Effect of Chronic Nicotine on
Brain Stimulation Reward:
II. An Escalating Dose Regimen
Michael A. Bozarth, Cindy M. Pudiak, & Rhonda KuoLee
Addiction Research Unit
Department of Psychology
State University of New York at Buffalo
Buffalo, New York 14260-4110
|This study examined whether repeated nicotine injections, using an escalating dose regimen, would produce brain stimulation reward facilitation indicative of a strong rewarding action. Male, Long-Evans rats with lateral hypothalamic stimulating electrodes were injected daily with escalating doses of nicotine bitartrate across 5-day cycles: 0.5, 1, & 2 mg/kg/day (dose expressed as freebase weight) were administered subcutaneously (s.c.) in consecutive 5-day cycles. Nicotine lowered thresholds across the first two 5-day cycles (i.e., 0.5 & 1 mg/kg/day doses), but thresholds returned to baseline levels during the last 5-day cycle (i.e., 2 mg/kg/day). The maximum threshold lowering produced by nicotine was similar to that previously reported for acute and chronic nicotine and for mild stimulants with a low addiction liability (i.e., caffeine and pseudoephedrine). Forty-eight hrs after terminating the nicotine injection regimen, thresholds were elevated revealing a nicotine withdrawal reaction. However, the high nicotine dose used during the last 5-day cycle is probably not pharmacologically relevant, thus making the significance of the withdrawal effect unclear. Overall, this study suggests that even under chronic administration using escalating doses, nicotineís profile in this animal model is that of a substance with a low addiction liability.|
The effect of a compound on brain stimulation reward (BSR) provides measure of the compoundís action on an important brain reward system. Both opiates and psychomotor stimulants activate the neural pathway that is activated by lateral hypothalamic brain stimulation [3,27,28]. This pharmacological activation enhances the rewarding impact of the electrical stimulation and produces BSR facilitation (e.g., stimulation-threshold lowering). BSR has been proposed as a model of the hedonic impact of a compound and is thought to reflect the compoundís intrinsic rewarding effect and therefore its potential addiction liability [11,22; see also 4]. This model has both face validity and considerable empirical support. Drugs that enhance the rewarding effects of electrical brain stimulation are generally highly addictive, and drugs that are not addictive usually fail to enhance BSR.
Nicotine has been shown to facilitate BSR [1,9,17,18,21], and these data are consistent with the proposed rewarding action of nicotine. However, recent work  supports an earlier suggestion  that quantitative aspects of a compoundís effect on BSR are important in estimating its potential addiction liability. Some compounds which reliably facilitate BSR have a low addiction liability (e.g., nalorphine; ). Therefore, the magnitude of the facilitation effect must be considered when using BSR to assess a substanceís potential addiction liability.
To distinguish the effects of compounds with high and low addiction liabilities, the facilitation produced by highly addictive compounds (e.g., cocaine) must be compared not only with inert substances (e.g., physiological saline) but with mildly psychoactive compounds that have a low addiction liability (e.g., pseudoephedrine). This avoids the problem of confusing substances that may have marginal rewarding effects with substances that have the potent rewarding effects characteristic of addictive drugs. Previous work has shown that nicotine produces BSR facilitation with the response profile of a compound with a low addiction liability , even when chronically administered [1,6]. This facilitation effect is quantitatively distinguishable from that obtained with prototypic addictive drugs. The possibility remains, however, that repeated, high-dose nicotine administration may enhance nicotineís potential rewarding action, and this enhanced rewarding impact may produce facilitation similar to that seen with prototypic addictive drugs. This study examined the effect of chronic nicotine on BSR using an escalating dose procedure designed to test the effect of repeated, high-dose nicotine administration.
Initial tobacco use by humans can produce aversive effects but tolerance quickly develops permitting continued, increasing tobacco use [26; see also 20]. Increasing tobacco use increases nicotine administration and may produce a strong rewarding action not present with initial, low-level tobacco use. Similarly, initial nicotine administration can produce apparent malaise in laboratory animals as shown by the development of conditioned taste aversions [e.g., 10,12] and by the disruption of operant behavior and locomotor activity. Tolerance quickly develops to the behaviorally disruptive effects of nicotine in laboratory animals [e.g., 6,25]. One strategy to investigate the maximum facilitatory action of nicotine is to mimic the escalating nicotine doses which occur during the acquisition of smoking behavior in humans. Nicotine doses are systematically increased, with repeated administration of each dose permitting the development of tolerance to nicotineís aversive effects. This allows testing larger nicotine doses with minimal disruptive effects. The initial nicotine dose selected for this study produces maximum BSR facilitation during acute administration. The nicotine dose then increases every 5 days until toxic reactions are noted.
Materials and Methods
Male, Long-Evans rats (Harlan Sprague-Dawley, Altamont, NY), weighting 225 to 275 g at the time of surgery, were implanted with monopolar stimulating electrodes aimed at the lateral hypothalamic level of the medial forebrain bundle. With the upper incisor bar 3.3 mm below the interaural plane, the coordinates were posterior 3.3 from bregma, lateral ± 1.8 from the midline mm, and 8.4 mm below dura. Electrodes were fabricated from 0.25 mm stainless steel wire insulated with Formvar except at the cross section of the tip. The stimulation ground was formed by wrapping 0.25 mm annealed stainless steel wire around two stainless steel screws (#80) anchored into the rostral aspect of the skull. Both the stimulating electrode and the ground terminated in gold-plated Amphenol pins that were connect to the stimulation lead during testing by mating Amphenol sockets.
Electrodes were implanted under sodium pentobarbital (65 mg/kg, i.p.) anesthetic, with atropine sulfate (0.4 mg/kg, i.p.) given to decrease mucosal secretions. Electrodes were anchored to the skull using three stainless steel screws embedded in dental acrylic. A single dose of penicillin-G (60,000 units, i.m.) was administered prophylactically following the completion of surgery. Animals were allowed a minimum of 5 days recovery from surgery before screening for BSR.
Rats were individually housed in stainless steel cages contained in a temperature and humidity controlled environment (23 ± °C, 40 to 60 %-RH). A 14-hour light/10-hour dark cycle of illumination was used, with all behavioral testing occurring during the light phase of this cycle. Subjects were given ad libitum access to food and water, except during behavior testing. At the end of the experiment, animals were sacrificed with an overdose of sodium pentobarbital (c. 80 mg/kg, i.p.) and were transcardially perfused with normal saline followed by phosphate-buffered formalin. The brains were removed and stored in formalin before sectioning with into 40 mm sections using a cryostat-microtome. The brain sections were stained using crystal violet, and electrode placements were verified at 10x magnification .
Stimulation pulses consisted of 300 msec trains of 300 msec cathodal pulses, with the electrode shunted to ground during the interpulse interval to prevent charge build-up in the stimulated tissue. Various current intensities (100 to 500 mA) and frequencies (32 to 126 Hz) were used. Stimulation pulses were controlled by a computer program, which determined all stimulation parameters except current intensity which was controlled by a constant-current stimulator . Current intensity was monitored by the voltage drop across a 1 kohm resistor in series with the stimulating electrode. Pulse form and current intensity were monitored throughout the test sessions using Textronic oscilloscopes.
Rats were tested in 26 x 47 x 38 cm operant chambers containing a lever located 8 cm above the floor. Each lever press produced a single train of stimulation. Subjects were connected to the stimulator with a flexible lead attached to an electrical commutator. Unrestricted movement of the subjects was maintained throughout the experimental sessions.
Rats were screened for BSR at 79 to 126 Hz using various current intensities (100 to 500 mA). Subjects showing vigorous lever-pressing were tested for several 30-min sessions at fixed stimulation parameters. After stable responding developed, testing with the threshold-tracking procedure  was begun using daily 30-min sessions. Stimulation frequencies were presented decreasing 0.1 log unit per minute until responding fell below criterion (i.e., 30 presses/min). Stimulation frequencies then increased 0.1 log unit per minute until responding met criterion (i.e., ³ 30 presses/min). Alternating descending and ascending thresholds were continuously determined throughout the test session. Threshold was defined as the average stimulation frequency that maintained criterion responding. Ascending and descending threshold were generally the same, producing response patterns that alternated vigorous pressing (at threshold) and nonresponding across successive 1-min periods.
Rats were tested daily with 30-min sessions. Mean frequency thresholds were calculated daily for each rat. Responding was considered stable when thresholds were within 5% of the previous 5-day mean. After frequency thresholds had stabilized (range 2 to 3 weeks), the experimental treatment was begun. Subjects were injected immediately before BSR testing and were tested continuously for 30 min following injections.
Nicotine bitartrate (Sigma Chemical, St. Louis) was dissolved in physiological saline, and the pH was adjusted to 7 ± 0.2 with sodium hydroxide. One group (n = 9) received a single subcutaneous (s.c.) injection of nicotine daily just before testing. Three successive nicotine doses were tested, each across 5 days: 0.5, 1, & 2 mg/kg/day (s.c., dose expressed as freebase weight). A second group (n = 9) was injected daily with physiological saline (1 ml/kg, s.c.) immediately before testing. Both groups were injected for 15 consecutive days, with BSR testing continuing for 7 days following termination of the injections. Data from the time period 16-30 min post injection were analyzed by comparing the effects of treatment with mean 5-day pretreatment baseline thresholds. Data are expressed as the percentage of baseline thresholds.
Figure 1 shows the threshold-lowering effect of nicotine across the
15-day escalating dose regimen. Daily nicotine injections lowered thresholds
across the first two 5-day cycles. The threshold-lowering effect of the
0.5 mg/kg/day and the 1 mg/kg/day nicotine doses were essentially the same.
When the nicotine dose was increased to 2 mg/kg/day during the last 5-day
cycle, thresholds unexpectedly returned to baseline levels. Animals administered
physiological saline daily showed little variation across the 15-day test.
|Figure 1: Effect of escalating nicotine doses on brain stimulation reward thresholds. Data shown are the mean (± SEM) percent of baseline thresholds for the time period 16-30 min post injections. The nicotine dose was increased from 0.5 to 1 to 2 mg/kg/day across 5-day cycles (indicated by the bars). Other animals were tested following daily injections of physiological saline. Symbols: open circles, saline; filled circles, nicotine.|
The mean threshold-lowering was somewhat less on the first two days than on subsequent days during the first 5-day cycle. A one-way analysis of variance (ANOVA) across the initial 5 days of testing when 0.5 mg/kg/day nicotine was administered revealed a significant effect of repeated testing [F(4,32) = 4.993, p = 0.003]. Post hoc comparisons using a Tukeyís test revealed that nicotineís threshold-lowering effect was less on Day-1 than on Days-3, 4, & 5 (pís < .05). ANOVAs conducted on the second [F(4,32) = 1.831, p = 0.147] and on the third [F(4,32) = 0.974, p = 0.436] 5-day cycles showed no significant variation within each cycle for the 1 and 2 mg/kg/day doses, respectively. A 3 x 5 ANOVA with repeated measures on both factors showed a significant effect of nicotine Dose [F(2,16) = 27.605, p < 0.001] and of Days within cycle [F(4,32) = 6.407, p < 0.001]. The Dose x Days interaction was not significant [F(8,64) = 1.082, p = 0.387], but this is not surprising considering the low power of the statistical test assessing the interaction (with a = 0.05, power = 0.070).
Nicotine injections also produced significant response suppression.
Figure 2 shows the response inhibition across days of repeated testing.
Tolerance developed rapidly to the inhibitory effect of the 0.5 mg/kg/day
dose [F(8,32) = 13.467, p < 0.001] as previously reported . Tolerance
developed more slowly to the 1 mg/kg/day dose [F(8,32) = 3.722, p <
0.013] and was not complete by the end of the second 5-day cycle. The 2
mg/kg/day dose produced profound response inhibition with partial tolerance
evident by the second day [F(8,32) = 2.849, p < 0.040] but with no further
tolerance developing throughout the remaining daily tests. The response
inhibition seen with the two lower nicotine doses is probably related to
a general sedative effect reported by other investigators [e.g., 25]. However,
the lack of continual tolerance development to the inhibitory effect of
the 2 mg/kg/day nicotine dose suggests that another mechanism is involved
in the disruptive effect of high-dose nicotine. Most of the subjects displayed
convulsions soon after receiving each high-dose nicotine injection, and
the latency to initiate responding is probably related to the after-effects
of seizure activity. Nonetheless, responding was generally stable during
the 16-30 min period when nicotine usually produces its peak BSR facilitation.
|Figure 2: Response inhibition from escalating nicotine doses. Data shown are the mean (± SEM) time (min) responding was inhibited following nicotine injections. The nicotine dose was increased from 0.5 to 1 to 2 mg/kg/day across 5-day cycles. Animals injected daily with physiological saline (data not shown) initiated responding within 30 sec post injections.|
BSR testing continued daily following termination of the nicotine injection regimen. Discontinuing nicotine treatment produced a slight increase in thresholds that was not statistically significant on the first day [Day-16: mean ± SEM = 9.4 ± 5.2%; t(8) = 1.774, p = 0.114] but increased further and was significant on the second day post nicotine withdrawal [Day-17: mean ± SEM = 15.8 ± 5.0%; t(8) = 2.983, p = 0.018]. Thresholds then returned to baseline levels for the remaining 5 days of testing (Days 18 to 22: range = -0.3 to 8.2% of baseline values) with no other significant differences.
The objective of the present study was to determine the maximum facilitation of BSR that can be produced by high-dose nicotine administration. The experimental strategy was to increase the nicotine dose across successive 5-day cycles. Each dose of nicotine was repeated for several days to permit the development of tolerance to the disruptive effect of nicotine at each dose level. This protocol was designed to mimic the increasing nicotine doses experienced during the acquisition of smoking behavior. Tolerance develops rapidly to the initial aversive effect of nicotine, and many investigators presume this permits increasing nicotine intake to a level whereby nicotine produces a potent rewarding action. If nicotine did produce a more pronounced rewarding action with increased doses, this should be reflected by nicotineís enhanced facilitation of BSR. In contrast to the predicted changes, nicotineís facilitation of BSR was not enhanced across days of repeated testing, even with increasing nicotine doses. In fact, there were few significant changes within the first two 5-day cycles, and the highest nicotine dose actually caused thresholds to return to baseline values. The potency of the high-dose nicotine injections, however, was confirmed by the response inhibition seen during the first 10 minutes of treatment.
The inability of the highest nicotine dose to modify BSR thresholds is puzzling and may reflect tachyphylaxis. Receptor desensitization could explain the apparent acute tolerance to nicotineís facilitation of BSR, and desensitization of nicotine-stimulated dopamine release can occur very quickly [e.g., 8,24]. In vivo studies showing desensitization to nicotine-stimulated dopamine release reveal a first-order process with a half-life of 42.7 sec . Although distribution kinetics from peripheral administration should slow the development of desensitization appreciably, it is possible that desensitization occurs rapidly enough to mask any initial facilitation of BSR. Although it might seem unlikely that absorption and distribution following a subcutaneous injection would produce a very rapid CNS action, the response inhibition produced by initial nicotine injections occurs within 30 sec post injection and is dissipated by 7 to 14 min post injection depending upon nicotine dose. Furthermore, total desensitization can occur with high nicotine concentrations, and this is consistent with the lack of effect following the 2 mg/kg nicotine dose.
Termination of the escalating dose regimen produced an apparent nicotine withdrawal reaction as shown by increased BSR thresholds. Previous work with cocaine has documented a similar withdrawal reaction following termination of cocaine administration, but the maximum threshold elevation seen following nicotine withdrawal (i.e., » 15%) is much less than the threshold elevation seen following cocaine withdrawal (i.e., » 100%; [14,15]). This is consistent with other quantitative differences between the effects obtained with nicotine and with cocaine (e.g., maximum facilitation; ). Furthermore, the relevance of high-dose nicotine administration to physiological levels normally reached during human tobacco use is unclear. The 2 mg/kg/day nicotine dose administered during the last 5-day cycle was obviously toxic; most subjects displayed convulsions shortly after receiving the nicotine injections. Thus, this dose is clearly not pharmacologically relevant for human tobacco use (i.e., cigarette smoking does not usually lead to convulsions). Finally, the time-course of the threshold elevations does not correspond with the time-course reported for spontaneous withdrawal signs. Significant threshold elevations were seen 48 hrs post nicotine withdrawal, whereas other work has shown significant withdrawal signs present 16 hrs post nicotine withdrawal but absent 40 hrs post withdrawal . Nonetheless, cessation of high-dose nicotine administration produces a significant elevation in BSR thresholds 48 hrs post nicotine withdrawal. This shows that a putative withdrawal reaction can be produced by repeated nicotine administration, even if the high doses are not pharmacologically relevant and the time-course is different than that seen with other withdrawal signs.
In conclusion, increasing nicotine doses up to a toxic level failed to enhance the facilitation of BSR beyond the level seen with acute nicotine administration. Thus, there is no evidence that the ability of nicotine to modulate brain reward mechanisms increases with repeated administration. If nicotine produced a potent rewarding action, then nicotineís BSR facilitation should be much stronger, comparable to that seen with prototypic addictive drugs such as cocaine. Indeed, the present data suggest that even under conditions of repeated, high-dose administration, nicotine has only a weak action on brain reward mechanisms.
Matt Morris is thanked for assistance in behavioral testing. Data reported in this paper are from the Nicotine Evaluation Program supported by a grant from the Philip Morris Research Center (Richmond, VA). The opinions expressed herein are those of the authors and not necessarily those of the sponsor.
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