Effect of Chronic Nicotine on
Brain Stimulation Reward:
I. Effect of Daily Injections
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
|Previous work has shown that nicotine facilitates BSR but that the maximum effect obtainable with nicotine is similar to that seen with nonaddictive compounds. This study examined whether repeated nicotine injections would enhance the facilitatory action of nicotine on BSR. Rats with lateral hypothalamic stimulating electrodes were tested using a threshold-tracking procedure. This procedure determined the minimum stimulation frequency (i.e., stimulation threshold) necessary to maintain ³30 presses/min during 30-min test sessions. Rats were injected daily with nicotine bitartrate (0.5 mg/kg, s.c., dose expressed as free-base weight) or physiological saline (1 ml/kg, s.c.) immediately before testing for 21 consecutive days. Nicotine lowered thresholds across the 21-day injection regimen. Neither tolerance nor sensitization to this effect was apparent. The magnitude of threshold lowering produced by nicotine was similar to that previously reported for acute nicotine and for mild stimulants with low addiction liabilities (i.e., caffeine and pseudoephedrine). This finding suggests that even under chronic administration, nicotine’s profile in this animal model is that of a substance with a low addiction liability.|
Considerable evidence suggests that a compound’s effect on brain stimulation reward (BSR) provides a useful assessment of its addiction liability [see 3,8,14,15]. Drugs that have a high addiction liability generally enhance the rewarding effects of electrical brain stimulation, while drugs that have a low addiction liability usually fail to enhance (or even inhibit) BSR. However, recent work suggests that quantitative aspects of a compound’s effect on BSR are important in accurately estimating its addiction liability [4; see also 2]. The simple determination of whether a compound facilitates BSR is insufficient to asses its addiction potential; rather, the magnitude of the facilitation effect must be considered.
The effect of a compound on BSR can be characterized as typical of addictive or nonaddictive substances. The response profile produced by prototypic addictive drugs (e.g., cocaine) establishes the facilitation effect expected for highly addictive compounds, while the response profile produced by mildly psychoactive but generally nonaddictive substances (e.g., pseudoephedrine) establishes the facilitation effect expected for compounds with a low addiction liability. Previous work has shown that nicotine produces BSR facilitation [1,4,10,11,13,16], but the response profile is that of a nonaddictive compound —nicotine’s facilitation effect is quantitatively distinguishable from that obtained with prototypic addictive drugs. The possibility remains, however, that repeated nicotine administration may enhance nicotine’s potential rewarding action and may produce facilitation similar to that seen with prototypic addictive drugs. This study examined the effect of chronic nicotine on brain stimulation reward. The optimal nicotine dose was selected from a previous dose-response analysis [4; see also 1,7], and animals were injected daily for 21 consecutive days.
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 mm from the midline, and 8.3 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. 100 mg/kg, i.p.) and were transcardially perfused with normal saline followed by 10% phosphate-buffered formalin. The brains were removed and stored in 10% formalin before sectioning 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 48 x 38 cm high 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 1min 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 = 10) received a single subcutaneous (s.c.) injection of 0.5 mg/kg nicotine (dose expressed as free-base weight) daily just before testing. A second group (n = 9) was injected daily with physiological saline (1 ml/kg, s.c.) immediately before testing. Both groups were injected for 21 consecutive days, with BSR testing continuing for 5 days following termination of the injections. Data from the time period 15-30 min post injection were analyzed by comparing the effects of treatment with mean 5-day pretreatment baseline thresholds. Previous determination of the time-course of nicotine’s facilitation effect had determined that nicotine’s effect peaks during the interval 15-30 min post injection (6). To minimize the influence of variance associated with different absolute baseline thresholds (i.e., range 27.6 to 94.4 Hz), data are expressed as the percentage of baseline thresholds.
Figure 1 shows the threshold-lowering effect of nicotine across the
21-day injection regimen. A 2 x 21 analysis of variance (ANOVA) with repeated
measures on one factor (unweighted-means solution; 18) revealed that nicotine
reliably lowered thresholds [F(1,17) = 72.776, p < .0001]. There was
no effect for Days of repeated testing [F(20,340)=1.588, p > .1]
nor was there a Treatment x Days interaction [F(20,340) =
0.828, p > .1]. A one-way within subjects ANOVA across the 21 days of nicotine
administration revealed no overall change in threshold lowering [F(20,180)
= 1.579, p > .06], despite the fact that Day-1 facilitation appeared somewhat
weaker than the facilitation seen on some other days of testing. The slightly
weaker initial facilitation may be related to the disruptive effect of
initial nicotine exposure on responding for BSR.
|Figure 1: Effect of chronic nicotine on brain stimulation reward. Data shown are the mean (± SEM) percent of baseline thresholds for the time period 15-30 min post injections. Nicotine reliably facilitated BSR during chronic administration, with no significant changes in its threshold-lowering effect across the 21-day injection regimen. Symbols: open circles, saline; filled circles, nicotine.|
Figure 2 shows the mean latency to initiate responding following the
nicotine injections across the first 7 days of testing. The first nicotine
injection suppressed responding for an average of 6 min, while tolerance
to this effect was seen by the second injection on Day-2 [t(9)= 2.782,p
< .021]. From Day-5 through Day21, animals generally initiated responding
within 1 min of the nicotine injection. The possibility that the slightly
weaker facilitation seen on Day-1 was related to the initial response suppression
was examined using a correlational analysis of response inhibition and
BSR thresholds. The Pearson product-moment correlation just missed statistical
significance (r = .625, p = .054), but a significant relationship
was seen between response-inhibition and threshold ranks (i.e., Spearman
on the first day of testing (r = .648, p = .0426).
This analysis shows that initial response inhibition was associated with
higher stimulation thresholds (i.e., less BSR facilitation) during the
first day of testing. No other significant correlations between response
inhibition and stimulation thresholds were found across the 21-day injection
regimen: the effect of response inhibition on stimulation thresholds appears
limited to the first day of nicotine exposure.
|Figure 2: Response inhibition from initial nicotine exposure. The figure shows the mean (± SEM) duration responding was inhibited during the first 7 days of testing. Tolerance developed rapidly to the disruptive effect of nicotine on responding for BSR. The mean latency to initiate responding after the daily nicotine injections was usually £ 1 min from Day-5 through Day-21.|
Initial nicotine administration inhibited responding for several minutes after the injection, but tolerance to this disruptive effect was apparent by the second nicotine injection. Partial tolerance to the locomotor suppressing effect of nicotine has been reported to develop very rapidly [e.g., 17], although rate-dependent measures of BSR sometimes show depressant effects for several days of continued nicotine administration [6,13]. The threshold-tracking procedure, used in the present study, revealed a significant BSR facilitation emerging soon after the animals reinitiated responding during the first day of testing.
Repeated nicotine injections reliably lowered stimulation thresholds across the 21-day injection regimen. Nicotine’s threshold-lowering effect was stable showing neither tolerance nor sensitization with repeated injections. This finding is consistent with Bauco and Wise  who examined several nicotine doses across 10 days using a frequency-rate measure of BSR. These two studies, using different experimental procedures, provide corroborative evidence that repeated nicotine injections do not produce cocaine-like facilitation of BSR.
If nicotine produced a potent rewarding action, then nicotine’s BSR facilitation would be expected to be much stronger and quantitatively similar to that seen with prototypic addictive drugs such as cocaine. In contrast, the BSR studies suggest that nicotine, like other mildly psychoactive substances, produces only weak actions on brain reward mechanisms. A potential limitation of the present study is that it used only a single nicotine dose, although this dose was shown to produce maximum facilitation from acute administration . It is possible that successively increasing nicotine doses might produce cocaine-like facilitation of BSR: tolerance develops rapidly to nicotine’s disruptive effect on behavior, and increasing the nicotine dose across repeated administrations might unmask stronger BSR facilitation. None-the-less, the present study contributes to an increasing body of literature suggesting that nicotine has only modest effects on brain reward systems and that nicotine’s effect in preclinical models is quantitatively different from the effects produced by prototypic addictive drugs which have strong effects on brain reward mechanisms.
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.
2. Bozarth, M. A., Intracranial Self-Stimulation as an Index of Opioid Addiction Liability: An Evaluation, unpublished master’s thesis, Rensselaer Polytechnic Institute, Troy, NY, 1978.
3. Bozarth, M. A. An overview of assessing drug reinforcement. In M.A. Bozarth (Ed.) Methods of Assessing the Reinforcing Properties of Abused Drugs, Springer-Verlag, New York, 1987, pp. 635-658.
4. Bozarth, M. A., Pudiak, C. M. and KuoLee, R., Predicting addiction liability from brain stimulation reward data: I. A comparison of the effects of cocaine, pseudoephedrine, nicotine, and caffeine, under editorial review.
5. Bozarth, M. A., Pudiak, C. M., Nocera, J. and Specht, C., A threshold-tracking procedure for studying brain stimulation reward, Soc. Neurosci. Abstr. 16 (1990) 591.
6. Clarke, P. B. S. and Kumar, R., Nicotine does not improve discrimination of brain stimulation reward by rats, Psychopharmacology 79 (1983) 271-277.
7. Huston-Lyons, D. and Kornetsky, C., Effects of nicotine on the threshold for rewarding brain stimulation in rats, Pharmacol. Biochem. Behav. 41 (1992) 755-759.
8. Kornetsky, C., Esposito, R. U., McLean, S. and Jacobson, J. O., Intracranial self-stimulation thresholds: A model for the hedonic effects of drugs of abuse, Arch. Gen. Psychia. 36 (1979) 289-292.
9. Mundl, W., A constant-current stimulator, Physiol. Behav. 24 (1980) 991-993.
10. Newman, L. M., Effects of cholinergic agonists and antagonists on self-stimulation behavior in the rat, J. Comp. Physiol. Psychol. 79 (1972) 394-413.
11. Olds, M. E. and Domino, E. F., Comparison of muscarinic and nicotine cholinergic agonists on self-stimulation behavior, J. Pharmacol. Exp. Ther. 166 (1969) 189-204.
12. Pellegrino, L. S., Pellegrino, A. S. and Cushman, A. J., A Stereotaxic Atlas of the Rat Brain, Plenum Press, New York, 1979.
13. Pradham, S. N. and Bowling, C., Effects of nicotine on self-stimulation behavior, J. Pharmacol. Exp. Ther. 176 (1971)229-243.
14. Reid, L. D., Tests involving pressing for intracranial stimulation as an early procedure for screening likelihood of addiction of opioids and other drugs. In M.A. Bozarth (Ed.), Methods of Assessing the Reinforcing Properties of Abused Drugs, Springer-Verlag, New York, 1987, pp. 391-420.
15. Reid, L. D.; Bozarth, M. A. Addictive agents and intracranial stimulation (ICS): The effects of various opioids on pressing for ICS. Problems of Drug Dependence (1978) 729-741.
16. Schaefer, G. J. and Michael, R. P., Interactions between alcohol and nicotine on intracranial self-stimulation and locomotor activity in rats, Drug Alcohol Depend. 30 (1992) 37-47.
17. Stolerman, I. P., Bunker, P. and Jarvik, M. E., Nicotine tolerance in rats: Role of dose and dose interval, Psychopharmacologia 34 (1974) 317-324.
18. Winer, B. J., Statistical Principles in Experimental Design, McGraw-Hill, New York, 1971.