Research Article

Reexposure to nicotine during withdrawal increases the pacemaking activity of cholinergic habenular neurons

Andreas Görlich, Beatriz Antolin-Fontes, Jessica L. Ables, Silke Frahm, Marta A. Ślimak, Joseph D. Dougherty, and Inés Ibañez-Tallon
PNAS October 15, 2013 110 (42) 17077-17082; https://doi.org/10.1073/pnas.1313103110

  1. Edited by Jean-Pierre Changeux, Centre National de la Recherche Scientifique, Institut Pasteur, Paris, France, and approved September 3, 2013 (received for review July 11, 2013)

Significance

According to the World Health Organization, tobacco consumption causes the death of close to 6 million people each year, yet successful attempts to quit smoking are very rare. The present study identifies a group of neurons in the brain that respond differently to nicotine after a period of abstinence, suggesting that altered activity of these neurons may contribute to difficulties with smoking cessation.

Abstract

The discovery of genetic variants in the cholinergic receptor nicotinic CHRNA5-CHRNA3-CHRNB4 gene cluster associated with heavy smoking and higher relapse risk has led to the identification of the midbrain habenula–interpeduncular axis as a critical relay circuit in the control of nicotine dependence. Although clear roles for α3, β4, and α5 receptors in nicotine aversion and withdrawal have been established, the cellular and molecular mechanisms that participate in signaling nicotine use and contribute to relapse have not been identified. Here, using translating ribosome affinity purification (TRAP) profiling, electrophysiology, and behavior, we demonstrate that cholinergic neurons, but not peptidergic neurons, of the medial habenula (MHb) display spontaneous tonic firing of 2–10 Hz generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels and that infusion of the HCN pacemaker antagonist ZD7288 in the habenula precipitates somatic and affective signs of withdrawal. Further, we show that a strong, α3β4-dependent increase in firing frequency is observed in these pacemaker neurons upon acute exposure to nicotine. No change in the basal or nicotine-induced firing was observed in cholinergic MHb neurons from mice chronically treated with nicotine. We observe, however, that, during withdrawal, reexposure to nicotine doubles the frequency of pacemaking activity in these neurons. These findings demonstrate that the pacemaking mechanism of cholinergic MHb neurons controls withdrawal, suggesting that the heightened nicotine sensitivity of these neurons during withdrawal may contribute to smoking relapse.

The tobacco epidemic kills nearly six million smokers a year, primarily from lung cancer. The success rate for smoking cessation without pharmacological treatment is only 3–5% and less than 30% with nicotine-replacement therapies (1). Nicotine, the addictive component of tobacco, mediates its action by activating nicotinic acetylcholine receptors (nAChRs), which respond to the endogenous neurotransmitter acetylcholine (ACh). Like other drugs of abuse, chronic nicotine exposure promotes adaptations in neuronal circuits that sustain the use of cigarettes (2, 3). Upon nicotine cessation in humans, a withdrawal syndrome—characterized by negative somatic and affective symptoms such as irritability, anxiety, depressed mood, and loss of concentration—develops (2, 3) and contributes to the high probability of smoking relapse. In rodents that have been chronically treated with nicotine, withdrawal can be induced either by pharmacological precipitation or by cessation of nicotine administration. Somatic and affective signs of withdrawal differentially manifest in these two cases (3, 4). Additionally, studies in knockout mice have revealed a distinct contribution of various nAChR subtypes to somatic and affective withdrawal signs (5, 6), illustrating the molecular complexity of nicotine dependence, withdrawal, and relapse.

Converging evidence from genome-wide association studies and animal models has strongly implicated the nicotinic receptor subunits α3, α5, and β4 with heavy tobacco use and decreased success in smoking-cessation therapy in humans (7, 8), and with nicotine aversion and withdrawal in laboratory animals (6, 9, 10). These nicotinic receptor subunits (with the exception of α5) are not present in the mesolimbic dopamine tract (typically implicated in addiction disorders) (2, 11) but are concentrated in the medial habenula (MHb) and interpeduncular nucleus (IPN), which together comprise a major cholinergic tract in the mammalian brain that conveys convergent information from the limbic forebrain to the midbrain via the fasciculus retroflexus (2, 12, 13).

A number of studies have begun to dissect the medial and lateral habenular nuclei in an effort to determine whether subpopulation differences in cytoarchitecture and connectivity could be correlated to molecular markers and electrophysiological properties (14⇓⇓–17). For instance, the dorsal and ventral parts of the MHb can be clearly divided based on their respective enrichment in the neuropeptide Tachykinin 1, also known as substance P (SP), or the ACh synthesizing enzyme choline acetyltransferase (ChAT), and the segregation of their efferents to different parts of the IPN (18). A recent study showed that MHb ChAT-positive neurons corelease glutamate and ACh upon different patterns of blue-light stimulation in transgenic ChAT-Channelrhodopsin mice (19), but that sufficient ACh to activate nAChRs in IPN neurons is detected only once a certain threshold of tonic firing is reached. Given that it has been reported that medial habenular neurons generate tonic trains of action potentials (15), similar to neurons in the mesolimbic dopamine tract, we wanted to address whether this property was restricted to cholinergic neurons, and whether this tonic firing could be modulated in vivo by nicotine. Here, we analyzed the pacemaking activity and translating ribosome affinity purification (TRAP) molecular profile of MHb neurons. By comparing ChAT and SP neurons, we found that cholinergic MHb neurons are equipped with pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that confer them with intrinsic pacemaking activity, and that infusion of the HCN antagonist ZD7288 into the MHb of nicotine naïve mice precipitates somatic and affective signs of withdrawal. This pacemaking activity is increased by nicotine via activation of α3β4-containing (α3β4*) nAChRs and is not changed in mice treated with chronic nicotine, but is further potentiated by exposure to nicotine in mice undergoing nicotine withdrawal.

Results

Cholinergic, but Not Peptidergic, Neurons in the MHb Show Spontaneous Action-Potential Firing.

We confirmed the clear segregation of cholinergic and peptidergic neurons in the two mouse models used in this study: ChAT-DW167 mice and Tabac mice. ChAT-DW167 mice are transgenic for a bacterial artificial chromosome (BAC) of the ChAT gene and regulatory regions driving expression of an enhanced green fluorescent protein (eGFP)-tagged L10a ribosomal subunit (eGFP-L10a) (20). Tabac (Transgenic α3β4α5 cluster) mice carry a BAC of the Chrnb4-Chrna3-Chrna5 gene cluster driving expression of eGFP in α3β4* nAChR-positive neurons (10). As shown in Fig. 1 A and B, cholinergic neurons are concentrated in the ventral two-thirds of the MHb whereas SP-containing cells are exclusively localized in the dorsal part of the nucleus. ChAT-DW167 and Tabac mice show colocalization of eGFP and ChAT immunoreactivity (Fig. 1B and Fig. S1), which indicates that both BAC vectors accurately drive transgene expression to cholinergic neurons of the MHb.

Fig. 1.
Fig. 1.

Cholinergic, but not peptidergic, neurons in the MHb show spontaneous action-potential firing. (A) Immunohistochemical detection of eGFP-tagged L10a ribosomal subunit in a coronal section from the ChAT-DW167 TRAP line. The boxed area indicates the MHb, shown in detail in B. (Scale bar: 1 mm.) (B) Immunofluorescent detection of eGFP, ChAT, and SP in MHb of the ChAT-DW167 mouse. The Insets demonstrate each individual channel. ChAT and eGFP colocalize in the ventral part (outlined right) of MHb whereas Substance P is expressed in the dorsal part (outlined left). (Scale bar: 100 μm.) ChAT, acetylcholine transferase; d, dorsal; MHb, medial habenula; 3V, third ventricle; v, ventral. (C) Patch-clamp recordings show spontaneous pacemaking activity in ChAT-positive neurons. (D) ChAT-negative neurons in the dorsal 1/3 of the MHb show no spontaneous activity. (E) ChAT-positive MHb neurons respond to 200 pA current injection with a train of APs. (F) ChAT-negative neurons have a very diminished response to 200 pA current injection. (G) Representative image of a double cell-attached recording from adjacent ChAT-positive neurons. (H) ChAT-positive neurons showed unsynchronized firing, but similar action-potential frequency.

We next used ChAT-DW167 mice for patch-clamping experiments in brain slices. We performed whole-cell recordings in cholinergic and peptidergic neurons of the MHb (Fig. 1 C–F). Eighty-five percent of the ChAT-positive neurons in the MHb showed tonic action-potential firing in the current clamp mode (58 out of 68 neurons) (Fig. 1C). Tonic firing could be recorded for up to 1 h. The action-potential frequency ranged from 2 to 10 Hz, with an average frequency of 4.2 ± 0.25 Hz. None of the patched GFP-negative, peptidergic neurons in the dorsal 1/3 of the MHb showed this pacemaking activity (n = 5) (Fig. 1D). Upon current injections (200 pA for 300 ms), eGFP–positive cholinergic neurons responded with three or more action potentials (APs) (Fig. 1E) whereas eGFP-negative peptidergic neurons in the dorsal 1/3 of the MHb showed only one or two APs (Fig. 1F). To address whether the spontaneous activity is synchronized within the MHb, we performed cell-attached recordings from two adjacent eGFP-positive cholinergic neurons (Fig. 1G). In all five of these double cell-attached recordings, neurons showed a similar firing frequency, but tonic firing was not synchronized (Fig. 1H). These results show that only ChAT-positive neurons in the MHb have pacemaking activity and that spontaneous APs are unsynchronized between different cells.

TRAP Analysis of Cholinergic MHb Neurons Reveals Enrichment of Channels and Receptors Involved in Pacemaking.

Given the clear distinction between cholinergic and peptidergic neurons in their pacemaking activity, we sought to identify the channels and receptors that could confer this property uniquely to the cholinergic subpopulation. We applied the TRAP methodology to habenular homogenates from the bacTRAP ChAT-DW167 mice targeting cholinergic neurons (20) (Fig. 2 A and B). This methodology employs affinity purification of eGFP-tagged ribosomes and the mRNAs that are being translated in the targeted cell population (20). Three replicates of RNA captured from cholinergic neurons (TRAP) and total RNA from the habenular dissection (Total) were subjected to amplification followed by hybridization with Affymetrix microarrays.

Fig. 2.
Fig. 2.

TRAP profiling of MHb cholinergic neurons reveals a unique transcriptional signature. (A) Scatterplot of microarray data comparing mean transcript levels in cholinergic cell TRAP (x axis) with total habenular dissection RNA (y axis), for all 17,000 available transcripts. Transcripts known to be found in cholinergic cells (blue) are robustly enriched in the TRAP sample whereas nonneuronal transcripts are depleted (red, glial genes). Biotinylated controls are unchanged between samples (green circles). Black lines at 0.5-, 1-, and 2-fold. Red line is background threshold as established by fold change of glial genes. Axes in log scale. (B) Hierarchical clustering of the transcriptional profile of cholinergic MHb neurons compared with all previously measured cell types reveals that these cells are highly unique. They are only loosely similar to other neuromodulatory populations and are clearly distinct from other cholinergic cells, such as motor neurons of spinal cord and hindbrain. bf, basal forebrain; BG, Bergman glia; cb, cerebellum; ctx, cortex; hb, habenula; hdb, hindbrain; hyp, hypothalamus; MSN, medium spiny neuron; N, Neurons; sc, spinal cord; str, striatum. (C) Quantification (mean ± SEM) of expression of the Hcn3 and Hcn4 isoforms and ChAT genes by qRT-PCR in ChAT-TRAP Immunoprecipitate (IP) versus total RNA. Hcn3, Hcn4, and ChAT are significantly enriched in the IP fraction compared with total. *P < 0.05, Bonferroni post hoc test.

As shown in Fig. 2A and Dataset S1, the TRAP samples show robust enrichment of known markers of MHb neurons, including Chrna3, Chrnb4, Tac2, and Gpr151, in agreement with other studies (14, 17), whereas nonneuronal transcripts (e.g., glial genes) are depleted (red in Fig. 2A). We identified transcripts for over 1,100 genes that were at least twofold enriched in MHb cholinergic neurons compared with total RNA (Dataset S2). Comparative analysis with all previously collected TRAP cell types (20, 21) identified 390 transcripts that were relatively enriched and/or specific to MHb cholinergic neurons as measured by the specificity index statistic (pSI < 0.01) (21), indicating that these cells have a highly unique transcriptional signature within the brain. Hierarchical clustering with all previous profiled cell types confirms the relative uniqueness of this cell type (Fig. 2B). These neurons fall only loosely near a neuron cluster with various neuromodulatory populations of cells, such as hypothalamic hypocretin neurons and cholinergic neurons of the basal forebrain, and they are quite distinct from other cholinergic populations such as spinal cord and hindbrain motor neurons. Conducting a Gene Ontologies analysis on the 390 transcripts that distinguish these cells reveals a remarkable preponderance of molecules with transcription-factor activity, ion-binding activity (mostly channels), and receptors, including Wnts (Fig. S2 and Dataset S3). This characteristic gene expression profile suggests that these cells have a unique physiological profile as well.

To better understand the physiology of these neurons, we further analyzed the ion channels and receptors enriched in these cells (Dataset S1). First, we observed extremely high expression levels of the big potassium (BK) channel (encoded by the potassium large conductance calcium-activated channel gene; Kcnma1) and the Cacnb3 (voltage-gated calcium channel auxiliary b3 subunit). In addition, the L-type voltage-gated calcium channel a1d (Cacna1d, also known as Cav1.3), although not present in the microarray, is highly expressed in these neurons (see Allen Brain Atlas: http://mouse.brain-map.org/experiment/show/68798037) and has been shown to drive pacemaker activity in dopamine (DA) neurons (22). We were also interested in the pacemaking HCN channels, which are differentially expressed in the habenula (16, 23). Our microarray data demonstrated a clear enrichment of the Hcn3 channel in these neurons whereas Hcn1 and Hcn2 were at or below the level of background. Because Hcn4 was not present in our microarrays but seems to be specific for the cholinergic part of the MHb (16, 23), we assessed its expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on TRAP samples [F(5,6) = 69.83, P < 0.0001, ANOVA] (Fig. 2C). Altogether, these data indicate that cholinergic MHb neurons differentially express high levels of Hcn3, Hcn4, BK, L-type Cav1.3, and α3β4* nAChRs that may endow these neurons with specific electrical properties that underlie their pacemaking capability.

Block of HCN Channel-Mediated Pacemaker Activity in MHb Neurons Results in a Withdrawal-Like Phenotype.

To further characterize the pacemaking activity of MHb neurons, we used pharmacological blockers. In five of five ChAT-positive neurons, the tonic AP-firing was unchanged by the application of a drug mixture containing AMPA, NMDA, GABAA, GABAB, and nAChR blockers [50 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), 50 µM D-(-)-2-amino-5-phosphonopentanoic acid (d-APV), 100 µM Picrotoxin, 1 µM CGP55845, and 3 µM mecamylamine, respectively] (Fig. 3A). Tonic AP-firing is therefore driven by intrinsic factors and independent of glutamatergic, GABAergic, or cholinergic inputs.

Fig. 3.
Fig. 3.

Block of HCN pacemaker channels mediating the autonomous tonic firing of MHb neurons precipitates withdrawal-like somatic and affective signs. (A) Application of 50 µM NBQX, 50 µM d-APV, 100 µM Picrotoxin, 1 µM CGP55845, and 3 µM Mecamylamine (drug mixture) to block glutamatergic, GABAergic, and cholinergic inputs had no effect on spontaneous action-potential firing in ChAT-positive MHb neurons. (B) The nonselective HCN blocker ZD7288 (20 µM) reduced tonic AP firing by 60%. (C) After microinjection of ZD7288 into the MHb, mice (n = 8) displayed increased number of withdrawal-like somatic signs, not present in mice microinfused with saline (n = 8). They were also less active in the center area, indicative of increased anxiety, an affective withdrawal sign. *P < 0.05, unpaired t test. (D) Schematic diagram showing injection sites. Image displays red-fluorescent dye infused into MHb. Tissue counterstained with DAPI. Hipp, hippocampus; LHb, lateral habenula; MHb, medial habenula; 3V, third ventricle. (Scale bar: 100 µm.)

HCN channels mediate spontaneous pacemaking activity in the heart and in other brain areas (23) and are enriched in MHb neurons (Fig. 2C). To test whether these channels regulate pacemaking in cholinergic neurons, we applied the nonselective HCN blocker ZD7288 (20 µM) to MHb slices. As shown in Fig. 3B, spontaneous firing was strongly reduced by 62 ± 10.5%. We also observed a smaller but significant contribution of the L-Type calcium (20 µM nifedipine increased tonic firing by 33 ± 3.9%) and BK channels (1 µM of paxilline, decreased tonic firing by 25 ± 2.0%.) to the pacemaking frequency (Fig. S3). These results support our findings from the TRAP analysis indicating that HCN, BK, and L-type calcium ionic currents are present in MHb cholinergic neurons and contribute to the generation and modulation of their intrinsic pacemaking activity.

To assess the behavioral relevance of the pacemaker activity of cholinergic neurons, ZD7288 was bilaterally microinjected into the MHb of mice through implanted cannulae (Fig. 3D). Immediately after the infusion, mice were observed for 15 min for any unique behavior. Microinjection of ZD7288 increased the number of typical somatic signs of withdrawal, including scratching, jumping, body and paw tremors, genital licking, and retropulsion (P = 0.031) (Fig. 3C). Mice injected with ZD7288 also showed reduced center area horizontal activity (P = 0.036) (Fig. 3C). This reduced center activity is indicative for increased anxiety, an affective sign of nicotine withdrawal. These data demonstrate that inhibition of MHb pacemaker activity in vivo precipitates a withdrawal-like phenotype even in mice that have not experienced nicotine.

Tonic Firing Frequency Increases upon Nicotine Application and Depends on α3β4* nAChRs.

Given the high concentration of nAChRs in the cholinergic subpopulation (Fig. S1, Fig. 2A, and Dataset S1) and their critical role in nicotine consumption and withdrawal (6, 9, 10), we next wanted to determine the contribution of nAChRs to the spontaneous pacemaking activity in the MHb. The 81 nM nicotine free base increased tonic AP firing by a factor of 1.6 (Fig. 4A), an effect that was abolished by the application of the nonspecific nAChR blocker mecamylamine (3 µM) (Fig. 4B). The nicotine-dependent increase in the number of spontaneous APs in the MHb is therefore mediated by nAChRs and is not caused by effects of nicotine on HCN (24), L-type calcium (25), or BK channels (26).

Fig. 4.
Fig. 4.

Tonic firing increases upon nicotine application and depends on α3β4* nAChRs. (A) Normalized number of APs per 20 s showing a 1.6-fold increase in tonic AP firing after nicotine application. Insets show representative cell-attached recordings from MHb neurons. Coapplication of the nAChR blocker mecamylamine (3 µM) (B) or the α3β4* nAChR blocker Conotoxin AuIB (C) abolished the effects of nicotine on AP activity. (D) Effects of nicotine upon coapplication with the indicated nAChR blockers. Percent frequency change was calculated as the change between baseline AP activity (first 5 min) and the last minute of nicotine application. The concentrations used were as follows: Nicotine, 81 nM; Mecamylamine, 3 µM; Conotoxin MII, 250 nM; Conotoxin AuIB, 10 µM; SR 16584, 25 µM; DHβE, 1 µM; DMAB, 10 µM; Bungarotoxin, 50 nM.

To determine which nAChR combination is responsible for the effect of nicotine on MHb pacemaking firing, we used a number of antagonists and peptide toxins. Application of 50 nM Bungarotoxin (α7*-blocker), 100 nM Conotoxin MII (α3β2*, β3*-blocker), 10 µM 3-(4)-dimethylaminobenzylidine anabaseine (DMAB) (α4β2*, α7*-blocker), or 1 µM dihydro-β-erythroidine hydrobromide (DHβE) (α4β2*, α4β4*-blocker) did not alter the effects of nicotine on tonic AP firing (Fig. 4D and Fig. S4) whereas preincubation with the selective blockers for α3β4* nAChRs conotoxin AuIB (Fig. 4 C and D) and SR16584 (Fig. 4D and Fig. S4) abolished the increase in tonic firing produced by nicotine. The specificity for α3β4 nAChRs was further supported by the use of Tabac mice in which Chrnb4 overexpression increases α3β4* nAChR levels (10). In these mice, nicotine led to a 3.27 ± 0.50-fold increase in spontaneous AP-activity in MHb neurons (Fig. S4). These results demonstrate that the nicotine-meditated increase in pacemaking activity is independent of α3β2*, α4β2*, α4β4*, α7*, or β3* nAChR subunits but is the result of α3β4* nAChRs activation.

Nicotine Withdrawal, but Not Chronic Nicotine, Potentiates Nicotine-Induced Pacemaking Activity.

We next sought to determine the functional consequences of chronic nicotine treatment and withdrawal on the spontaneous AP activity in the MHb. To analyze tolerance to chronic nicotine, we used a previously described protocol (4, 27). Mice were given nicotine in the drinking water for 28 d and recorded on the last day or withdrawn from nicotine and recorded on the next day (see experimental paradigm in Fig. S5). The water contained 2% saccharin to mask the bitter taste of nicotine, which was increased from 65 mg/L to 163 mg/L nicotine free base during the first week, and maintained at 163 mg/L nicotine free base during the following weeks. Based on the volume intake (Fig. S5), mice consumed 26.2 mg⋅kg−1⋅day−1 of nicotine free base. In C57BL/6J mice, this dose correlates to plasma cotinine (the major metabolite of nicotine) levels of 43 ng/mL (27). This concentration is comparable with nicotine plasma levels measured in the afternoon in smokers (10-50 ng/mL of cotinine, or 60–310 nM of nicotine) (28). Control mice were given 2% saccharin. We prepared acute brain slices from these mice and analyzed MHb ChAT-positive neurons. Recordings in these spontaneously active neurons showed that the basal pacemaking frequency was indistinguishable between nicotine-treated and saccharin-control groups (Fig. 5A) and that bath application of nicotine resulted in a similar increase of firing frequency in both groups (fold increase for saccharin-control, 1.63 ± 0.11; nicotine-treated, 1.71 ± 0.08) (Fig. 5 B and C). This fold increase was the same as that observed in untreated mice (Fig. 4A), indicating that chronic treatment with nicotine does not change either the basal or the nicotine-enhanced pacemaking.

Fig. 5.
Fig. 5.

Nicotine withdrawal, but not chronic nicotine treatment, increases the response to nicotine. (A) Nicotine-treated mice show similar basal AP frequencies compared with control animals. (B) The effects of 81 nM nicotine on slices of control and treated mice were indistinguishable. (C) The fold increase in tonic firing after nicotine application was not different between the control and the treated group. (D) The basal AP frequencies in slices from treated mice withdrawn from nicotine 14–18 h before slice preparation (withdrawal group) and control animals were indistinguishable. (E) Nicotine effects on AP firing were enhanced in the withdrawal group compared with the control group. (F) The fold increase in tonic firing after nicotine application was significantly higher in the withdrawal group, compared with the control group (P = 0.0014). (G) Coapplication of 3 µM Mecamylamine and 81 nM nicotine in slices from mice withdrawn from nicotine completely blocks the nicotine-induced increase in spontaneous AP firing. (H) Quantification of spontaneous somatic signs of withdrawal 14–18 h after nicotine (n = 9) and saccharin (n = 10) treatment cessation. *P < 0.05, unpaired t test.

In a second group, mice were treated as described before, but given water only to drink for the 14–18 h before preparation of the acute brain slices. The basal AP frequency before nicotine application was unchanged between the groups, as in the previous experiments (control withdrawal, 4.07 ± 0.39 Hz; nicotine withdrawal, 3.49 ± 0.28 Hz; P = 0.234) (Fig. 5D). However, when nicotine was bath applied, the fold increase of spontaneous AP frequency was significantly higher in mice undergoing nicotine withdrawal compared with the saccharin-withdrawal control group (control, 1.58 ± 0.12; nicotine withdrawal, 2.07 ± 0.17; P = 0.0014) (Fig. 5 E and F). This increase was blocked by the nAChR antagonist mecamylamine (Fig. 5G), demonstrating that this potentiation depends on the activity of nAChRs. Behaviorally, mice demonstrate spontaneous somatic signs of withdrawal during the same time frame as when the nicotine recordings were performed (that is 14–18 h after removing nicotine from the drinking bottle following 4 wk chronic nicotine exposure) (Fig. 5H). We conclude that chronic exposure to nicotine alters the activity of nAChRs in cholinergic neurons of the MHb such that, during withdrawal, these neurons display significantly increased pacemaking activity upon reexposure to nicotine.

Discussion

The cellular and molecular mechanisms underlying how the brain signals nicotine withdrawal and recognizes its previous exposure to nicotine remain poorly understood. The studies presented here establish that cholinergic habenular neurons possess intrinsic pacemaking activity that is mediated by HCN pacemaker channels and that inhibition of pacemaking activity, by infusion of HCN antagonists in the MHb, precipitates withdrawal-like behavior. In addition we find that fast fluctuations in nicotine levels, but not chronic nicotine, accelerate the pacemaking frequency of these neurons via α3β4* nAChR. Strikingly, mice undergoing withdrawal from nicotine double the pacemaking activity of these neurons upon reexposure to nicotine. Given the genetic association of α3β4* receptors with smoking relapse, our data suggest that altered pacemaking activity in medial habenular neurons upon nicotine withdrawal may contribute to difficulties with smoking cessation.

It is intriguing that the pacemaking activity in the MHb is restricted to cholinergic neurons when one considers the mechanisms of ACh release from habenular neurons and the signaling pathways that may modulate this release. For example, it was recently demonstrated that cholinergic MHb neurons corelease glutamate and ACh, but, only upon tonic photostimulation (≥20 Hz), the amount of released ACh is sufficient to activate nAChRs in postsynaptic IPN neurons (19). Interestingly, both in this study and in recordings from rat MHb neurons in vivo (29), this frequency threshold for ACh release is not reached under basal conditions. However, given the large α3β4*-dependent increase in pacemaking activity that we have observed in response to acute nicotine, it seems probable that elevation of ACh levels in the MHb as a consequence of physiological input activity or because of tobacco smoking will be important for the regulation of ACh release in the IPN. The fact that HCN, L-type Cav1.3, and BK channels cooperate to generate spontaneous pacemaking activity in MHb neurons suggests that additional pathways impinging on these channels can also alter tonic firing and thus ACh release in the IPN. For example, the identification of “cAMP-dependent protein kinase regulator activity” as a major functional category in cholinergic MHb neurons is interesting in light of the central role of cAMP in dopaminergic signaling (30) and the regulation of neuronal activity in reward circuitry (31). We anticipate that further studies of this pathway and of other molecular components identified in the TRAP studies reported here can lead to an improved understanding of tonic firing in the MHb and its regulation in response to the many inputs to this important midbrain circuit.

Given the complex behavioral effects of nicotine, and recent studies demonstrating a critical role for the MHb in nicotine intake, aversion, and withdrawal (6, 9, 10), the demonstration that pacemaking activity in the MHb responds to nicotine and that this modulation is altered during withdrawal is important. It is evident from these studies that the activity of HCN channels is positively modulated by nicotine and that this effect is mediated by nAChRs. However, whether this modulation is occurring directly by altering HCNs (i.e., phosphorylation or cAMP changes) or indirectly through other signal transduction events initiated by nAChRs will require further investigation. Our finding that the basal pacemaking of these α3β4*-enriched neurons is not changed is consistent with studies indicating that chronic nicotine treatment preferentially up-regulates α4β2* receptors (32) but not α3β4* receptors (33, 34). During a single withdrawal episode, 3H nicotine binding sites increase in cerebral cortex and midbrain (35). In contrast, repeated cycles of nicotine treatment and withdrawal result in progressive up-regulation of 3H epibatidine sites in striatum and hippocampus but not in the cortex (36). Given these studies, and the cell-specific response we have demonstrated here, it would be interesting to compare cytisine-resistant radioligand binding studies under a variety of withdrawal conditions to distinguish the responses of α4β2* and α3β4* receptors. It seems probable that cell-specific regulation of particular nAChR combinations may underlie the different behavioral and physiological effects of chronic nicotine exposure, withdrawal, and relapse. This specificity for nAChR subtypes is particularly important in light of recent human genetic association studies showing that smokers with the high genetic risk variants in the CHRNA5-CHRNA3-CHRNB4 locus not only have a predisposition to heavy smoking but will also respond differently to pharmacological treatments for smoking cessation (7, 8).

Finally, it is important to consider the pacemaking activity of cholinergic neurons in the MHb and its modulation by nicotine in the context of the widespread neural circuitry mediating nicotine dependence and withdrawal (37). Nicotine, like many other drugs of abuse, is known to have an impact in the mesocorticolimbic DA reward center (2, 38). Interestingly, DA neurons show both spontaneous tonic firing (with a frequency of 1–5 Hz, similar to MHb neurons) (39) and phasic firing. The existence of these two modes of firing is thought to contribute to the malleability of the DA system (38), and it follows that this could also be the case in the MHb. Phasic burst firing induced by afferent stimuli causes transient DA release and signals acute positive reinforcement by nicotine (38). Tonic firing maintains constant DA levels and is known to decrease during nicotine withdrawal (40). The actions of nicotine in this system are complex because it coactivates DA neurons and GABAergic interneurons, which both express nAChRs (2). For instance, nicotine greatly increases the frequency of the tonic discharge of DA neurons by activation of β2* nAChRs (41) but also contributes to burst firing by activation of β2* in GABAergic interneurons (42). Furthermore, subcellular compartmentalization of the nAChRs plays an important role in their physiological activities (2). Given our demonstration that tonic activity in cholinergic neurons in the MHb is modulated by nicotine, and the complex distribution of nAChRs in the MHb–IPN axis, we anticipate that mechanisms regulating the output of this circuitry will also be complex. Consequently, a detailed understanding of the Hb–IPN mechanisms that control nicotine withdrawal and relapse will require further cell-specific molecular and electrophysiological studies of additional cell types in this interesting and ancient midbrain circuit.

Materials and Methods

Details for all materials and methods can be found in SI Materials and Methods.

Mice.

Transgenic ChAT-DW167 and Tabac mice have been previously described (SI Materials and Methods). All studies were done in accordance with National Institutes of Health and Institutional Animal Care and Use guidelines. All procedures and protocols were approved by the Animal Care Committee of The Rockefeller University and the Max Delbrück Center.

Slice Preparation and Electrophysiological Recordings.

Patch pipettes had resistances of 4–8 MΩ when filled with a solution containing (in mM): 105 K-gluconate, 30 KCl, 10 Hepes, 10 phosphocreatine, 4 ATP-Mg2+, 0.3 GTP (pH adjusted to 7.2 with KOH). Electrophysiological responses were recorded at 33–36 °C.

Translating Ribosome Affinity Purification and Quantitative RT-PCR.

Translating ribosome affinity purification (TRAP) was conducted in triplicate as previously described using the TRAP line ChAT-DW167 (SI Materials and Methods). Immunoprecipitated (IP) and total mRNA fractions purified for TRAP were subjected to RT-PCR.

Histology and Confocal Imaging.

Staining of ChAT-DW167 and Tabac mice with anti-GFP, anti-ChAT, and anti-SP antibodies was done as described in SI Materials and Methods.

Chronic Nicotine Treatment and Nicotine Withdrawal.

All nicotine doses are reported as free base. Mice were given nicotine in the drinking water for 28 d, and withdrawal studies were conducted using the spontaneous nicotine withdrawal model as previously described (4, 27).

Surgical Procedures for Microinjections.

Mice were implanted with cannula guides into the MHb (coordinates, anteroposterior, −1.7 mm from bregma; mediolateral, ±0.3 mm from midline; dorsoventral, −2.9 mm from skull level). ZD7288 or saline was microinjected at a volume of 0.5 μL bilaterally for over 90 s.

Statistical Analysis.

Sets of data are presented by their mean values and SEMs. The unpaired two-tailed Student t test was used when comparing two sets of data with normal distribution.

Acknowledgments

We thank Cuidong Wang, Syed Samin Shehab, Monika Schwarz-Harsi, and Julio Santos-Torres for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant GO 2334/1-1 (to A.G.), by National Institute of Neurological Disorders and Stroke (NINDS) Grant 4R00NS067239-03 (to J.D.D.), by Helmholtz Association Grant 31-002 and Sonderforschungsbereich (SFB) Grant SFB665 (to I.I.T.), by the Irma L. and Abram S. Croll Charitable Trust (J.L.A.), and by the National Institutes of Health/National Institute of Mental Health Conte Center Grant MH074866.

Footnotes

  • ↵1To whom correspondence should be addressed. E-mail: iibanez{at}rockefeller.edu.

  • Author contributions: A.G., B.A.-F., and I.I.-T. designed research; A.G., B.A.-F., J.L.A., S.F., M.A.Ś., J.D.D., and I.I.-T. performed research; A.G., B.A.-F., J.D.D., and I.I.-T. analyzed data; and A.G. and I.I.-T. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE43164).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1313103110/-/DCSupplemental.

References

  1. ↵
    1. Stead LF,
    2. Perera R,
    3. Bullen C,
    4. Mant D,
    5. Lancaster T
    (2008) Nicotine replacement therapy for smoking cessation. Cochrane Database Syst Rev (1):CD000146.
  2. ↵
    1. Changeux JP
    (2010) Nicotine addiction and nicotinic receptors: Lessons from genetically modified mice. Nat Rev Neurosci 11(6):389–401.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Paolini M,
    2. De Biasi M
    (2011) Mechanistic insights into nicotine withdrawal. Biochem Pharmacol 82(8):996–1007.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Grabus SD,
    2. et al.
    (2005) Nicotine physical dependence and tolerance in the mouse following chronic oral administration. Psychopharmacology (Berl) 178(2-3):183–192.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Jackson KJ,
    2. Martin BR,
    3. Changeux JP,
    4. Damaj MI
    (2008) Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs. J Pharmacol Exp Ther 325(1):302–312.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Salas R,
    2. Sturm R,
    3. Boulter J,
    4. De Biasi M
    (2009) Nicotinic receptors in the habenulo-interpeduncular system are necessary for nicotine withdrawal in mice. J Neurosci 29(10):3014–3018.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Thorgeirsson TE,
    2. et al.
    (2008) A variant associated with nicotine dependence, lung cancer and peripheral arterial disease. Nature 452(7187):638–642.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Bierut LJ,
    2. et al.
    (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum Mol Genet 16(1):24–35.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Fowler CD,
    2. Lu Q,
    3. Johnson PM,
    4. Marks MJ,
    5. Kenny PJ
    (2011) Habenular α5 nicotinic receptor subunit signalling controls nicotine intake. Nature 471(7340):597–601.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Frahm S,
    2. et al.
    (2011) Aversion to nicotine is regulated by the balanced activity of β4 and α5 nicotinic receptor subunits in the medial habenula. Neuron 70(3):522–535.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Exley R,
    2. McIntosh JM,
    3. Marks MJ,
    4. Maskos U,
    5. Cragg SJ
    (2012) Striatal α5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum. J Neurosci 32(7):2352–2356.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Grady SR,
    2. et al.
    (2009) Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the alpha3beta4* and alpha3beta3beta4* subtypes mediate acetylcholine release. J Neurosci 29(7):2272–2282.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Sutherland RJ
    (1982) The dorsal diencephalic conduction system: A review of the anatomy and functions of the habenular complex. Neurosci Biobehav Rev 6(1):1–13.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Aizawa H,
    2. Kobayashi M,
    3. Tanaka S,
    4. Fukai T,
    5. Okamoto H
    (2012) Molecular characterization of the subnuclei in rat habenula. J Comp Neurol 520(18):4051–4066.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kim U,
    2. Chang SY
    (2005) Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus. J Comp Neurol 483(2):236–250.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Poller WC,
    2. et al.
    (2011) Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels. Neuroscience 193:205–216.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Quina LA,
    2. Wang S,
    3. Ng L,
    4. Turner EE
    (2009) Brn3a and Nurr1 mediate a gene regulatory pathway for habenula development. J Neurosci 29(45):14309–14322.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Contestabile A,
    2. et al.
    (1987) Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat: An immunocytochemical and microchemical approach. Neuroscience 21(1):253–270.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Ren J,
    2. et al.
    (2011) Habenula “cholinergic” neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes. Neuron 69(3):445–452.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Doyle JP,
    2. et al.
    (2008) Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell 135(4):749–762.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Dougherty JD,
    2. et al.
    (2013) The disruption of Celf6, a gene identified by translational profiling of serotonergic neurons, results in autism-related behaviors. J Neurosci 33(7):2732–2753.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Putzier I,
    2. Kullmann PH,
    3. Horn JP,
    4. Levitan ES
    (2009) Cav1.3 channel voltage dependence, not Ca2+ selectivity, drives pacemaker activity and amplifies bursts in nigral dopamine neurons. J Neurosci 29(49):15414–15419.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Santoro B,
    2. et al.
    (2000) Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS. J Neurosci 20(14):5264–5275.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Griguoli M,
    2. et al.
    (2010) Nicotine blocks the hyperpolarization-activated current Ih and severely impairs the oscillatory behavior of oriens-lacunosum moleculare interneurons. J Neurosci 30(32):10773–10783.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Stevens TR,
    2. Krueger SR,
    3. Fitzsimonds RM,
    4. Picciotto MR
    (2003) Neuroprotection by nicotine in mouse primary cortical cultures involves activation of calcineurin and L-type calcium channel inactivation. J Neurosci 23(31):10093–10099.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Kuhlmann CR,
    2. Trümper JR,
    3. Tillmanns H,
    4. Alexander Schaefer C,
    5. Erdogan A
    (2005) Nicotine inhibits large conductance Ca(2+)-activated K(+) channels and the NO/-cGMP signaling pathway in cultured human endothelial cells. Scand Cardiovasc J 39(6):348–352.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Sparks JA,
    2. Pauly JR
    (1999) Effects of continuous oral nicotine administration on brain nicotinic receptors and responsiveness to nicotine in C57Bl/6 mice. Psychopharmacology (Berl) 141(2):145–153.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Matta SG,
    2. et al.
    (2007) Guidelines on nicotine dose selection for in vivo research. Psychopharmacology (Berl) 190(3):269–319.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Zhao H,
    2. Rusak B
    (2005) Circadian firing-rate rhythms and light responses of rat habenular nucleus neurons in vivo and in vitro. Neuroscience 132(2):519–528.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Girault JA,
    2. Greengard P
    (2004) The neurobiology of dopamine signaling. Arch Neurol 61(5):641–644.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Nestler EJ
    (2004) Molecular mechanisms of drug addiction. Neuropharmacology 47(Suppl 1):24–32.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Gaimarri A,
    2. et al.
    (2007) Regulation of neuronal nicotinic receptor traffic and expression. Brain Res Brain Res Rev 55(1):134–143.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Marks MJ,
    2. et al.
    (2004) Subsets of acetylcholine-stimulated 86Rb+ efflux and [125I]-epibatidine binding sites in C57BL/6 mouse brain are differentially affected by chronic nicotine treatment. Neuropharmacology 46(8):1141–1157.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Nguyen HN,
    2. Rasmussen BA,
    3. Perry DC
    (2004) Binding and functional activity of nicotinic cholinergic receptors in selected rat brain regions are increased following long-term but not short-term nicotine treatment. J Neurochem 90(1):40–49.
    OpenUrlPubMed
  35. ↵
    1. Pietilä K,
    2. Lähde T,
    3. Attila M,
    4. Ahtee L,
    5. Nordberg A
    (1998) Regulation of nicotinic receptors in the brain of mice withdrawn from chronic oral nicotine treatment. Naunyn Schmiedebergs Arch Pharmacol 357(2):176–182.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Hilario MR,
    2. Turner JR,
    3. Blendy JA
    (2012) Reward sensitization: Effects of repeated nicotine exposure and withdrawal in mice. Neuropsychopharmacology 37(12):2661–2670.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Volkow ND,
    2. Wang GJ,
    3. Fowler JS,
    4. Tomasi D
    (2012) Addiction circuitry in the human brain. Annu Rev Pharmacol Toxicol 52:321–336.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Sesia T,
    2. Grace AA
    (2012) Shifting pharmacology of nicotine use and withdrawal: Breaking the cycle of drug abuse. Proc Natl Acad Sci USA 109(8):2697–2698.
    OpenUrlFREE Full Text
  39. ↵
    1. Grace AA
    (1991) Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: A hypothesis for the etiology of schizophrenia. Neuroscience 41(1):1–24.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Grieder TE,
    2. et al.
    (2012) Phasic D1 and tonic D2 dopamine receptor signaling double dissociate the motivational effects of acute nicotine and chronic nicotine withdrawal. Proc Natl Acad Sci USA 109(8):3101–3106.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Picciotto MR,
    2. et al.
    (1998) Acetylcholine receptors containing the beta2 subunit are involved in the reinforcing properties of nicotine. Nature 391(6663):173–177.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Tolu S,
    2. et al.
    (2013) Co-activation of VTA DA and GABA neurons mediates nicotine reinforcement. Mol Psychiatry 18(3):382–393.
    OpenUrlCrossRefPubMed
View Abstract
Back to top
Article Alerts
Email Article
Citation Tools
Request Permissions
Habenular pacemaking in nicotine withdrawal
Andreas Görlich, Beatriz Antolin-Fontes, Jessica L. Ables, Silke Frahm, Marta A. Ślimak, Joseph D. Dougherty, Inés Ibañez-Tallon
Proceedings of the National Academy of Sciences Oct 2013, 110 (42) 17077-17082; DOI: 10.1073/pnas.1313103110
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Proceedings of the National Academy of Sciences: 116 (49)
Current Issue

Submit

Article Classifications

Jump to section

You May Also be Interested in

Adaptations in heart structure and function likely enabled endurance and survival in preindustrial humans. Image courtesy of Pixabay/Skeeze.
Human heart evolved for endurance
Adaptations in heart structure and function likely enabled endurance and survival in preindustrial humans.
Image courtesy of Pixabay/Skeeze.
QnAs with NAS member and plant biologist Sheng Yang He. Image courtesy of Sheng Yang He.
Featured QnAs
QnAs with NAS member and plant biologist Sheng Yang He
Image courtesy of Sheng Yang He.

Similar Articles