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Unaided attempts to quit smoking commonly fail during the first 2 weeks of the nicotine withdrawal syndrome. Alterations in dopamine (DA) signaling correlate with withdrawal from chronic nicotine exposure, but those changes have not been well-characterized.
Mice were administered nicotine in their drinking water for 4 or 12 weeks. Then nicotine was withheld for 1 to 10 days while DA signaling was characterized with in vivo microdialysis or fast-scan cyclic voltammetry.
Upon withdrawal of nicotine, the basal DA concentration in the nucleus accumbens decreased as measured by microdialysis. The length of time that the low basal DA state lasted depended on the length of the chronic nicotine treatment. Microdialysis indicated that acute re-exposure to nicotine during withdrawal temporarily reversed this hypodopaminergic state. Voltammetry measurements supported the microdialysis results by showing that nicotine withdrawal decreased tonic and phasic DA release. The basal DA concentration and tonic DA signals, however, were disproportionately lower than the phasic DA signals. Therefore, the phasic/tonic DA signaling ratio was increased during the withdrawal period.
The relative increase in the sensitivity of DA release to phasic stimulation suggests an increase in the signal-to-noise relationship of DA signaling during the withdrawal period. Therefore, the DA signal produced by acute nicotine re-exposure produces a DA response that might reinforce relapse to drug use (i.e., smoking). Because the basal DA concentration is low during withdrawal, therapies aimed at elevating the background DA signal represent a reasonable treatment strategy for nicotine-dependent individuals attempting to quit.
Acute nicotine exposure enhances DA release through a dynamic balance of activation and desensitization of nicotinic acetylcholine receptors (nAChRs) located mainly in the ventral tegmental area (VTA) and in the striatum (
). These studies support the hypothesis that a low DA state might induce drug seeking to reverse the nicotine-induced DA deficiencies. In agreement with this hypothesis, the majority of people who attempt unaided to quit smoking relapse within the first 2 weeks (
), suggesting that the early period after nicotine withdrawal is a critical time for relapse and, potentially, for intervention.
In this study, we examined changes in DA signaling 1–10 days after withdrawal from chronic nicotine exposure. Nicotine withdrawal significantly lowered the baseline DA concentration in vivo compared with control subjects, and acute re-exposure to nicotine temporarily increased the absolute DA to the control levels. The persistence of these alterations depended on the duration of the chronic nicotine treatment. Voltammetric recordings in the nucleus accumbens (NAc) showed that tonic DA signals were inhibited to a greater degree than phasic DA signals after nicotine withdrawal, increasing the relative phasic-to-tonic response during the withdrawal period.
Methods and Materials
Wild-type C57BL/6J mice 2–3 months old (Jackson Laboratory, Bar Harbor, Maine) had free access to food and water and were housed in accordance with the guidelines specified by the Institutional Animal Care and Use Committee at Baylor College of Medicine and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Chronic Nicotine Administration and Withdrawal
Nicotine hydrogen tartrate at a final concentration of 300 μg/mL was administered chronically to the mice in their home cage water supply with 2% saccharin with previously established methods (
) (Figure 1A). The gray and black filled circles in Figure 1A indicate the experiments occurred after 4 or 12 total weeks of nicotine treatment. Nicotine was withdrawn for 1, 5, and 10 days before the experiments.
). The concentrations of urinary cotinine were monitored by an enzyme-linked immunosorbent assay (Bio-Quant, San Diego, California). The urinary cotinine concentration was adjusted for the urinary creatinine concentration to improve the accuracy (
). Guide cannulae were aimed at the medial NAc (Figure S1 in Supplement 1). The stereotaxic coordinates (relative to bregma) were 1.4 mm anterior posterior, .55 mm lateral, and 3.3 mm dorsal-ventral. After 3–5 days recovery from surgery, the microdialysis probe (CMA/7) (CMA/Microdialysis, Solna, Sweden) was perfused with artificial cerebral spinal fluid (149 mmol/L sodium chloride, 2.8 mmol/L potassium chloride, 1.2 mmol/L calcium dichloride, 1.2 mmol/L magnesium chloride, and .25 mmol/L ascorbic acid, and 5.4 mmol/L D-glucose) and implanted at least 14 hours before the experiment. The perfusion flow rate was .5 μL/min overnight and then 2.0 μL/min beginning at least 1 hour before baseline sampling.
The DA content of the microdialysates was determined by high-pressure liquid chromatography: pump (Model 582; ESA, Chelmsford, Massachusetts), autosampler (Model 542; ESA), and an HR-80 × 3.2 mm column (3-μm particle size; ESA). A coulometric cell (5014B; ESA) was connected to an ESA Coulochem II detector. Quantification of dialysate DA concentration was estimated relative to external standards (0–2 nmol/L). The slope and the y-intercept were calculated for the standard curve, and then the microdialysate concentration was interpolated from the regression with the slope-intercept equation.
Fast-Scan Cyclic Voltammetry in Mouse Striatal Brain Slices
Horizontal slices (350 μm) containing the NAc shell were cut from nicotine-treated or control mice and studied at 34 ± 1°C in (mmol/L) 125 sodium chloride, 2.5 potassium chloride, 1.3 magnesium dichloride, 2.5 calcium dichloride, 26 sodium dihydrogen phosphate, 1.25 sodium bicarbonate, and 10.0 glucose saturated with 95% oxygen and 5% carbon dioxide (
). Scans of the microelectrode potential (10 Hz) were from 0 mV to −400 mV to 1000 mV to −400 mV to 0 mV against a silver/silver chloride reference electrode at a rate of 300 mV/msec. An oxidation peak between 400 and 600 mV and a reduction peak between −200 and −250 mV was used to identify the DA signal. Intra-accumbens stimuli (1 msec duration, .6 mA constant current) were delivered with bipolar tungsten electrodes with 120–240 sec between stimuli to ensure recovery of DA release unless trains of stimulation were used as indicated. The tip of the carbon-fiber recording electrode was centered approximately 200 μm away from the tips of the stimulating electrode. The voltammetric DA signal was obtained by digital subtraction and calibrated against standards of .5 to 5 μmol/L DA. The DA signals were determined as amplitude (μmol/L) or the area under the curve (μmol/L × sec).
Dialysate DA concentrations (in nanomolar or percent of baseline values) were analyzed with analysis of variance (ANOVA) with repeated measures. The DA concentrations were log transformed to maintain homogeneity of variance. The average of the three 20-min basal samples defined the baseline DA response, which we used to test between-subject and within-subject effects. The DA concentrations measured by voltammetry were assessed by paired t test or one-way ANOVA. The ANOVA was performed with the multivariate analysis of variance in SPSS for Windows (SPSS, Chicago, Illinois). Significance for all analyses was determined at p < .05.
Alterations in Basal and Nicotine-Induced DA Signals After Nicotine Withdrawal
Mice self-administered nicotine (plus saccharin) via their drinking water for 4 or 12 weeks followed by withdrawal (Figure 1A), whereas the control subjects only drank saccharin water. The ratio of urinary cotinine concentration to creatinine concentration was used to estimate the nicotine exposure levels over the chronic treatment period (
). The cotinine/creatinine ratio reached peak values (approximately 24 μg/mg) after 3 weeks of nicotine treatment and remained stable thereafter (Figure 1B). This estimated level of nicotine exposure is comparable to what a chronic smoker might experience (
To examine the effect of the chronic nicotine treatment on basal and nicotine-induced DA signals during early withdrawal (1 day), we measured DA concentrations in the medial NAc (mainly the NAc shell) with in vivo microdialysis (Figure S1 in Supplement 1). The timeline for chronic nicotine treatment, nicotine withdrawal, and the microdialysis experiments are shown in Figure 2A. The mean basal DA concentration (Figures 2B and 2C) was significantly lower (p < .05) after 4 weeks (.22 ± .03 nmol/L, n = 7) and 12 weeks (.24 ± .01 nmol/L, n = 7) of nicotine treatment compared with the control (.31 ± .02 nmol/L, n = 9). There was no difference in basal DA levels between the 4-week and 12-week nicotine treatments after 1 day of withdrawal [F(1,12) = .73, p > .05].
Acute administration of nicotine (1 mg/kg, intraperitoneal injection) after 1 day of withdrawal increased the absolute DA concentrations to similar levels within 20 min of the injection across all treatments (Figure 2B and 2D). Because the nicotine-treated mice had lower baseline DA concentrations than the control mice, we normalized to compare the relative amplitudes of the nicotine-induced DA concentration changes (Figure 2E). Both the 4-week and 12-week withdrawal groups showed a significantly higher relative nicotine-induced DA response compared with the control mice (Figure 2E and 2F).
Modulation of Tonic and Phasic DA Signals After Nicotine Withdrawal
Fast-scan cyclic voltammetry measurements have shown that the effect of nicotine on DA release depends on neuronal firing patterns (
). To examine the influence of nicotine withdrawal, we used voltammetry to measure DA release evoked by stimulus trains applied to mouse brain slices containing the NAc shell. To mimic tonic firing (approximately 3 Hz) and phasic burst firing (approximately 20 Hz) of DA neurons in vivo, we applied a stimulus train with a combination of tonic (7 pulses delivered at 3 Hz) and phasic (5 pulses delivered at 20 Hz) stimulation. In control brain slices, tonic stimulation produced regular DA responses that elevated the baseline DA concentration, and phasic stimulation evoked a larger DA response that merged into a single transient (control, Figure 3A). In brain slices from mice chronically treated with nicotine (12 weeks), there was a general reduction in DA release evoked by tonic and phasic stimulation after withdrawal (1 day) compared with the control subjects (Figure 3B). However, by normalizing the DA signals to the tonic plateau response, we found that the percent change in phasic DA signaling was enhanced after nicotine withdrawal, compared with the control subjects (Figure 3C). This decrease in (tonic) DA release and the relative enhancement of phasic responses after withdrawal was consistent with the microdialysis data (Figure 2).
Ratio of Phasic-to-Tonic DA Signals Increases After Nicotine Withdrawal
Previous studies demonstrated that acute nicotine regulates the phasic-to-tonic relationship of DA release (
). To determine whether withdrawal from chronic nicotine exposure influences the DA signals evoked by tonic and phasic firing activity, we applied single stimulus pulses (1p) and phasic stimulation (5p at 20 Hz). In control NAc slices (Figure 4A), a single pulse (1p) stimulation evoked a smaller DA signal (.21 ± .02 μmol/L × sec, n = 51) compared with phasic stimulation (.70 ± .06 μmol/L × sec, n = 51). The ratio between these two measurements (i.e., the ratio of phasic-to-tonic DA release) was 3.4 ± .13 in control mice, indicating a potentiation of phasic evoked DA responses. After chronic nicotine treatment, the DA signals evoked by the single-pulse stimulus (.13 ± .01 μmol/L × sec, n = 51) and the 5-pulse stimulus train (.54 ± .05, n = 51) were both smaller than the control response (p < .01) (Figure 4A). However, normalization of the single-pulse evoked DA signal to the control 1p response revealed a robust frequency-dependent facilitation (Figure 4B) as well as an increase in the ratio of phasic-to-tonic DA release (4.2 ± .15, p < .05). Thus, nicotine withdrawal revealed an overall decrease in DA release, but the relative response to phasic stimulation was greater when compared with the control.
The enhanced phasic-to-tonic DA responses (Figures 4A and 4B) after nicotine withdrawal indicate an alteration in the release properties. To examine the probability of evoked DA release before and after withdrawal, we applied paired-pulse stimulations (Figure 4C). Paired pulses evoked smaller DA signals (.18 ± .03, n = 17) in the 12-week group after withdrawal (1 day) compared with the control group (.32 ± .03, n = 45, p < .05). However, the paired pulse (P2/P1) ratio was higher after nicotine withdrawal (.89 ± .12) compared with the control (.59 ± .05; p < .001) (Figure 4C), suggesting lower initial probability of release and greater facilitation of release after withdrawal. In the NAc shell, the DA signal in the control was linearly dependent on the number of pulses given during a 20-Hz stimulus train (Figure 4D, gray data). Nicotine withdrawal increased the slope of this relationship (Figure 4D, black data), indicating an increased sensitivity to phasic burst length. That is, phasic bursts induced relatively more DA release during the withdrawal period, and the longer the burst the greater the enhancement of DA release.
Alterations in DA Signals After Extended Periods of Nicotine Withdrawal
To determine the duration of the nicotine-induced withdrawal adaptations, we measured dialysate DA concentrations 1, 5, and 10 days after withdrawal from chronic nicotine treatment. After 4 weeks of nicotine treatment, the baseline DA levels returned to control levels by day 5 of withdrawal (Figure 5A). In contrast, after 12 weeks of nicotine treatment, the baseline DA levels did not return to normal until 10 days after withdrawal (Figure 5B). The baseline DA concentrations (Figure 5C) and the peak DA response to 1 mg/kg-nicotine (Figure 5D) are shown for each group at 1, 5, and 10 days after withdrawal. Interestingly, the baseline DA concentration (Figure 5C) was inversely related to the percent increase in DA concentration produced by the nicotine challenge (Figure 5D).
Voltammetric recordings further confirmed that 1 day and 5 days after withdrawal (from 12 weeks of nicotine) DA signals were lower across all stimulation frequencies (1p, and 5p at 5–20 Hz) (Figure 6A). By day 10, the DA signals returned near to control levels (Figure 6B). During those periods when tonic (1p) DA signals were inhibited, there was a higher ratio of phasic-to-tonic DA release compared with the control (Figures 6B and 6C). After DA release to a single stimulus (1p) returned to control levels, then the phasic/tonic ratio also returned to normal (Figure 6B and 6C). This result suggests that the background DA signal indicates the sensitivity of DA release to phasic burst firing.
High-Affinity nAChRs Influence DA Release After Nicotine Withdrawal
The high-affinity nAChRs (containing the β2 subunit) are the predominant nicotinic receptor subtype in both rodents and humans, and they influence DA neuron firing and DA release in the striatum (
). We examined the influence of the high-affinity nAChRs over tonic and phasic DA release during nicotine withdrawal. Inhibition of high-affinity nAChRs with dihydro-β-erythroidine (DHβE) (100 nmol/L) in control slices decreased the single-pulse evoked DA signal by 57 ± 5%, n = 16 (Figures 7A and 7B). By contrast, in slices from nicotine withdrawn mice, DHβE only reduced the single-pulse evoked signal by 19 ± 8%, n = 17 (Figures 7A and 7B), which was significantly less than the control response (p < .01). Blockade of the high-affinity (mainly β2*) nAChRs only reduced the phasic evoked DA signal by 29 ± 7% (Figures 7C and 7D) in the control subjects but had almost no effect on phasic DA signals (1 ± 10%) after nicotine withdrawal. These findings indicate that the influence of high-affinity nAChRs over NAc DA release decreased after chronic nicotine.
In the present study, nicotine was self-administered via home cage drinking water. Nicotine was maintained, on the basis of the stable cotinine/creatinine measurements, at a consistent level that was comparable to that obtained by chronic daily smoking (60–300 nmol/L) (
). Withdrawal from chronic nicotine treatment decreased the basal DA concentration in the NAc and decreased tonic and phasic DA release in the NAc. The DA concentration decrease measured by microdialysis lasted longer after 12 weeks of nicotine treatment than after 4 weeks of treatment. Re-exposure to acute nicotine during the withdrawal period normalized the DA concentration to control levels for a short time. Voltammetric measurements in the NAc shell supported this finding by showing that tonic DA release was more strongly inhibited than phasic release during the withdrawal period.
After chronic nicotine, the stronger inhibition of tonic DA release enhanced the contrast between tonic and phasic DA signaling. It also switched the pattern of DA release so that it was more highly dependent on the number of spikes within a burst. That change increased the dependence of NAc DA release on bursts. Therefore, during the withdrawal period, phasic DA neuron activity of the kind induced by nicotine (
The alterations in DA function after nicotine withdrawal were partly due to a decrease in the influence of the high-affinity (mainly β2*) nAChRs over DA release in the NAc. Normally, β2 in combination with α6 and α4 (
). After chronic nicotine treatment, nAChR regulation of DA release in the target area (i.e., NAc) decreased. The following hypothesis might be reasonable, on the basis of the literature. After chronic nicotine there is upregulation of mainly high-affinity nAChRs of the α4β2* subtype (
). Nashmi et al. showed that, consistent with these findings, repeated nicotine exposure causes specific upregulation of high-affinity nAChRs that are located on inhibitory γ-aminobutyric acid (GABA) inputs at midbrain DA neurons but not on DA neurons themselves (
). Increased GABA inhibition would result in an overall decrease in the basal spontaneous firing of DA neurons (i.e., tonic release would particularly decrease). Coupled with this hypothesized decrease in DA neuron firing, our voltammetric recordings showed a disproportionate inhibition of tonic release compared with release evoked by phasic bursts. The present study and others indicate that blockade of β2* nAChRs caused less inhibition of phasic DA signals than tonic signals (
). Nevertheless, it remains possible that upregulation of less-common β2* nAChRs or perhaps modifications in subunit composition, as a result of chronic nicotine exposure, could contribute to alterations in DA release during withdrawal.
Alternatively, the reduction in basal DA after chronic nicotine exposure could also arise from an increase in DA uptake from the extracellular space through changes in DA transporter function (
). Tonic background DA levels might be regulated more readily by DA transporter activity compared with phasic-evoked responses. However, we estimated reuptake changes by quantifying the rate of the falling phase for the evoked DA signals. We found that this measure (t90%) was not significantly different before (control, .63 ± .44 sec) and after chronic nicotine treatment (.71 ± .36 sec), which is consistent with the work of others (
Alterations in Tonic and Phasic Dopamine Signals After Nicotine Withdrawal
In chronic nicotine-treated mice, both tonic and phasic DA release were significantly lower than the control mice. This general decrease in DA function might contribute to motivational deficits such as anhedonia (
). However, an acute nicotine challenge during the withdrawal period produced a DA response that was relatively large, returning DA levels to near control values for a short time. Due to lower background DA levels during withdrawal, the DA target areas, such as the NAc shell, experience a greater relative change in DA concentration. Thus, the signal-to-noise relationship of the DA signal is higher after nicotine withdrawal than in the control subjects. Therefore, despite these deficits in basal DA function, the salience of the drug experience upon re-exposure might be greater.
Adaptations in DA signaling caused by chronic nicotine exposure alter the responses to nicotine (the drug) and possibly to nicotine-related stimuli. Previous studies demonstrate an inverse relationship between single action potential-evoked release and the degree of frequency-dependent facilitation (
). In the present study, we found that long-term nicotine exposure especially decreased the tonic DA signals. However, there was a steeper increase in DA release associated with the burst length. That is, the positive correlation between the evoked DA concentration and the number of pulses within a burst was amplified during the withdrawal period.
Nicotine increases the average firing rate of DA neurons in vivo, but the major effect is an increase in burst firing (
). Acute nicotine administration increases both the number and length of DA neuron phasic bursts. During the nicotine withdrawal period, the acute nicotine-induced bursts will produce a significant DA signal because the release process is more responsive to facilitation produced by burst firing. This result might explain why the low basal DA signal could rapidly increase in response to nicotine re-exposure. Thus, the reinforcing effects of nicotine during abstinence are particularly strong because nicotine burst firing causes an even stronger relative response than usual. This hypothesis is supported by recent studies showing that self-administration of nicotine, after chronic exposure, enhances burst firing activity in DA neurons in vivo (
). This study was performed in Long-Evans rats, whereas our study employed C57 mice. Species-related quantitative differences in DA function between rats and mice might account for the differences in the results (
In addition to changes in DA function, nicotine exposure modulates a variety of other neurotransmitter and neuropeptide systems that influence DA transmission directly but could also act in concert through independent mechanisms (
). Alterations in GABAergic and glutamatergic drive during nicotine withdrawal likely contribute to changes in tonic versus phasic DA neuron firing. In addition, the widespread action of nicotine within the central nervous system also recruits cholinergic, serotonin, and opioid systems (
). Thus, the behavioral reinforcement and withdrawal effects induced by nicotine arise from complex interactions between neurotransmitter systems, with DA having a key role.
These findings have implications for understanding how exposure to drug-associated cues or drug relapse might act upon and exploit existing pathological changes in DA function arising from long-term nicotine use. Future therapies aimed at restoring low tonic DA levels might represent an important therapeutic target for the treatment of nicotine addiction.
The authors are supported by grants from the National Institutes of Health, National Institute on Drug Abuse DA09411 and National Institute of Neurological Disorders and Stroke NS21229 , Cancer Prevention and Research Institute of Texas, and the Diana Helis Henry Medical Research Foundation through its direct engagement in the continuous active conduct of medical research in conjunction with Baylor College of Medicine and the project, Genomic, Neural, Preclinical Analysis for Smoking Cessation, and the Cancer Program.
The authors reported no biomedical financial interests or potential conflicts of interest.