Activation of GABAergic neurons in the interpeduncular nucleus triggers physical nicotine withdrawal symptoms

Abstract

Background: Chronic exposure to nicotine elicits physical dependence in smokers, yet the mechanism and neuroanatomical bases for withdrawal symptoms are unclear. As in humans, rodents undergo physical withdrawal symptoms after cessation from chronic nicotine characterized by increased scratching, head nods, and body shakes.

Results: Here we show that induction of physical nicotine withdrawal symptoms activates GABAergic neurons within the interpeduncular nucleus (IPN). Optical activation of IPN GABAergic neurons via light stimulation of channelrhodopsin elicited physical withdrawal symptoms in both nicotine-naive and chronic-nicotine-exposed mice. Dampening excitability of GABAergic neurons during nicotine withdrawal through IPN-selective infusion of an NMDA receptor antagonist or through blockade of IPN neurotransmission from the medial habenula reduced IPN neuronal activation and alleviated withdrawal symptoms. During chronic nicotine exposure, nicotinic acetylcholine receptors containing the β4 subunit were upregulated in somatostatin interneurons clustered in the dorsal region of the IPN. Blockade of these receptors induced withdrawal signs more dramatically in nicotine-dependent compared to nicotine-naive mice and activated nonsomatostatin neurons in the IPN.

Conclusions: Together, our data indicate that therapeutic strategies to reduce IPN GABAergic neuron excitability during nicotine withdrawal, for example, by activating nicotinic receptors on somatostatin interneurons, may be beneficial for alleviating withdrawal symptoms and facilitating smoking cessation.

Figure 1. Mecamylamine precipitates withdrawal in nicotine-dependent mice

A) Experimental strategy for inducing nicotine dependence/withdrawal in C57Bl/6J mice, quantifying symptoms, and perfusing brains for immunohistochemistry experiments illustrated in Figure 1. B) Averaged total somatic withdrawal signs in control and nicotine-treated animals after saline or mecamylamine (1 mg/kg, i.p., n = 5 mice/treatment) injection. Two-way ANOVA: Significant effect of chronic treatment (F_{1, 16} = 25.79, p < 0.0001), drug (F_{1, 16} = 18.16, p < 0.001), and a significant interaction (F_{1, 16} = 9.69, p < 0.01). C) Averaged number of individual nicotine withdrawal symptoms from (A). Two-way ANOVA: Scratches, significant effect of drug (F_1,16 = 17.39, p < 0.001), and chronic treatment (F_{1, 16} = 13.2, p < 0.01); Body Shakes, significant effect of drug (F_1,16 = 26.64, p < 0.001), chronic treatment (F_{1, 16} = 24.63, p < 0.001) and interaction (F_{1, 16} = 12.77, p < 0.01); Head Nods, significant effect of drug (F_1,16 = 31.74, p < 0.001), chronic treatment (F_{1, 16} = 29.93, p < 0.001) and interaction (F_{1, 16} = 24.81, p < 0.001). D) Average number of marbles buried in the MBT in control and nicotine-treated animals after saline or mecamylamine injection (n = 7–24 mice/treatment). Two-way ANOVA: Significant effect of chronic treatment (F_{1, 57} = 7.93, p < 0.01), not drug, and a significant interaction (F_{1, 57} = 8.47, p < 0.01). E) Average time spent in the open arms of the EPM in mice (n = 7–10) treated as in (D). Two-way ANOVA: Significant effect of drug (F_{1, 31} = 12.79, p < 0.01). F) Average total arm entries in the EPM in mice from (E). Error bars indicate standard error of the mean (SEM). * p < 0.05, **p < 0.01, ***p < 0.001. Bonferroni post-hoc test.

Figure 2. Nicotine withdrawal activates IPN GABAergic neurons

A) Representative photomicrographs of IPN slices immunolabeled for GAD2/1 (green, top panels) and c-Fos (red, middle panels) from control or nicotine-treated mice that received saline (Sal) or mecamylamine (Mec) injections as indicated. Bottom panels depict merged signals. Insets show 630x images. Co-localization of c-Fos in GAD immunopositive neurons was apparent after mecamylamine injections. Note that a similar pattern of c-Fos labeling after mecamylamine challenge was observed in the IPN of mice that self-administer nicotine in a 24-hr two-bottle choice assay (data not shown). B) Average number of c-Fos immunoreactive (ir) IPN neurons under each condition from panel (A) (n = 5 mice/treatment). Two-way ANOVA, Bonferroni post-hoc test. C) Representative whole-cell recordings illustrating sEPSCs from IPN neurons of control mice (left) and nicotine-withdrawn mice (right). D) Average sEPSC frequency (left) and amplitude (right) in IPN neurons of control (n=26 neurons/treatment) and nicotine-withdrawn mice (n=45 neurons/treatment). Error bars indicate SEM. Student’s t-test with Welch’s correction. *** p < 0.001.

Figure 3. Activation of GABAergic neurons triggers somatic withdrawal symptoms

A) Top, Schematic of experimental strategy and neuronal activation protocol. Bottom, averaged individual withdrawal symptom behaviors elicited by light exposure in chronic nicotine-treated GAD2-Cre mice infected with AAV2-eGFP (Control, white bars) or AAV2-ChR2-eYFP (black bars, n = 7 mice/condition). Unpaired two-tailed t-tests with Welch’s correction revealed that light stimulation induced a significant increase in Scratching (t_5.22 = 2.96), Body Shakes (t_6.54 = 18.2), Head Nods (t_5.52 = 26.2), Backings (t_6.14 = 3.24) and Other symptoms (t_4.27 = 3.49) in AAV2-CHR2-eYFP-infected mice compared to AAV2-eGFP-infected mice (n = 4–6 mice/group). Inset, total signs during light stimulation (unpaired two-tailed t-test, t₇ = 6.58). B) Average time spent in the open arms (top, left) and closed arms (top, right), and total arm entries (bottom, left) in the EPM during light stimulation in nicotine-treated GAD2-Cre mice infected with AAV2-eGFP or AAV2-CHR2-eYFP (n = 7–10 mice/group). Average number of marbles buried in the MBT in AAV2-CHR2-eYFP-infected GAD2-Cre mice at baseline prior to light stimulation and during light stimulation (bottom, right, n = 11). C) Representative IPN sections from AAV2-eGFP- and AAV2 ChR2-eYFP-infected mice after light stimulation. Sections were immunolabeled for c-Fos (red). Insets show 630x images. Co-localization of c-Fos in eYFP expressing neurons was apparent after light stimulation. D) Averaged total number of IPN c-Fos-ir neurons after light stimulation (t₁₀ = 5.18). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Glutamatergic signaling from MHb projections is critical for IPN neuronal activation during withdrawal and expression of somatic withdrawal symptoms

A) Representative IPN sections from control or chronic nicotine-treated mice after IPN infusion of Sal + Mec, or AP5 + Mec. Sections were immunolabeled for GAD2/1 (red, top panels) and c-Fos (green, middle panels). Bottom panels depict merged signals. Insets show 630x images and co-localization of c-Fos in GAD immunopositive neurons. B) Averaged total number of c-Fos-ir IPN neurons for each condition in panel A (control mice, n = 3 mice/treatment; chronic nicotine-treated mice, n=4 mice/treatment). C) Averaged number of individual spontaneous withdrawal signs in nicotine-dependent mice at baseline (prior to cessation), after IPN-infusion of saline, or after IPN-infusion of AP5 (n = 6 mice/treatment). One-way repeated measure ANOVA for each symptom indicated a significant main effect of infusion on Scratches (F_2,10 = 13.65, p < 0.01), Body Shakes (F_2,10 = 33.5, p < 0.001), (Head Nods, F_2,10 = 27.34, p < 0.001), (Backing, F_2,10 = 4.15, p < 0.05) and Others (F_{2, 10} = 4.96, p < 0.05). Inset, average total number of withdrawal symptoms. One-way repeated measures ANOVA indicated a significant main effect of infusion on total number of symptoms (F_2,10 = 30.73, p < 0.001). D) Averaged number of individual spontaneous withdrawal signs in nicotine-dependent mice undergoing spontaneous withdrawal after saline infusion (white bars) or lidocaine infusion (black bars) into the MHb (n = 6 mice/group). Unpaired two-tailed t-tests revealed that lidocaine induced a significant reduction in Scratching (t₁₀= 5.08), Body Shakes (t₁₀= 5.16), Head Nods (t₁₀= 8.64), and Other symptoms (t₁₀= 10.33) compared to saline infusion. Inset, total withdrawal symptoms after MHb saline or lidocaine infusions (unpaired two-tailed t-test, t₁₀ = 8.71). Error bars indicate SEM. * p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. A role for Sst interneuron nAChRs containing theβ4 subunit in somatic nicotine withdrawal symptoms

A) Comparison of nAChR subunit gene expression in Sst-immunopositive (top) and -immunonegative (bottom) neurons from the IPN of nicotine-naïve and chronic nicotine-treated mice. Fold change was calculated using the 2^−ΔΔCt method. Red dotted line represents equal gene expression between nicotine-naïve and chronic nicotine-treated animals (n = 3 mice/group). Insets, representative photomicrographs of IPN sections immunolabeled for Sst. White dotted lines represent regions where neurons were laser-captured for each analysis. * p < 0.05 gene expression of nAChR subunit from nicotine treated compared to control mice (Unpaired t-tests: α7: t₄ = 3.13; β3: t₄ = 3.85; β4: t₄ = 2.94). B) Top, representative whole-cell currents in response to 10 μM nicotine (bath-applied, black bars) in IPN Sst neurons from nicotine-naïve (left) and chronic nicotine-treated mice (right). Bottom, average peak currents from nicotine whole-cell responses in Sst neurons from nicotine-naïve and chronic nicotine-treated mice in the absence (white bar) and presence (black bar) of SR16584. ^ p < 0.05 compared to neurons from nicotine-naïve mice (t₂₆ = 1.89). ** p < 0.01 compared to control responses in neurons from nicotine-treated mice (t₂₀ = 3.02). C) Averaged number of individual spontaneous withdrawal signs in control and chronic nicotine-treated mice after IPN infusion of saline or SR16584 (n = 5–8 mice/group). Two-way ANOVA: Scratches, significant effect of antagonist (F_1,19 = 7.52, p < 0.05), and chronic treatment (F_{1, 19} = 5.14, p < 0.05); Body Shakes, significant effect of antagonist (F_1,19 = 22.02, p < 0.001), chronic treatment (F_{1, 19} = 9.18, p < 0.01) and interaction (F_{1, 19} = 7.63, p < 0.05); Head Nods, significant effect of antagonist (F_1,19 = 34.38, p < 0.001), chronic treatment (F_{1, 19} = 96.64, p < 0.001) and interaction (F_{1, 19} = 44.78, p < 0.001); Rearing, significant effect of drug (F_1,19 = 33.95, p < 0.001); Other symptoms, significant effect of antagonist (F_1,19 = 62.72, p < 0.001). D) Average total withdrawal symptoms from (C). Two-way ANOVA indicated a statistically significant effect of antagonist (F_1,19 = 9.09, p < 0.01) and chronic treatment (F_1,19 = 77.77, p < 0.001). Error bars indicate SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Bonferroni post-hoc.

Figure 6. Mechanistic model of somatic nicotine withdrawal symptom expression

A) The IPN receives glutamatergic input (Glu) from the MHb. Presynaptic glutamate release is controlled, in part, by Sst neurons which express nAChRs. Nicotine activates Sst neurons through nAChRs containing the β3 and β4 subunits (β3β4* nAChRs) inhibiting MHb glutamate release (Red arrow). With chronic nicotine exposure these nAChRs are upregulated. B) During nicotine withdrawal, decreased nAChR signaling either through nicotine cessation or via nAChR blockade with an antagonist, reduces Sst neuron activation, disinhibiting glutamate release from MHb presynaptic terminals. The increased glutamate release stimulates activity of IPN GAD2-expressing (presumably projection) neurons eliciting somatic withdrawal signs. C) In the optogenetic experiments, ChR2 is expressed in GAD2-expressing neurons only. Light stimulation ultimately drives activation of these neurons triggering somatic withdrawal signs.