-like behavior induced by CHAT knockdown was not reversed by chronic administration of the selective serotonin reuptake inhibitor fluoxetine. To determine whether habenular cholinergic signaling is associated with regulation of dopamine neurons in the ventral tegmental area (VTA) and serotonin neurons in the dorsal raphe nucleus (DRN), we used CHAT::cre transgenic mice expressing the Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Pharmacogenetic activation of habenular cholinergic neurons induces the excitation of dopamine neurons in the VTA and reduces the immunoreactivity of 5-hydroxytryptamine (5-HT) in the DRN. Habenular cholinergic gene down-regulation was recapitulated in the postmortem habenula of suicide victims diagnosed with major depressive disorder (MDD).

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Effect of chronic restraint stress on the expression of cholinergic system genes in the rat habenula. (a) Schematic diagram of a generalized cholinergic synapse. The choline transporter protein (CHT) delivers choline into the cytoplasm, where choline acetyltransferase (CHAT) catalyze the transfer of an acetyl group from the coenzyme, acetyl Co-A, to choline, generating acetylcholine, and the vesicular acetylcholine transporter (VACHT) packs acetylcholine into the vesicle. Presynaptic nicotinic acetylcholine receptors (nAChR; CHRNA3, CHRNB3 and CHRNB4) modulate the release of neurotransmitter. (bh) qRT-PCR was used to quantify mRNA expression levels of six cholinergic system genes in the habenula of rats exposed to CRS. The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were significantly reduced by CRS. Values for each individual gene were normalized to the mean of the reference gene GAPDH. CAMK2B was not changed by CRS. Data represent mean ± SEM. NS, n = 4; CRS, n = 4; *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney U-test.
Figure 2
Figure 2
Effects of CHAT knockdown in medial habenula on depression-like behaviors. (a) Schematic representation of the AAV vector engineered to induce CHAT knockdown. (b) Experimental paradigm for behavioral testing of rats infected with the virus. (c) Representative photomicrographs of Hb slices from the rats injected with AAV-sh-SCR (control) or AAV-sh-CHAT (CHAT knockdown). CHAT protein expression was visualized as red immunofluorescence. Cell nuclei and viral infection were visualized using Hoechst staining (blue) and GFP (green), respectively. The inset box shows a higher magnification of an infected MHb region. None of the neurons infected with the AAV-sh-CHAT showed CHAT expression. Scale bar, 50 μm. (d) VACHT expression is not affected by AAV-sh-CHAT. Hb neurons injected with AAV-sh-CHAT were immunostained for VACHT (red) and EGFP (green). Scale bar, 50 μm. (e) CHAT and VACHT western blots from the Hb of rats injected with AAV-sh-CHAT. Rats were injected with AAV-sh-SCR (control) or AAV-sh-CHAT, which were expressed for 3 weeks before western blot analysis. (f) Quantification of the effects of shRNA-mediated CHAT knockdown. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4; *P < 0.05, Student’s t-test). (g) VACHT expression is not affected by AAV-sh-CHAT. Data represent mean ± SEM (sh-SCR, n = 4 rats; sh-CHAT, n = 4) (hj) Behavioral effects of expressing AAV-sh-CHAT in the MHb on sucrose preference (h), total fluid intake during SPT test (i), and forced swim (j). Data represent mean ± SEM (sh-SCR, n = 20; sh-CHAT, n = 27; **P < 0.01, Student t-test).
Figure 3
Figure 3
Fluoxetine does not reverse the anhedonia-like behavior induced by CHAT knockdown. (a) Rats were infused with AAV-sh-SCR or AAV-sh-CHAT and chronically treated with saline or fluoxetine (5 mg/kg) from one week after AAV infections throughout the experiment. Chronic administration of fluoxetine did not recover the effects of AAV-sh-CHAT infusion in the sucrose preference test (b), though no changes of total fluid consumption (c) or immobile time in the FST (d). Data represent mean ± SEM (sh-SCR + Sal, AAFV-sh-SCR + saline, n = 9 rats; sh-CHAT + Flx, AAV-sh-CHAT + fluoxetine, n = 15; **P < 0.01, Student’s t-test).
Figure 4
Figure 4
In vivo activation of MHb cholinergic neurons induces the excitation of dopamine neurons in the ventral tegmental area. (a) Pharmacogenetic stimulation by DREADD mice. We injected ChAT::cre mice with Cre-dependent hM3Dq virus (AAV-hM3Dq-mCherry) after 1 week habituation. (b) The mCherry-fused hM3Dq protein was expressed in the CHAT-positive habenula cholinergic neurons. (c and d) c-Fos expression (green) was induced in habenula cholinergic neurons expressing hM3Dq-mCherry (red) following pharmacogenetic selective stimulation for 60 min using i.p. injection of CNO. (eh) CNO administration induced the increase of c-Fos immunoreactivity (green) in the TH-positive cells (i.e. dopamine neurons; white) and the decrease of c-Fos immunoreactivity in the TH-negative cells. Yellow arrow heads indicate the TH-positive and c-Fos-positive cells. (i) In CNO-injected DREADD mice, 64.5% of the dopamine neurons (n = 49 cells from 2 mice) and 35.5% of non-dopamine cells were c-Fos-positive. In contrast, saline-injected DREADD mice, 11.1% of the dopamine neurons (n = 10 cells from 2 mice) and 88.9% of non-dopamine cells were c-Fos-positive. Scale bar, (bf) 100 μm; (g and h), 10 μm.
Figure 5
Figure 5
MHb cholinergic neurons regulate the population of 5-HT-immunoreactive neurons in the dorsal raphe nucleus. (a and b) DREADD mice expressing Cre-dependent hM3Dq were pharmacogenetically stimulated for 60 min using i.p. injection of CNO. Cell nuclei and 5-HT were visualized using Hoechst staining (blue) and Cy3 (red), respectively. CNO administration reduced the number of 5-HT-immunoreactive neurons in the DRN. Data represent mean ± SEM (PBS, n = 7 mice; CNO, n = 8; ***P < 0.001, Student’s t-test). Scale bar, 50 μm. (c and d) CHAT knockdown effect on the number of 5-HT-immunoreactive neurons in the DRN. Immunohistochemical analysis was performed using rats that were used for behavioral tests in Fig. 2. Cell nuclei and 5-HT were visualized using Hoechst staining (blue) and Cy3 (red), respectively. CHAT knockdown increased the number of 5-HT-immunoreactive neurons in the DRN Data represent mean ± SEM (sh-SCR, n = 5 rats; sh-CHAT, n = 5; **P < 0.01, Student’s t-test). Scale bar, 100 μm.
Figure 6
Figure 6
Expression of cholinergic system genes in habenula tissue obtained from persons with MDD who died by suicide. (a) The human brain was separated into two hemispheres by slicing through the center of the brain stem and cerebellum. Each hemisphere was sliced coronally into 18–20 slabs (slab 1, frontal lobe; slab 18, occipital lobe) and these slabs were frozen. The habenula is nearly adjacent to the pineal gland and is contained in the 11th of 18 slabs. To isolate habenula samples, a carving tool with a round tip was used to make long grooves one at a time. (ci) The mRNA expression levels of CHAT, VACHT, CHT, CHRNA3, CHRNB3, and CHRNB4 were evaluated using TaqMan probe-based qRT-PCR. All cholinergic system genes examined were down-regulated in suicide, but not the control CAMK2B gene (b), an excitatory neuronal marker. GAPDH and β-Actin were used as reference genes to normalize qRT-PCR data. Data represent mean ± SEM (CON, Control, n = 11; MDD, n = 12 subjects; *P < 0.05, Mann–Whitney U-test).

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