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Adrenocortical hormone regulation of nicotine sensitivity in mice
Physiology & Behavior, Vol. 44, pp. 109-116. Copyright © Pergamon Press plc, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Adrenocortical Hormone Regulation of Nicotine Sensitivity in Mice J A M E S R. P A U L Y , E L I Z A B E T H
A. ULLMAN
AND ALLAN
C. C O L L I N S 1
Institute Jbr Behavioral Genetics and School of Pharmacy, University of Colorado, Boulder, CO 80309 R e c e i v e d 13 A p r i l 1988 PAULY, J. R., E. A. ULLMAN AND A. C. COLLINS. Adrenocorticalhormone regulationof nicotine sensitivityin mice. PHYSIOL BEHAV 44(1) 109-116, 1988.--The possibility that nicotine-induced corticosterone (CCS) release regulates nicotine sensitivity was investigated in female mice of the C3H strain. Adrenalectomy (ADX) resulted in an increase in nicotine sensitivity as measured in a number of physiological and behavioral tests. In ADX animals, chronic CCS (100 /.~g/ml) administered in the drinking solution normalized nicotine sensitivity. Dexamethasone (DEX), a potent synthetic glucocorticoid which interacts with a distinct population of CNS steroid receptors, did not reverse the effects of ADX. Unoperated animals administered CCS (200/xg/ml) were protected from the effects of nicotine for several test battery parameters. ADX had no effect on the number of brain nicotinic cholinergic receptors and also did not alter nicotine metabolism. These data support the hypothesis that CCS secretion modulates nicotine sensitivity in the mouse; however, the mechanisms by which this regulation occurs are unknown. Nicotine
Adrenalectomy
Corticosterone
Dexamethasone
TOBACCO users tend to smoke when in stressful situations, and high nicotine-containing cigarettes have been shown to reduce emotionality to a greater extent than do low nicotine cigarettes (16,39). Consequently, it may be that one of the reinforcing properties of nicotine is its ability to reduce anxiety. The mechanisms by which nicotine reduces emotionality are not well understood but is has been shown that nicotine, administered via injection or smoke inhalation, causes a dose-dependent increase in plasma concentrations of adrenocortical hormones (9,21) and adrenocorticotrophic hormone (ACTH) (35,46). This effect is probably mediated by central nervous system (CNS) nicotinic receptors since mecamylamine, a CNS nicotinic receptor antagonist, has been shown to block the actions of nicotine on CCS release in mice (12). Furthermore, the intracerebroventricular injection of antibodies raised against the nicotinic cholinergic receptor from myasthenia gravis patients blocks stressinduced increases in CCS (8,51). Hillhouse et al. (19) have demonstrated that acetylcholine induces the release of corticotrophin releasing hormone from an isolated rat hypothalamic preparation. Thus, a central mode of action for nicotine-induced CCS release is present in rodents. Tolerance to nicotine-induced CCS release develops if animals are chronically treated with nicotine (3,48). The possibility that corticosterone (CCS) released in response to nicotine plays a role in the determination of nicotine's physiological and behavioral effects has not been
investigated. One method of determining whether or not CCS release is of consequence to nicotine response is to deprive animals of their primary source of CCS, the adrenal gland. Mice are readily adrenalectomized (ADX) and can be maintained for long periods of time if provided with 0.9% saline as a drinking solution. The purpose of the present study was to test the hypothesis that CCS release in response to nicotine is an important factor in the determination o f nicotine response. Adrenalectomy resulted in an increase in acute nicotine sensitivity that could be reversed by chronic administration of CCS. These data indicate that adrenal responses may modulate nicotine sensitivity in rodents. METHOD Female mice of the C3H strain were used for these studies. Animals were raised in the colony at the Institute for Behavioral Genetics and maintained on a 12 hour light/dark cycle with lights on at 0700. Food (Wayne Lab Blox) and water were provided ad lib. All behavioral testing was performed between 0800 and 1300 on adult animals (60-90 days of age).
Adrenalectomy Animals were anesthetized with pentobarbital (54 mg/kg) administered intraperitoneally. A small mid-dorsal incision was made on each side of the animal's back and the kidneys
'Requests for reprints should be addressed to Dr. Allan C. Collins, Institute for Behavioral Genetics, University of Colorado, Campus Box 447, Boulder, CO 80309.
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NICOTINE DOSE ( m g / k g ) FIG. 1. Dose-response curves for nicotine effects on several test battery parameters for adrenaleetomized (ADX) and sham-operated animals. ADX animals were significantly more sensitive to nicotine for all tests [startle response, F(1,36)=9.01; Y-maze crosses, F(1,36)=6.20; heart rate, F(1,36)=9.79; body temperature, F(1,36)=25.54].
exposed. The adrenal glands were removed using a pair of fine curved forceps. Surgery for sham-operated animals was identical to that for ADX animals except the adrenal glands were not removed. Following surgery, all animals were housed individually and ADX animals were provided with 0.9% saline as a drinking solution. Animals were tested for nicotine sensitivity in a battery of tests, usually one week following surgery. Nicotine was administered via intraperitoneal injection of drug dissolved in physiological saline.
Nicotine Response Nicotine sensitivity was measured in a multifactorial test battery that included acoustic startle response, Y-maze activity (crosses and rearings), heart rate and body temperature. Previous studies from our laboratory have demonstrated that no significant interest interactions occur between these tests (34). The timing of these tests was determined from the results of a time course study on the effects of nicotine on several components of the test battery (32). Each test in the battery was conducted as follows: Acoustic Startle Response--A Columbus Instruments Startle Reflex Monitor (Columbus Instruments, Columbus, OH) was used to quantitate the startle response elicited by a short auditory stimulus. A mouse was placed in a Plexiglas cage inside a sound-attenuated box and 10 auditory stimuli were delivered (frequency, 6250 Hz; intensity, 120 dB; duration, 50 msec). For each stimulus both response amplitude and response latency were recorded. This test was initiated 2.5 rain following nicotine injection. Y-Maze Activity--The maze is a symmetrical Y-shaped runway with each arm being 26.0 cm long, 6.1 cm high and 10.2 cm wide. Each arm of the maze was divided into two sections; crosses from one section/arm to another were counted for 3 min. The number of rearings that occurred during the test period was also recorded. This test was conducted 4.5 rain after nicotine administration.
Heart Rate--After completion of the Y-maze test, mice were placed in a restrainer and needle electrodes were inserted through the skin (one behind the left foreleg and the other in front of the right hindleg). The electrodes were connected through a preamplifier to an E and M Physiograph (Narco Biosystems, Houston, TX). Heart rate was monitored for 6 sec beginning 8.5 rain after nicotine injection. Body Temperature--Rectal temperature was measured using a Bailey Instruments (Saddlebrook, N J) digital thermometer. The probe was lubricated with peanut oil before it was inserted 2.5 cm into the rectal cavity. Body temperature was measured 15 min following nicotine injection. Steroid Replacement In some experiments, ADX or sham-operated animals were provided with exogenous sources of adrenocortical hormones. Steroid hormones (CCS or dexamethasone) (Sigma Chemical Company, St. Louis, MO) were dissolved in a small volume of absolute ethanol (1.0% final concentration) and administered chronically in the animal's drinking solution. Control animals received a drinking solution of 1.(1% ethanol. Following behavioral testing, plasma CCS levels were measured using a modification of the radioimmunoassay described by Gwosdow-Cohen et al. (15).
Nicotine Levels In order to test for differences in the absorption, distribution and/or elimination of nicotine following ADX, levels of nicotine in blood and brain were measured one week postsurgery in ADX or sham-operated animals. Nicotine content was assayed using an extraction procedure that has been described in detail in previously published reports (10, 17, 18). Nicotine levels were estimated in blood and brain 10 min after administration of a 1.5 mg/kg dose of nicotine that had been spiked with 2.0 izCi of [3H]-nicotine (New England Nuclear, Boston, MA) as a tracer. Animals were decapi-
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NICOTINE DOSE ( m g / k g ) FIG. 2. Dose-response curves for nicotine effects on several test battery parameters in ADX animals tested one day or one week followingsurgery. Lines were significantly different only for the body temperature measurement, F(1,43)=5.12.
tated, trunk blood collected (100-300/zl) and the brains removed, blotted and weighed. After homogenization and centrifugation of the samples, aliquots were added to 1.0 ml of 0.1 N NaOH and 7.5 ml of purified heptane. After these samples were shaken for 20 min, the nicotine was extracted by removing 5.0 ml of the organic phase (supernatant) and placing it into a tube containing 0.1 N HCI. The contents were shaken for 5 min and centrifuged. After removal of the organic phase by aspiration, 1.0 ml aliquot of the acid phase was removed and counted by liquid scintillation spectrometry. Previous studies from our laboratory have demonstrated that this procedure separates nicotine from its more polar metabolites (I 7,18).
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Nicotinic Receptor Binding The brains of some animals were assayed for CNS nicotinic cholinergic receptor binding to determine if ADX affected the number or affinity of these receptors. Following the completion of behavioral testing, animals were decapitated and the brains were dissected into the following eight anatomical regions: cortex, striatum, midbrain, hypothalamus, hippocampus, colliculi, cerebellum and hindbrain. Tissue regions were placed in 10 volumes of HEPES-buffered Ringer's solution (NaCI, 118 mM; KCI, 4.8 mM; CaC12, 2.5 mM; MgSO4, 1.2 mM; HEPES, 20 mM; pH adjusted to 7.5 with NaOH) and then frozen at -70°C. The particulate fraction from these samples was assayed for nicotinic receptor binding. The binding of L-pH]-nicotine was measured using a modification of the procedure of Romano and Goldstein (43) as previously described (33). All brain regions were assayed using a single concentration of radiolabeled nicotine. The binding of alpha-[*~5I]-bungarotoxin (BTX) (New England Nuclear, Boston, MA) was performed as described by Marks and Collins (33) using a single concentration of labeled BTX. Protein determinations were made using the method of Lowry et al. (23), using bovine serum albumin as a standard.
FIG. 3. Effects of steroid hormone replacement therapy on nicotine sensitivity for acoustic startle response. A significant effect of treatment was determined, F(3,111)=42.92. Startle response was significantly dampened by chronic administration of both corticosterone (CCS) (100/xg/ml for 7 days) and dexamethasone (DEX) (100 p.g/ml for 7 days).
Data Analysis Results from behavioral testing were analyzed using two-way analysis of variance (treatment by dose). Newman-Keuls post hoe tests were used to analyze the data when overall effects of dose or treatment were identified. t-Tests were used for all other statistical comparisons. RESULTS One week following surgery, nicotine sensitivity was measured in a battery of tests in ADX and sham-operated animals. Preliminary experiments (data not shown) demonstrated that there was no difference in nicotine sensitivity between unoperated and sham-operated animals. Ad-
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NICOTINE DOSE ( m g / k g ) FIG. 4. Effects of steroid hormone replacement therapy on nicotine sensitivity for Y-maze crosses. A significant effect of treatment was determined, F(3,111)= 3.22. Chronic CCS normalized nicotine sensitivity (i.e., line not different from that of sham-operated animals). Dexamethasone replacement however did not significantly normalize nicotine response.
FIG. 5. Effects of steroid hormone replacement therapy on nicotine sensitivity for Y-maze rears. A significant effect of treatment was determined, F(3,111)=4.82. Chronic CCS administration normalized nicotine sensitivity whereas chronic DEX did not.
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renalectomy, however, clearly induced an increase in nicotine sensitivity in C3H mice. The effects of ADX on nicotine sensitivity for startle response, Y-maze crosses, heart rate and body temperature measures are shown in Fig. 1. Increases in nicotine sensitivity were statistically significant for all test battery parameters. In some experiments, animals were tested for nicotine sensitivity on the day following ADX. Data from day 1 and day 7 ADX animals are shown in Fig. 2. N o significant differences in nicotine sensitivity in day 1 and day 7 ADX animals were present except for the body temperature parameter. Day 1 animals were significantly less sensitive to nicotine's hypothermic response than were animals tested one week postADX, The adrenal gland releases many substances in response to stressful stimuli. In order to test for the hormonespecificity of ADX effects it was necessary to measure nicotine sensitivity in ADX animals receiving chronic ad-
FIG. 7. Effects of steroid hormone replacement therapy on nicotine sensitivity for body temperature. A significant effect of treatment was determined, F(3,1 ! 1)= 20.55. Both CCS and DEX replacement therapy normalized nicotine sensitivity. renocortical hormone therapy. Corticosterone or dexamethasone were administered in the drinking saline of ADX animals at a concentration of 100 /xg/ml. Previous studies have demonstrated that CCS administered at this concentration effectively causes down regulation of hippocampal CCS receptors (45,47). Consumption of the steroid-containing solution was monitored for one week following ADX and animals were then tested for nicotine sensitivity. Animals that received steroid supplement in their drinking solution consumed a significantly larger volume of fluid (/9<0.01) than did animals drinking only saline. Consumption of the CCS-containing solution averaged 13.94 +0.66 ml/day and was not signifmantly different from consumption of the dexamethasone-contaming solutton (14.85+0.76 mFday). ADX animals receiving only saline drank an average of 7.65-+0.54 nil/day. Plasma CCS levels were determined by radioimmunoassay in ADX animals and ADX animals receiving CCS in their drinking water. CCS therapy resulted in a significant elevation in plasma CCS levels (p<0.01) (155.1-+34.9 ng/ml in
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NICOTINE DOSE ( m g / k g ) FIG. 8. Dose-response curves for nicotine effects on several test battery parameters in naive animals and animals given chronic CCS (200/.tg/ml for 10 days). Lines were significantly different for the following tests: startle response, F(1,39)=73.13; heart rate, F(1,39)= 11.51; body temperature, F(1,39)=38.66; and Y-maze rears, F(1,39)= 10.40.
CCS-treated animals vs. 21.8_+4.5 ng/ml in saline-treated animals). Chronic CCS administration prevented the ADX-induced increase in nicotine sensitivity for all test battery parameters. Data for startle response, Y-maze crosses, Y-maze rears, heart rate and body temperature are shown in Figs. 3 through 7 respectively. Dose-response curves for shamoperated animals and ADX animals receiving CCS were not significantly different (except for startle response which was dampened by CCS administration). Dexamethasone replacement was not equivalent to CCS replacement in terms of ADX-effects reversal. Chronic administration of this potent synthetic glucocorticoid normalized the effects of ADX only on the nicotine doseresponse curve for body temperature (Fig. 7). All other test battery parameters were unchanged by dexamethasone supplementation (Figs. 3-6). Because ADX animals that received chronic CCS appeared to be somewhat protected from the effects of nicotine on test battery parameters, unoperated animals were given chronic CCS (200/~g/ml) in order to test for possible nicotine response subsensitivity. Figure 8 demonstrates such a desensitization of nicotine response. For startle response, Y-maze rears, heart rate and body temperature, intact animals that received chronic CCS were significantly less sensitive to the effects of nicotine than were intact animals that received vehicle only as a drinking solution (1% ethanol). Startle response in animals that received chronic CCS was almost completely absent. Adrenalectomy could induce an increase in nicotine sensitivity through a change in nicotine metabolism or it may be that ADX could cause changes in tissue sensitivity. Using standard biochemical receptor binding assays we have determined that ADX does not significantly alter the number of CNS binding sites for nicotinic cholinergic receptors. The binding of [SH]-nicotine was significantly lower in the midbrain of ADX animals but no differences were measured in
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five other brain regions analyzed (Fig. 9). [12H]-BTXbinding was not significantly altered by ADX in any brain region (Fig. 10). Blood and brain levels of nicotine in ADX and shamoperated animals 10 minutes following nicotine administration are shown in Fig. 11. No significant effect of ADX on nicotine distribution was detected. Thus it is unlikely that ADX-induced increases in nicotine sensitivity are a result of alterations in nicotine metabolism. DISCUSSION
The results of this study demonstrate that alterations in the brain-pituitary-adrenal axis can induce changes in acute
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sensitivity to nicotine. Following ADX, mice of the C3H strain had significant changes in nicotine sensitivity for all test battery parameters. The changes induced by ADX were shown to be specific for the primary adrenocortical hormone in the mouse, corticosterone (CCS). Furthermore, we have also demonstrated that intact animals that received chronic CCS in their drinking water were significantly less responsive to nicotine than were control animals. Several groups of investigators have previously demonstrated that nicotine, administered by injection or through inhalation of cigarette smoke, induces a dose-dependent increase in plasma CCS (1, 2, 20, 52). The results obtained in our experiments demonstrate for the first time that CCS released in response to nicotine may modulate the behavioral and physiological actions of this drug. It is somewhat paradoxical that nicotine, a drug that has been shown to increase the activity of the autonomic nervous system, also has been reported to reduce emotion and increase tranquillity. Several investigators have shown that nicotine reduces negative affect and that humans use nicotine in stressful situations to achieve pleasurable relaxation (14,37). The mechanisms by which nicotine elicits these effects are not known and are obviously very complex. However, it is conceivable that nicotine-induced CCS release may be critically involved. Several studies have demonstrated that CCS and some of its metabolites decrease neuronal excitability through an enhancement of brain GABA-ergic neurotransmission (30,31). It is possible that an important factor in antistress responses is a reduction in neuronal excitability. Such a reduction in excitability may be present in animals that received chronic CCS. Nicotine does not induce CCS release via a direct action on the adrenal gland; this response is probably regulated through nicotinic receptors in the central nervous system. Mecamylamine, a CNS nicotinic receptor blocker, pretreatment prevents the nicotine-induced increase in CCS release (12) and several studies (8,51) have demonstrated that intracerebroventricular administration of antibodies raised against the nicotinic cholinergic receptor from Torpedo electric organ blocks stress-induced increases in CCS. Acetylcholine (ACh) has been shown to stimulate the
FIG. 11. Estimation of nicotine levels in the blood and brains of ADX and sham-operated animals 10 min following a challenge dose of nicotine spiked with a tracer. No significant differences between treatment groups were present. activity of CNS systems that regulate adrenocortical responses (1,13). Using an isolated rat hypothalamus preparation, Hillhouse et al. (19) demonstrated that ACh stimulates the release of corticotropin releasing factor (CRF). CRF in turn regulates the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary which stimulates the release of adrenocortical hormones. In rats, 2 min following systemic nicotine administration, plasma ACTH levels are significantly elevated (35,46). The mechanism by which ADX increases sensitivity to acute nicotine administration is not known. Nicotinic receptor binding and nicotine metabolism were not significantly affected by adrenalectomy. Changes in nicotine sensitivity following ADX could be due to the lack of adrenocortical hormones or, alternatively, changes could be due to increases in brain concentrations of ACTH and/or CRF. Several studies have demonstrated that basal and stress-induced levels of CRF and ACTH are elevated in ADX animals due to the lack of negative feedback (24,28). In the absence of adrenocortical hormones there is an up-regulation of neuronal receptors for these hormones (47). In the central nervous system there are at least two types of receptors for adrenocortical hormones; the distribution of these receptor subtypes in the CNS is heterogeneous. Type I, the CCS receptor (CR), is localized predominantly in the hippocampus and to a lesser extent in the septum. Glucocorticoid receptors (GR), or Type II receptors, are more uniformly distributed among neurons and glial ceils (11, 25-28, 49, 50). The use of synthetic glucocorticoids such as dexamethasone has greatly aided the identification of adrenocortical receptor subtypes. GR is sensitive only to high concentrations of CCS (e.g., stress-induced CCS release), whereas CR is sensitive to lower concentrations of CCS and detects smaller changes in CCS levels such as those which occur throughout the circadian cycle (40-42). In receptor binding assays CR can be identified using CCS, whereas GR is labeled using dexamethasone or other synthetic glucocorticoids. Corticosterone receptors have a low affinity for dexamethasone. Chronic DEX administration has been shown to have no effect on the number of CCS receptors (CR) (44). Thus, in ADX animals, it is possible to selectively prevent ADX-induced increases in these receptors through the chronic administration of CCS or DEX (44). Dexamethasone prevents the release of ACTH from the anterior pituitary through feedback inhibition. ADX animals that receive chronic DEX have increased numbers of CCS
CORTICOSTERONE AND NICOTINE SENSITIVITY receptors but increases in DEX receptors (GR) are prevented. Chronic DEX also prevents ADX-induced increases in ACTH levels (5,6). Thus a first test as to whether ADX effects are due to increased levels of ACTH can be performed using chronic DEX versus chronic CCS hormone therapy in ADX animals. If CCS therapy reverses the effects of ADX but DEX therapy (which normalizes ACTH concentrations) does not, then it can be inferred that increased nicotine responsiveness subsequent to ADX is not a direct result of increases in ACTH. In our battery of tests, we determined that CCS reversed the effects of ADX on nicotine sensitivity for all parameters. DEX therapy reversed the effects of ADX only for the body temperature measure. Therefore, increased sensitivity of the hypothermic response elicited by nicotine in ADX animals could be due to increased ACTH levels. This possibility is further supported by our finding that day 1 ADX animals were not as sensitive as day 7 ADX animals in terms of nicotine-induced hypothermia. At this time point, ACTH levels may not be significantly elevated and this may explain why these animals are not as sensitive as day 7 ADX animals to nicotine's hypothermic response. In rodents, ACTH has been shown to induce hypothermia when administered centrally (22). Our finding that DEX and CCS replacements do not have equivalent effects on locomotor activity (Y-maze crosses and rears) is not surprising. Several groups of investigators (6,29) have demonstrated that ADX causes a reduction in open field activity that is normalized by CCS but not DEX.
115 Other measures that are affected by ADX and normalized by CCS but not DEX include sensitivity to epinephrine in a passive avoidance response (7) and extinction of an appetitive runway task (6,36). As is the case with nicotine sensitivity, these behaviors are probably not due to ADX-induced changes in CNS concentrations of ACTH. The pituitary/adrenal system plays an essential role in the physiological control of adaptive behavior through the secretion of adrenocortical and other hormones. Corticosterone has been shown to be a potent modulator of central nervous system function. The present study demonstrates that alterations in adrenal status cause marked changes in sensitivity to nicotine, a drug that induces a variety of physiological and behavioral responses. The precise mechanism by which ADX causes an increase in nicotine sensitivity is not known. However, other studies in our laboratory (38) have demonstrated that this phenomenon is regulated by genetic factors. Future studies in our laboratory will investigate further the relationship between nicotine sensitivity and adrenocortical responses in various strains of mice. These studies may help elucidate the complex relationship between tobacco (nicotine) use, adrenocortical responses and anxiety reduction. ACKNOWLEDGEMENTS The authors gratefully acknowledge the advice and counsel of Dr. Michael J. Marks. This research was supported by the NICHD training grant HD-07289 and also by a grant from the R. J. Reynolds Tobacco Company.
REFERENCES 1. Balfour, D. J. K.; Khullar, A. K.; Longden, A. K. Effects of nicotine on plasma corticosterone and brain amines in stressed and unstressed rats. Pharmacol. Biochem. Behav. 3:179--184; 1975. 2. Balfour, D. J. K.; Morrison, C. F. A possible role for the pituitary-adrenal system in the effects of nicotine on avoidance behavior. Pharmacol. Biochem. Behav. 3:349--354; 1975. 3. Benwell, M. E. M. ; Balfour, D. J. K. Effects of nicotine administration and its withdrawal on plasma corticosterone and brain 5-hydroxyindoles. Psycopharmacology (Berlin) 63:7-11; 1979. 4. Benwell, M. E. M.; Balfour, D. J. K. Effects of chronic nicotine administration on the response and adaptation to stress. Psychopharmacology (Berlin) 76:160--162; 1982. 5. Bohus, B.; De Kloet, E. R. Adrenal steroids and extinction: antagonism by progesterone, deoxycorticosterone and dexamethasone of a specific effect of corticosterone. Life Sci. 28:433 a~9; 1981. 6. Bohus, B.; De Kloet, E. R.; Veldhuis, H. D. Adrenal steroids and behavioral adaptation: relationship to brain corticoid receptors. In: Ganten, D.; Pfaff, D., eds. Current topics in neuroendocrinology. New York: Springer-Verlag; 1982:108--148. 7. Borrell, B.; De Kloet, E. R.; Bohus, B. Corticosterone decreases the efficacy of adrenaline to affect passive avoidance retention of adrenalectomized rats. Life Sci. 34:99-105; 1984. 8. Brenner, T.; Mizrachi, R.; Bodoff, M.; Weindenfeld, J. Evidence that central nicotinic-acetylcholine receptors are involved in the modulation of basal and stress-induced adrenocortical responses. Exp. Neuroi. 94:735-743; 1986. 9. Cam, G. R.; Bassett, J. R. The effects of acute nicotine administration on plasma levels of thyroid hormones and corticosterone in the rat. Pharmacol. Biochem. Behav. 19:559-561; 1983.
10. De Fiebre, C. M.; Medhurst, L. J.; Collins, A. C. Nicotine response and nicotinic receptors in long-sleep and short-sleep mice. Alcohol 4:493-501; 1987. 11. De Kloet, E. R.; Wallach, G.; McEwen, B. S. Differences in corticosterone and dexamethasone binding to rat brain and pituitary. Endocrinology 96:598--609; 1975. 12. Freund, R. K.; Martin, B. J.; Jungschaffer, D. A.; Ullman, E. A. ; Collins, A. C. Genetic differences in plasma corticosterone levels in response to nicotine injection. Pharmacol. Biochem. Behav. 30:1059-1064; 1988. 13. Gilad, G. M.; Rabey, J. M.; Gilad, V. H. Presynaptic effects of glucocorticoids on dopaminergic and cholinergic synaptosomes. Implications for rapid endocrine-neural interactions in stress. Life Sci. 40:2401-2408; 1987. 14. Gilbert, D. G. Paradoxical tranquilizing and emotion-reducing effects of nicotine. Psychol. Bull. 86(4):643-661; 1979, 15. Gwosdow-Cohen, A.; Chen, C. L.; Besch, E. L. Radioimmunoassay of serum corticosterone in rats. Proc. Soc. Exp. Biol. Med. 170:29-34; 1982. 16. Hall, G. H.; Morrison, C. F. New evidence for a relationship between tobacco smoking, nicotine dependence and stress. Nature 243:199-201; 1973. 17. Hatchell, P. C.; Collins, A. C. Influences of genotype and sex on behavioral tolerance to nicotine in mice. Pharmacol. Biochem. Behav. 6:25-30; 1977. 18. Hatchell, P. C.; Collins, A. C. The influence of genotype and sex on behavioral sensitivity to nicotine in mice. Psychopharmacology (Berlin) 71:45--49; 1980. 19. Hillhouse, E. W.; Burden, J.; Jones, M. T. The effect of various putative neurotransmitters on the secretion of corticotrophinreleasing hormones from the rat hypothalamus in vitro--a model of the neurotransmitters involved. J. Endocrinol. 69:110; 1975.
116 20. Kershbaum, A. ; Pappajohn, D. J.; Bellet, S.; Hirabayashi, M.; Shafiha, H. Effect of smoking and nicotine on adrenocortical secretion. JAMA 203(4): 113-116; 1968. 21. Levine, S.; Treiman, D. M. Differential plasma corticosterone response to stress in four inbred strains of mice. Endocrinology 75:142-144; 1964. 22. Lipton, J. M.; Clark, W. G. Neurotransmitters in temperature control. Annu. Rev. Physiol. 48:613-623; 1986. 23. Lowry, O. H.; Rosebrough, N. H.; Farr, A. C.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. 24. McEwen, B. S. Influences of adrenocortical hormones on pituitary and brain function. In: Baxter, J. D.; Rousseau, G. G., eds. Glucocorticoid hormone action. New York: Springer-Verlag; 1979:467-492. 25. McEwen, B. S. Glucocorticoids and hippocampus: receptors in search of a function. In: Ganten, D.; Pfaff, D., eds. Current topics in neuroendocrinology. New York: Springer-Verlag; 1982:1-22. 26. McEwen, B. S. Glucocorticoid-biogenic amine interactions in relation to mood and behavior. Biochem. Pharmacol. 36(11): 1755-1763; 1987. 27. McEwen, B. S.; Davis, P. G.; Parsons, B.; Pfaff, D. W. The brain as a target for steroid hormone action. Annu. Rev. Neurosci. 2:65--112; 1979. 28. McEwen, B. S.; DeKloet, E. R.; Rostene, W. Adrenal steroid receptors and actions in the nervous system. Physiol. Rev. 66(4): 1121-1188; 1986. 29. Mclntyre, D. C. Adrenalectomy: Protection from kindled emulsion-induced amnesia in rats. Physiol. Behav. 17:78%795; 1976. 30. Majewska, M. D. Steroids and brain activity: essential dialogue between brain and body. Biochem. Pharmacol. 36(22):37813788; 1987. 31. Majewska, M. D.; Bisserbe, J. C.; Eskay, R. L. Glucocorticoids are modulators of GABAa receptors in brain. Brain Res. 339:178-182; 1985. 32. Marks, M. J.; Burch, J. B.; Collins, A. C. Effect of chronic nicotine infusion on tolerance development and nicotinic receptors. J. Pharmacol. Exp. Ther. 226(3):817-825; 1983. 33. Marks, M. J.; Collins, A. C. Characterization of nicotine binding in mouse brain and comparison with the binding of a-bungarotoxin and quinuclidinyl benzilate. Mol. Pharmacol. 22:554-564; 1982. 34. Marks, M. J.; Stitzel, J. A.; Collins, A. C. Time course study of the effects of nicotine infusion on drug response and brain receptors. J. Pharmacol. Exp. Ther. 235(3):619--628; 1985. 35. Matta, S. G.; Beyer, H. S.; McAllen, K. M.; Sharp, B. M. Nicotine elevates rat plasma ACTH by a central mechanism. J. Pharmacol. Exp. Ther. 243(1):217-226; 1987. 36. Micco, D. J.; McEwen, B. S. Glucocorticoids, the hippocampus and behavior: interactive relation between task activation and steroid binding specificity. J. Comp. Physiol. Psychol. 94(4):624--633; 1980. 37. Nesbitt, P. D. Smoking, physiological arousal and emotional response. J. Pers. Soc. Psychol. 25:137-144; 1973.
PAULY, ULLMAN AND COLLINS 38. Pauly, J. R.; Ullman, E. A.; Collins, A. C. A strain comparison of nicotine sensitivity in adrenalectomized mice. Pharmacol. Biochem. Behav., submitted for publication; 1988. 39. Pomerleau, O. F.; Turk, D. C.; Fertig, J. B. The effects of cigarette smoking on pain and anxiety. Addict. Behav. 9:265271; 1984. 40. Reul, J. M. H. M.; DeKloet, E. R. Two receptor systems tor corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117(6):2505--2511 ; 1985. 41. Reul, J. M. H. M.; DeKloet, E. R. Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradiography and computerized image analysis. J. Steroid Biochem. 24(1):26%272; 1986. 42. Reul, J. M. H. M.; van den Bosch, F. R.; DeKloet, E. R. Relative occupation of type I and type II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrinol. 115:45%467; 1987. 43. Romano, C.; Goldstein, A. Stereospecific nicotine receptors on rat brain membranes. Science 210:647-650; 1980. 44. Sapolsky, R. M.; McEwen, B. S. Down-regulation of neural corticosterone receptors by corticosterone and dexamethasone. Brain Res. 339:161-165; 1985. 45. Sarrieau, A.; Vail, M.; McEwen, B.; Broer, Y.; Dussaillant, M.; Philibert, D.; Moguilewsky, M.; Rostene, W. Corticosteroid receptors in rat hippocampal sections: effect of adrenalectomy and corticosterone replacement. J. Steroid Biochem. 24(3):721-724; 1986. 46. Sharp, B. M.; Beyer, H. S. Rapid desensitization of the acute stimulatory el/'ects of nicotine on rat plasma adrenocorticotropin and prolactin. J. Pharmacol. Exp. Ther. 238(2):486-491; 1986.
47. Tornello, S.; Orti, E.; DeNicola, A. F.; Rainbow, T. C.; McEwen, B. S. Regulation of glucocorticoid receptors in brain by corticosterone treatment of adrenalectomized rats. Neuroendocrinology 35:411-417; 1982. 48. UIIman, E. A.; Pauly, J. R.; Collins, A. C. Chronic nicotine infusions induce tolerance to adrenocortical responses to nicotine. FASEB 2(4):2858; 1988. 49. Veldhuis, H. D.; DeKloet, E. R. The adrenal steroid receptor system in rat hippocampus. In: Endroczi, E.; DeWeid, D.; Angelucci, L.; Scapagnini, V., eds. Integrative neurohumoral mechanisms. New York: Elsevier Science Publications: 1983:157-164. 50. Veldhuis, H. D.; van Hoppen, C.; van Ittersum, M.; DeKloet, E. R. Specificity of the adrenal steroid receptor system in rat hippocampus. Endocrinology 110(5):2044-2051; 1982. 51. Weidenfeld, J.; Siegel, R.; Conforti, N.; Mizrachi, R. ; Brenner, T. Effect of intracerebroventricular injections of nicotinic acetylcholine receptor antibodies on ACTH, corticosterone and prolactin secretion in the male rat. Brain Res. 265:152-156; 1983. 52. Wilkins, J. N.; Carlson, H. E.; Vanakis, H. U.; Hill, M. A. Gritz, E.; Jarvik, M. E. Nicotine from cigarette smoking increases circulating levels of cortisol, growth hormone and prolactin in male chronic smokers. Psychopharmacology (Berlin) 78:305-308; 1982.
0031-9384/88 $3.00 + .00
Adrenocortical Hormone Regulation of Nicotine Sensitivity in Mice J A M E S R. P A U L Y , E L I Z A B E T H
A. ULLMAN
AND ALLAN
C. C O L L I N S 1
Institute Jbr Behavioral Genetics and School of Pharmacy, University of Colorado, Boulder, CO 80309 R e c e i v e d 13 A p r i l 1988 PAULY, J. R., E. A. ULLMAN AND A. C. COLLINS. Adrenocorticalhormone regulationof nicotine sensitivityin mice. PHYSIOL BEHAV 44(1) 109-116, 1988.--The possibility that nicotine-induced corticosterone (CCS) release regulates nicotine sensitivity was investigated in female mice of the C3H strain. Adrenalectomy (ADX) resulted in an increase in nicotine sensitivity as measured in a number of physiological and behavioral tests. In ADX animals, chronic CCS (100 /.~g/ml) administered in the drinking solution normalized nicotine sensitivity. Dexamethasone (DEX), a potent synthetic glucocorticoid which interacts with a distinct population of CNS steroid receptors, did not reverse the effects of ADX. Unoperated animals administered CCS (200/xg/ml) were protected from the effects of nicotine for several test battery parameters. ADX had no effect on the number of brain nicotinic cholinergic receptors and also did not alter nicotine metabolism. These data support the hypothesis that CCS secretion modulates nicotine sensitivity in the mouse; however, the mechanisms by which this regulation occurs are unknown. Nicotine
Adrenalectomy
Corticosterone
Dexamethasone
TOBACCO users tend to smoke when in stressful situations, and high nicotine-containing cigarettes have been shown to reduce emotionality to a greater extent than do low nicotine cigarettes (16,39). Consequently, it may be that one of the reinforcing properties of nicotine is its ability to reduce anxiety. The mechanisms by which nicotine reduces emotionality are not well understood but is has been shown that nicotine, administered via injection or smoke inhalation, causes a dose-dependent increase in plasma concentrations of adrenocortical hormones (9,21) and adrenocorticotrophic hormone (ACTH) (35,46). This effect is probably mediated by central nervous system (CNS) nicotinic receptors since mecamylamine, a CNS nicotinic receptor antagonist, has been shown to block the actions of nicotine on CCS release in mice (12). Furthermore, the intracerebroventricular injection of antibodies raised against the nicotinic cholinergic receptor from myasthenia gravis patients blocks stressinduced increases in CCS (8,51). Hillhouse et al. (19) have demonstrated that acetylcholine induces the release of corticotrophin releasing hormone from an isolated rat hypothalamic preparation. Thus, a central mode of action for nicotine-induced CCS release is present in rodents. Tolerance to nicotine-induced CCS release develops if animals are chronically treated with nicotine (3,48). The possibility that corticosterone (CCS) released in response to nicotine plays a role in the determination of nicotine's physiological and behavioral effects has not been
investigated. One method of determining whether or not CCS release is of consequence to nicotine response is to deprive animals of their primary source of CCS, the adrenal gland. Mice are readily adrenalectomized (ADX) and can be maintained for long periods of time if provided with 0.9% saline as a drinking solution. The purpose of the present study was to test the hypothesis that CCS release in response to nicotine is an important factor in the determination o f nicotine response. Adrenalectomy resulted in an increase in acute nicotine sensitivity that could be reversed by chronic administration of CCS. These data indicate that adrenal responses may modulate nicotine sensitivity in rodents. METHOD Female mice of the C3H strain were used for these studies. Animals were raised in the colony at the Institute for Behavioral Genetics and maintained on a 12 hour light/dark cycle with lights on at 0700. Food (Wayne Lab Blox) and water were provided ad lib. All behavioral testing was performed between 0800 and 1300 on adult animals (60-90 days of age).
Adrenalectomy Animals were anesthetized with pentobarbital (54 mg/kg) administered intraperitoneally. A small mid-dorsal incision was made on each side of the animal's back and the kidneys
'Requests for reprints should be addressed to Dr. Allan C. Collins, Institute for Behavioral Genetics, University of Colorado, Campus Box 447, Boulder, CO 80309.
109
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exposed. The adrenal glands were removed using a pair of fine curved forceps. Surgery for sham-operated animals was identical to that for ADX animals except the adrenal glands were not removed. Following surgery, all animals were housed individually and ADX animals were provided with 0.9% saline as a drinking solution. Animals were tested for nicotine sensitivity in a battery of tests, usually one week following surgery. Nicotine was administered via intraperitoneal injection of drug dissolved in physiological saline.
Nicotine Response Nicotine sensitivity was measured in a multifactorial test battery that included acoustic startle response, Y-maze activity (crosses and rearings), heart rate and body temperature. Previous studies from our laboratory have demonstrated that no significant interest interactions occur between these tests (34). The timing of these tests was determined from the results of a time course study on the effects of nicotine on several components of the test battery (32). Each test in the battery was conducted as follows: Acoustic Startle Response--A Columbus Instruments Startle Reflex Monitor (Columbus Instruments, Columbus, OH) was used to quantitate the startle response elicited by a short auditory stimulus. A mouse was placed in a Plexiglas cage inside a sound-attenuated box and 10 auditory stimuli were delivered (frequency, 6250 Hz; intensity, 120 dB; duration, 50 msec). For each stimulus both response amplitude and response latency were recorded. This test was initiated 2.5 rain following nicotine injection. Y-Maze Activity--The maze is a symmetrical Y-shaped runway with each arm being 26.0 cm long, 6.1 cm high and 10.2 cm wide. Each arm of the maze was divided into two sections; crosses from one section/arm to another were counted for 3 min. The number of rearings that occurred during the test period was also recorded. This test was conducted 4.5 rain after nicotine administration.
Heart Rate--After completion of the Y-maze test, mice were placed in a restrainer and needle electrodes were inserted through the skin (one behind the left foreleg and the other in front of the right hindleg). The electrodes were connected through a preamplifier to an E and M Physiograph (Narco Biosystems, Houston, TX). Heart rate was monitored for 6 sec beginning 8.5 rain after nicotine injection. Body Temperature--Rectal temperature was measured using a Bailey Instruments (Saddlebrook, N J) digital thermometer. The probe was lubricated with peanut oil before it was inserted 2.5 cm into the rectal cavity. Body temperature was measured 15 min following nicotine injection. Steroid Replacement In some experiments, ADX or sham-operated animals were provided with exogenous sources of adrenocortical hormones. Steroid hormones (CCS or dexamethasone) (Sigma Chemical Company, St. Louis, MO) were dissolved in a small volume of absolute ethanol (1.0% final concentration) and administered chronically in the animal's drinking solution. Control animals received a drinking solution of 1.(1% ethanol. Following behavioral testing, plasma CCS levels were measured using a modification of the radioimmunoassay described by Gwosdow-Cohen et al. (15).
Nicotine Levels In order to test for differences in the absorption, distribution and/or elimination of nicotine following ADX, levels of nicotine in blood and brain were measured one week postsurgery in ADX or sham-operated animals. Nicotine content was assayed using an extraction procedure that has been described in detail in previously published reports (10, 17, 18). Nicotine levels were estimated in blood and brain 10 min after administration of a 1.5 mg/kg dose of nicotine that had been spiked with 2.0 izCi of [3H]-nicotine (New England Nuclear, Boston, MA) as a tracer. Animals were decapi-
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tated, trunk blood collected (100-300/zl) and the brains removed, blotted and weighed. After homogenization and centrifugation of the samples, aliquots were added to 1.0 ml of 0.1 N NaOH and 7.5 ml of purified heptane. After these samples were shaken for 20 min, the nicotine was extracted by removing 5.0 ml of the organic phase (supernatant) and placing it into a tube containing 0.1 N HCI. The contents were shaken for 5 min and centrifuged. After removal of the organic phase by aspiration, 1.0 ml aliquot of the acid phase was removed and counted by liquid scintillation spectrometry. Previous studies from our laboratory have demonstrated that this procedure separates nicotine from its more polar metabolites (I 7,18).
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Nicotinic Receptor Binding The brains of some animals were assayed for CNS nicotinic cholinergic receptor binding to determine if ADX affected the number or affinity of these receptors. Following the completion of behavioral testing, animals were decapitated and the brains were dissected into the following eight anatomical regions: cortex, striatum, midbrain, hypothalamus, hippocampus, colliculi, cerebellum and hindbrain. Tissue regions were placed in 10 volumes of HEPES-buffered Ringer's solution (NaCI, 118 mM; KCI, 4.8 mM; CaC12, 2.5 mM; MgSO4, 1.2 mM; HEPES, 20 mM; pH adjusted to 7.5 with NaOH) and then frozen at -70°C. The particulate fraction from these samples was assayed for nicotinic receptor binding. The binding of L-pH]-nicotine was measured using a modification of the procedure of Romano and Goldstein (43) as previously described (33). All brain regions were assayed using a single concentration of radiolabeled nicotine. The binding of alpha-[*~5I]-bungarotoxin (BTX) (New England Nuclear, Boston, MA) was performed as described by Marks and Collins (33) using a single concentration of labeled BTX. Protein determinations were made using the method of Lowry et al. (23), using bovine serum albumin as a standard.
FIG. 3. Effects of steroid hormone replacement therapy on nicotine sensitivity for acoustic startle response. A significant effect of treatment was determined, F(3,111)=42.92. Startle response was significantly dampened by chronic administration of both corticosterone (CCS) (100/xg/ml for 7 days) and dexamethasone (DEX) (100 p.g/ml for 7 days).
Data Analysis Results from behavioral testing were analyzed using two-way analysis of variance (treatment by dose). Newman-Keuls post hoe tests were used to analyze the data when overall effects of dose or treatment were identified. t-Tests were used for all other statistical comparisons. RESULTS One week following surgery, nicotine sensitivity was measured in a battery of tests in ADX and sham-operated animals. Preliminary experiments (data not shown) demonstrated that there was no difference in nicotine sensitivity between unoperated and sham-operated animals. Ad-
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FIG. 5. Effects of steroid hormone replacement therapy on nicotine sensitivity for Y-maze rears. A significant effect of treatment was determined, F(3,111)=4.82. Chronic CCS administration normalized nicotine sensitivity whereas chronic DEX did not.
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renalectomy, however, clearly induced an increase in nicotine sensitivity in C3H mice. The effects of ADX on nicotine sensitivity for startle response, Y-maze crosses, heart rate and body temperature measures are shown in Fig. 1. Increases in nicotine sensitivity were statistically significant for all test battery parameters. In some experiments, animals were tested for nicotine sensitivity on the day following ADX. Data from day 1 and day 7 ADX animals are shown in Fig. 2. N o significant differences in nicotine sensitivity in day 1 and day 7 ADX animals were present except for the body temperature parameter. Day 1 animals were significantly less sensitive to nicotine's hypothermic response than were animals tested one week postADX, The adrenal gland releases many substances in response to stressful stimuli. In order to test for the hormonespecificity of ADX effects it was necessary to measure nicotine sensitivity in ADX animals receiving chronic ad-
FIG. 7. Effects of steroid hormone replacement therapy on nicotine sensitivity for body temperature. A significant effect of treatment was determined, F(3,1 ! 1)= 20.55. Both CCS and DEX replacement therapy normalized nicotine sensitivity. renocortical hormone therapy. Corticosterone or dexamethasone were administered in the drinking saline of ADX animals at a concentration of 100 /xg/ml. Previous studies have demonstrated that CCS administered at this concentration effectively causes down regulation of hippocampal CCS receptors (45,47). Consumption of the steroid-containing solution was monitored for one week following ADX and animals were then tested for nicotine sensitivity. Animals that received steroid supplement in their drinking solution consumed a significantly larger volume of fluid (/9<0.01) than did animals drinking only saline. Consumption of the CCS-containing solution averaged 13.94 +0.66 ml/day and was not signifmantly different from consumption of the dexamethasone-contaming solutton (14.85+0.76 mFday). ADX animals receiving only saline drank an average of 7.65-+0.54 nil/day. Plasma CCS levels were determined by radioimmunoassay in ADX animals and ADX animals receiving CCS in their drinking water. CCS therapy resulted in a significant elevation in plasma CCS levels (p<0.01) (155.1-+34.9 ng/ml in
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CCS-treated animals vs. 21.8_+4.5 ng/ml in saline-treated animals). Chronic CCS administration prevented the ADX-induced increase in nicotine sensitivity for all test battery parameters. Data for startle response, Y-maze crosses, Y-maze rears, heart rate and body temperature are shown in Figs. 3 through 7 respectively. Dose-response curves for shamoperated animals and ADX animals receiving CCS were not significantly different (except for startle response which was dampened by CCS administration). Dexamethasone replacement was not equivalent to CCS replacement in terms of ADX-effects reversal. Chronic administration of this potent synthetic glucocorticoid normalized the effects of ADX only on the nicotine doseresponse curve for body temperature (Fig. 7). All other test battery parameters were unchanged by dexamethasone supplementation (Figs. 3-6). Because ADX animals that received chronic CCS appeared to be somewhat protected from the effects of nicotine on test battery parameters, unoperated animals were given chronic CCS (200/~g/ml) in order to test for possible nicotine response subsensitivity. Figure 8 demonstrates such a desensitization of nicotine response. For startle response, Y-maze rears, heart rate and body temperature, intact animals that received chronic CCS were significantly less sensitive to the effects of nicotine than were intact animals that received vehicle only as a drinking solution (1% ethanol). Startle response in animals that received chronic CCS was almost completely absent. Adrenalectomy could induce an increase in nicotine sensitivity through a change in nicotine metabolism or it may be that ADX could cause changes in tissue sensitivity. Using standard biochemical receptor binding assays we have determined that ADX does not significantly alter the number of CNS binding sites for nicotinic cholinergic receptors. The binding of [SH]-nicotine was significantly lower in the midbrain of ADX animals but no differences were measured in
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five other brain regions analyzed (Fig. 9). [12H]-BTXbinding was not significantly altered by ADX in any brain region (Fig. 10). Blood and brain levels of nicotine in ADX and shamoperated animals 10 minutes following nicotine administration are shown in Fig. 11. No significant effect of ADX on nicotine distribution was detected. Thus it is unlikely that ADX-induced increases in nicotine sensitivity are a result of alterations in nicotine metabolism. DISCUSSION
The results of this study demonstrate that alterations in the brain-pituitary-adrenal axis can induce changes in acute
114
PAULY, U L L M A N AND C O L L I N S
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BRAIN REGION
FIG. 10. Effects of ADX on the binding of alpha-[1251]-bungarotoxin to mouse brain membranes from regionally dissected brain regions. No significant effects of ADX were present. Brain regions are designated as in Fig. 9.
sensitivity to nicotine. Following ADX, mice of the C3H strain had significant changes in nicotine sensitivity for all test battery parameters. The changes induced by ADX were shown to be specific for the primary adrenocortical hormone in the mouse, corticosterone (CCS). Furthermore, we have also demonstrated that intact animals that received chronic CCS in their drinking water were significantly less responsive to nicotine than were control animals. Several groups of investigators have previously demonstrated that nicotine, administered by injection or through inhalation of cigarette smoke, induces a dose-dependent increase in plasma CCS (1, 2, 20, 52). The results obtained in our experiments demonstrate for the first time that CCS released in response to nicotine may modulate the behavioral and physiological actions of this drug. It is somewhat paradoxical that nicotine, a drug that has been shown to increase the activity of the autonomic nervous system, also has been reported to reduce emotion and increase tranquillity. Several investigators have shown that nicotine reduces negative affect and that humans use nicotine in stressful situations to achieve pleasurable relaxation (14,37). The mechanisms by which nicotine elicits these effects are not known and are obviously very complex. However, it is conceivable that nicotine-induced CCS release may be critically involved. Several studies have demonstrated that CCS and some of its metabolites decrease neuronal excitability through an enhancement of brain GABA-ergic neurotransmission (30,31). It is possible that an important factor in antistress responses is a reduction in neuronal excitability. Such a reduction in excitability may be present in animals that received chronic CCS. Nicotine does not induce CCS release via a direct action on the adrenal gland; this response is probably regulated through nicotinic receptors in the central nervous system. Mecamylamine, a CNS nicotinic receptor blocker, pretreatment prevents the nicotine-induced increase in CCS release (12) and several studies (8,51) have demonstrated that intracerebroventricular administration of antibodies raised against the nicotinic cholinergic receptor from Torpedo electric organ blocks stress-induced increases in CCS. Acetylcholine (ACh) has been shown to stimulate the
FIG. 11. Estimation of nicotine levels in the blood and brains of ADX and sham-operated animals 10 min following a challenge dose of nicotine spiked with a tracer. No significant differences between treatment groups were present. activity of CNS systems that regulate adrenocortical responses (1,13). Using an isolated rat hypothalamus preparation, Hillhouse et al. (19) demonstrated that ACh stimulates the release of corticotropin releasing factor (CRF). CRF in turn regulates the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary which stimulates the release of adrenocortical hormones. In rats, 2 min following systemic nicotine administration, plasma ACTH levels are significantly elevated (35,46). The mechanism by which ADX increases sensitivity to acute nicotine administration is not known. Nicotinic receptor binding and nicotine metabolism were not significantly affected by adrenalectomy. Changes in nicotine sensitivity following ADX could be due to the lack of adrenocortical hormones or, alternatively, changes could be due to increases in brain concentrations of ACTH and/or CRF. Several studies have demonstrated that basal and stress-induced levels of CRF and ACTH are elevated in ADX animals due to the lack of negative feedback (24,28). In the absence of adrenocortical hormones there is an up-regulation of neuronal receptors for these hormones (47). In the central nervous system there are at least two types of receptors for adrenocortical hormones; the distribution of these receptor subtypes in the CNS is heterogeneous. Type I, the CCS receptor (CR), is localized predominantly in the hippocampus and to a lesser extent in the septum. Glucocorticoid receptors (GR), or Type II receptors, are more uniformly distributed among neurons and glial ceils (11, 25-28, 49, 50). The use of synthetic glucocorticoids such as dexamethasone has greatly aided the identification of adrenocortical receptor subtypes. GR is sensitive only to high concentrations of CCS (e.g., stress-induced CCS release), whereas CR is sensitive to lower concentrations of CCS and detects smaller changes in CCS levels such as those which occur throughout the circadian cycle (40-42). In receptor binding assays CR can be identified using CCS, whereas GR is labeled using dexamethasone or other synthetic glucocorticoids. Corticosterone receptors have a low affinity for dexamethasone. Chronic DEX administration has been shown to have no effect on the number of CCS receptors (CR) (44). Thus, in ADX animals, it is possible to selectively prevent ADX-induced increases in these receptors through the chronic administration of CCS or DEX (44). Dexamethasone prevents the release of ACTH from the anterior pituitary through feedback inhibition. ADX animals that receive chronic DEX have increased numbers of CCS
CORTICOSTERONE AND NICOTINE SENSITIVITY receptors but increases in DEX receptors (GR) are prevented. Chronic DEX also prevents ADX-induced increases in ACTH levels (5,6). Thus a first test as to whether ADX effects are due to increased levels of ACTH can be performed using chronic DEX versus chronic CCS hormone therapy in ADX animals. If CCS therapy reverses the effects of ADX but DEX therapy (which normalizes ACTH concentrations) does not, then it can be inferred that increased nicotine responsiveness subsequent to ADX is not a direct result of increases in ACTH. In our battery of tests, we determined that CCS reversed the effects of ADX on nicotine sensitivity for all parameters. DEX therapy reversed the effects of ADX only for the body temperature measure. Therefore, increased sensitivity of the hypothermic response elicited by nicotine in ADX animals could be due to increased ACTH levels. This possibility is further supported by our finding that day 1 ADX animals were not as sensitive as day 7 ADX animals in terms of nicotine-induced hypothermia. At this time point, ACTH levels may not be significantly elevated and this may explain why these animals are not as sensitive as day 7 ADX animals to nicotine's hypothermic response. In rodents, ACTH has been shown to induce hypothermia when administered centrally (22). Our finding that DEX and CCS replacements do not have equivalent effects on locomotor activity (Y-maze crosses and rears) is not surprising. Several groups of investigators (6,29) have demonstrated that ADX causes a reduction in open field activity that is normalized by CCS but not DEX.
115 Other measures that are affected by ADX and normalized by CCS but not DEX include sensitivity to epinephrine in a passive avoidance response (7) and extinction of an appetitive runway task (6,36). As is the case with nicotine sensitivity, these behaviors are probably not due to ADX-induced changes in CNS concentrations of ACTH. The pituitary/adrenal system plays an essential role in the physiological control of adaptive behavior through the secretion of adrenocortical and other hormones. Corticosterone has been shown to be a potent modulator of central nervous system function. The present study demonstrates that alterations in adrenal status cause marked changes in sensitivity to nicotine, a drug that induces a variety of physiological and behavioral responses. The precise mechanism by which ADX causes an increase in nicotine sensitivity is not known. However, other studies in our laboratory (38) have demonstrated that this phenomenon is regulated by genetic factors. Future studies in our laboratory will investigate further the relationship between nicotine sensitivity and adrenocortical responses in various strains of mice. These studies may help elucidate the complex relationship between tobacco (nicotine) use, adrenocortical responses and anxiety reduction. ACKNOWLEDGEMENTS The authors gratefully acknowledge the advice and counsel of Dr. Michael J. Marks. This research was supported by the NICHD training grant HD-07289 and also by a grant from the R. J. Reynolds Tobacco Company.
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