Increased CRF signalling in a ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety during nicotine withdrawal

Abstract

Increased anxiety is a prominent withdrawal symptom in abstinent smokers, yet the neuroanatomical and molecular bases underlying it are unclear. Here we show that withdrawal-induced anxiety increases activity of neurons in the interpeduncular intermediate (IPI), a subregion of the interpeduncular nucleus (IPN). IPI activation during nicotine withdrawal was mediated by increased corticotropin releasing factor (CRF) receptor-1 expression and signalling, which modulated glutamatergic input from the medial habenula (MHb). Pharmacological blockade of IPN CRF1 receptors or optogenetic silencing of MHb input reduced IPI activation and alleviated withdrawal-induced anxiety; whereas IPN CRF infusion in mice increased anxiety. We identified a mesointerpeduncular circuit, consisting of ventral tegmental area (VTA) dopaminergic neurons projecting to the IPN, as a potential source of CRF. Knockdown of CRF synthesis in the VTA prevented IPI activation and anxiety during nicotine withdrawal. These data indicate that increased CRF receptor signalling within a VTA-IPN-MHb circuit triggers anxiety during nicotine withdrawal.

Figure 1. The IPN is a neuroanatomical substrate of anxiety during nicotine withdrawal

a. Average number of marbles buried in control and chronic nicotine-treated mice after saline (n = 13 mice/group) or mecamylamine (1 µg, n = 13 mice/group, and 3 µg, n = 12 and 13, respectively) infusion into the IPN. b. Average time spent in the open arms of the EPM in control or chronic nicotine-treated mice after an IPN infusion of saline (n = 10 mice/group) or mecamylamine (1 µg, n = 10 and 8 mice, respectively, and 3 µg, n = 10 mice/group). c. Average total arm entries in control or chronic nicotine-treated animals after IPN infusion of saline or mecamylamine from mice in panel b. d. Average number of marbles buried in control and chronic nicotine-treated mice after saline (n = 11 and 10, respectively) or nicotine (100 ng, n = 9 mice/group) infusion into the IPN. Behavior was measured 24 hr after control or nicotine solution was replaced with water. e. Average time spent in the open arms of the EPM in control or chronic nicotine-treated mice during spontaneous withdrawal after an IPN infusion of saline (n = 15 and 11, respectively) or nicotine (n = 9 and 10, respectively). f. Average total arm entries in withdrawn control or chronic nicotine-treated animals after IPN infusion of saline or nicotine from mice in panel e. Data are expressed as mean ± SEM. Two-way ANOVA, Bonferroni post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 for saline compared to drug infusion within groups. ^p < 0.05, ^^^p < 0.001 for drug infusion in nicotine-treated animals compared to infusions in control-treated animals.

Figure 2. CRF receptor signaling in the IPN mediates anxiety during nicotine withdrawal

a. Average number of marbles buried in control and chronic nicotine-treated, non-withdrawn mice after infusion of saline (n = 14 and 9, respectively) or 50 ng (n = 17 and 10, respectively) CRF into the IPN. b. Average time spent in the open arms of the EPM in control or chronic nicotine-treated, non-withdrawn mice after an IPN infusion of saline (n = 20 and 15 mice/respectively) or 50 ng (n = 17 and 10, respectively) CRF into the IPN. c. Average total arm entries in control or chronic nicotine-treated, non-withdrawn animals after IPN infusion of saline or CRF from panel b. d. Average number of marbles buried in control and chronic nicotine-treated mice after pre-infusion of Vehicle (Veh, 5% DMSO in saline, n=10 mice/group), the CRF1 receptor antagonist, antalarmin (1 µg, n = 12 and 13, respectively), or the CRF2 receptor antagonist, antisauvagine 30 (2 µg, n = 10 mice/group) prior to an infusion of mecamylamine (to precipitate withdrawal, 3 µg,) into the IPN. e. Average time spent in the open arms of the EPM in control or chronic nicotine-treated mice after an IPN pre-infusion of saline (n = 10 mice/group), antalarmin (n = 9 and 10) or antisauvagine 30 (n = 10 mice/group) prior to an infusion of mecamylamine. f. Average total arm entries in control or chronic nicotine-treated animals after IPN infusions as above. Data are expressed as mean ± SEM. Two-way ANOVA, Bonferroni post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 for saline compared to drug infusion within groups. ^p < 0.05, ^^p < 0.01, ^^^p < 0.001 for drug infusion in nicotine-treated animals compared to infusions in control-treated animals.

Figure 3. Mecamylamine-precipitated withdrawal activates neurons within the IPI of the IPN through a CRF1 receptor-dependent mechanism

a. Photomicrographs from coronal sections immunolabeled for c-Fos (green). Sections are taken from control or chronic nicotine-treated mice given i.p. injections of saline, mecamylamine (Mec, 3 mg kg⁻¹) or antalarmin (Ant, 10 mg kg⁻¹) prior to mecamylamine as indicated in the left-hand column. IPN sub-regions are indicated by dotted lines. b. Each bar graph represents the average total number of c-Fos(+) IPI neurons after a mecamylamine i.p. injection or an injection of antalarmin 15 min prior to mecamylamine in control-treated (n = 4 mice/treatment, 16 slices/mouse) or chronic nicotine-treated mice (n = 3 and 5 mice/treatment, 16 slices/mouse). c. Photomicrograph from representative coronal sections after FISH with a control (left) or CRF1 receptor probe. Note a robust CRF1 receptor signal is predominantly localized to the IPI sub-region of the IPN. d. Photomicrograph of coronal sections from chronic nicotine-treated mice given a CRF infusion (300 ng) into the IPN. Sections are double-labeled for CRF1 receptor mRNA (using FISH, red) and c-Fos protein (using immunohistochemistry, green). Merged sections imaged at 10X magnification are shown (middle). Scale bar: 200 µm. Outside sections shown at 63X magnification illustrate localization of c-Fos in CRF1 receptor-expressing IPI neurons. Scale bar: 20 µm. Data are expressed as mean ± SEM. Two-way ANOVA, Bonferroni post-hoc test. ***p < 0.001 compared to saline pre-injection within groups. ^^p < 0.01 chronic nicotine-treated compared to control-treated mice.

Figure 4. CRF modulates glutamatergic activity in a subset of IPI neurons

Representative whole-cell voltage clamp recordings from Type 1 (a.) and Type 2 (d.) neurons within the IPI in response to CRF. Average sEPSC frequency and amplitude at baseline, after a 5-minute application of CRF, and after washout from Type 1 (b., c., respectively, n = 8 neurons) and Type 2 (e., f., respectively: One-way ANOVAs with repeated measures: Frequency, F_{2, 12} = 19.2, p = 0.0002; Amplitude, F_{2, 12} = 10.49, p = 0.0023, n = 7) IPI neurons. Representative whole-cell voltage clamp recordings from Type 1 (g.) and Type 2 (j.) nicotine-withdrawn IPI neurons in response to antalarmin. Average sEPSC frequency and amplitude at baseline, after a 5-minute application of antalarmin, and after washout from Type 1 (h., i., respectively, n = 8) and Type 2 (k., l., respectively: Frequency, F_{2, 14} = 17.22, p = 0.0002; Amplitude, F_{2, 14} = 13.66, p = 0.0005, n = 8) IPI neurons. Neurons were recorded in slices from nicotine-dependent mice. Scale bar: y = 20 pA, × = 5 s. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01.

Figure 5. Glutamatergic signaling in the IPN is critical for expression of anxiety-like behavior during nicotine withdrawal

a. Average number of marbles buried in control and chronic nicotine-treated mice after infusion of saline prior to saline (n = 8 mice/group), saline prior to mecamylamine (3 µg, n = 10 mice/group), or AP5 (0.3 µg) prior to mecamylamine (n = 9 and 10, respectively) into the IPN. b. Average time spent in the open arms of the EPM in control or chronic nicotine-treated mice after infusion of saline (n = 12 mice/group) or AP5 (n = 9 and 10, respectively) prior to mecamylamine into the IPN. c. Average total arm entries in control or chronic nicotine-treated animals after infusion of saline or AP5 prior to mecamylamine from mice in panel b. d. Representative IPI sections from control or chronic nicotine-treated mice after IPN infusion of Sal + Mec, or AP5 + Mec. Sections were immunolabeled for GAD2/1 (red, left panels) and c-Fos (green, middle panels). Right panels depict merged signals. Scale bar: 100 µm. e. Averaged total number of c-Fos immunopositive IPI neurons for each condition in panel d (control mice, n = 3 mice/treatment, 16 slices/mouse; chronic nicotine-treated mice, n = 4 mice/treatment, 16 slices/mouse). Data are expressed as mean ± SEM. Two-way ANOVA, Bonferroni post-hoc test. **p < 0.01, ***p < 0.001. ^p < 0.05, ^^^p < 0.001 for drug infusion in nicotine-treated animals compared to infusions in control-treated animals.

Figure 6. IPN neurotransmission from the MHb is necessary for anxiety during nicotine withdrawal and activation of IPI neurons

In vivo halorhodopsin stimulation paradigm for analysis of the effect of MHb input on activation of IPI neurons during mecamylamine-precipitated nicotine withdrawal for light non-stimulated (a.) and light-stimulated (b.) mice expressing eNpHR-eYFP. Middle, photomicrographs from representative MHb and IPN coronal sections from each group illustrating eNpHR-eYFP expression (yellow). Bottom, representative coronal sections immunostained for c-Fos (left panel, red) and imaged for detection of eYFP (middle panel, yellow). Merged signals are depicted in the right-hand panel. c. Average number of c-Fos immunopositive IPI neurons after mecamylamine-precipitated nicotine withdrawal in light non-stimulated (Light-off, 16 slices/mouse, n = 4) and light-stimulated (Light on, 16 slices/mouse, n = 5) mice expressing eNpHR-eYFP. ***p < 0.001, two-tailed student’s t-test. d. In vivo halorhodopsin stimulation paradigm for analysis of the effect of MHb input on MBT and EPM assays during spontaneous nicotine withdrawal. e. Number of marbles buried in nicotine-dependent, non-withdrawn (Nicotine) or nicotine-dependent, spontaneous withdrawn ChAT-cre mice (Withdrawal) expressing either control virus (Control, n = 9) or halorhodopsin (eNpHR-eYFP, n = 9 and 12, respectively). Behaviors in all groups were done during light stimulation. e. Time spent on the open arms of the EPM in nicotine-dependent, non-withdrawn (Nicotine) or nicotine-dependent, spontaneous withdrawn ChAT-cre mice (Withdrawal) expressing either control virus (Control, n = 8) or halorhodopsin (eNpHR-eYFP, n = 9 and 12, respectively). Behaviors in all groups were done during light stimulation. f. Total arm entries in the EPM for each group. Data are expressed as mean ± SEM. Two-way ANOVA, Bonferroni post-hoc test. **p < 0.01, ***p < 0.001 compared within groups. ^p < 0.05, ^^^p < 0.001 withdrawn, control virus-expressing mice compared to non-withdrawn control virus-expressing mice.

Figure 7. Knock-down of CRF synthesis in the VTA reduces activity of IPI neurons and anxiety during nicotine withdrawal

a. Representative photomicrographs from coronal sections containing the VTA (top). C57Bl/6J mouse brains were infected with control shRNA (ctrlRNA, left columns) or CRF shRNA (right columns) and were analyzed for viral infection (green). Bottom, qRT-PCR comparing CRF (left) or TH (right) gene expression in ctrlRNA- and shRNA-infected VTA (n = 6 mice/group). Gene expression was normalized to GAPDH. ***p < 0.001, two-tailed student’s t-test. b. Top, photomicrographs depicting the IPI c-Fos pattern (green) induced by mecamylamine-precipitated withdrawal in chronic nicotine-treated mice expressing ctrlRNA (left) or CRF shRNA (right) in the VTA. Bottom, quantitative analysis of the number of IPI c-Fos immunopositive neurons during chronic nicotine before withdrawal and after mecamylamine-precipitated nicotine withdrawal from mice infected with ctrlRNA (n = 3 and 4 mice/group, 14 slices/mouse) or CRF shRNAs (n = 3 mice/group, 14 slices/mouse). c. Top, photomicrographs depicting the IPR c-Fos pattern (green) induced by mecamylamine-precipitated withdrawal in chronic nicotine-treated mice expressing ctrlRNA (left) or CRF shRNA (right) in the VTA. Bottom, quantitative analysis of the number of IPR c-Fos immunopositive neurons during chronic nicotine before withdrawal and after mecamylamine-precipitated nicotine withdrawal from mice infected with ctrlRNA (n = 3 and 4 mice/group) or CRF shRNAs (n = 3). d. Number of marbles buried in mice expressing ctrlRNA or CRF shRNA in the VTA during chronic nicotine exposure prior to withdrawal (n = 15 and 18, respectively) and during spontaneous nicotine withdrawal (n = 11 mice/group). e. Time spent on the open arms of the EPM for mice expressing ctrlRNA or CRF shRNA in the VTA during chronic nicotine exposure prior to withdrawal (n = 15 and 20, respectively) and during spontaneous nicotine withdrawal (n = 11 mice/group). f. Total arm entries in the EPM for each group in panel e. Data are expressed as mean ± SEM. Scale Bar: 100 µm. Two-way ANOVA, Bonferroni post-hoc test. **p < 0.01, **p < 0.001. ^p < 0.05, ^^p < 0.01 for between group comparisons.

Figure 8. Mechanistic model of increased anxiety during nicotine withdrawal

a. The IPN receives glutamatergic input (Glu) from the MHb. Chronic nicotine induces CRF synthesis in a population of VTA DAergic neurons that innervate the ventral IPN including the IPI. In addition, chronic nicotine increases expression of the CRF1 receptor in type II neurons of the IPI. b. During nicotine withdrawal, CRF from the VTA is released and activates the upregulated CRF1 receptors on Type II IPI neurons, increasing signaling (speaker icon) and amplitude of sEPSCs (presumably through NMDA/AMPA receptors). CRF also increases sEPSC frequency indicating increased glutamate release, which stems from MHb inputs. We speculate that MHb terminals may also express the CRF1 receptor, which would underlie increased glutamate release during withdrawal.