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i m ~ m m ELSEVIER i i m W Life Seience~ Vol. 61, No. 1~ pp. PL 18~.190, 1997 Published by Elsevie~ ~Sc~enc~ Inc. P~in~ i~ ~1~ USA. All. ri~lat~ re~od 0024.3205~7 $17.00 + .00 PII S007,4-320S(~r~ 00~-6 ~/~ICOLOGF/~ 7TERS .~celer~ed ~cat~on SEX DIFFERENCE IN UP-REGULATION OF NICOTINIC ACETYLCHOLINE RECEPTORS IN RAT BRAIN Ersin Koylu~, Serdar Demirg6ren~, Edythe D. London2, Sakire P6~n~ ~Centcr for Brain Research and Department of Physiology, Ego University School of Medicine; TUBITAK Basic Neuroseience Research Unit, Bornova 35100 lzmir, Turkey zintramural Research Program, National Institute on Drug Abuse, NtH, Baltimore, MD 21224, USA (Submitted Ma~ch 21, 1997; accepted April 22, 1997; re.~dv~ in final form June 10, 1997) Abstract: This study tested for sex differences in the effects of chronic nicotine administration and withdrawal on nicotinic acetyleholine receptor binding in brain. Kats received nicotine (0.6 mg/kg, sc) or saline once daily for 15 days, and were sacrificed 1 or 20 days after termination of treatment. Saturation studies of nAChR, binding were performed using [3H]cytisine as the radioligand in whole brain minus cerebellum taken from animals in the chronic treatment groups and from naive rats. Male but not female rats that received chronic nicotine had higher receptor densities than corresponding control groups; up-regulation of nAChR was not seen 20 days a~er withdrawal. Furthermore, in groups that showed no up-regulation (controls and rats withdrawn for 20 days), nAChR, densities were higher in female rats than males. The findings underscore the importance of sex differences in pharmacological responses as well as in basal neurochemicai parameters, l'ubaslusd~ El~-vier geien~ Inc. Words: nicotine acctylchoHnc re~pto¢, ey~6n~ n~eotin~ ~x differences, up-regulatlon, withdrawal Introduction Although the prevalence of tobacco smoking has declined over the past thirty years, the decline has been less pronounced in women than in men (1). As gender influences a variety of neurotransmitter systems, it seems possible that sex differences in smoking cessation cmlld, to some extent, reflect differences in how chronic nicotine interacts with nACb.R, the substrate of the biological effects of nicotine. Postmortem assay of [~H]nicotine binding in brains from smokers and nonsmokers indicated an up- regulation ofnAChRs in Brodmann areas 11 and 27, cerebellar cortex, hippocampal formation and ,the median raphe nuetei of the midbrain but not in the medulla oblongata; no significant interactions between smoking and sex or age were observed. However, in the hippocampus, where Correspondence: Prof. Dr. ~akire P6~in, Director, Center for Brain Research, Ege University School of Medicine, Department of Physiology Bornova 35100, Izmir, Turkey, Tel: +90-232-388- 2868 Fax: +90-232-374-6597, E-Mail: pogun @bornova.ege.edu.tr This article is tbr individual use only and may not be further reproduced or stored elcctromcally without written permission from the copyright holder.

Sex D~erences ~n Nk~,in/c Receptors VoL 6I, No. 1~ 1~ [3H]nicotine binding was highest, binding at higher [3H]nicotine concemrations (25 nM compared to 5-20 nM) was significantly higher in tissue prepared from brains of men than in brains from women (2). In this study, the age of the subjects varied between 55 and 88, and subjects who had given up smoking at least 5 years prior to death were considered as "nonsmokers" (i.e. some of them had been exposed to nicotine). As animal studies facilitate control over such variability, the aim of the present study was to investigate possible sex differences in alterations of neuronal nAChR binding following chronic nicotine administration and withdrawal in rats. Materials and Methods Animals: Experimental subjects were sexually mature, three-month-old male and female Sprague Dawley rats, maintained on a 12-hour light/dark cycle with food and water provided ad [ibitum. Animals were assigned to five treatment groups (5-8 rats in each group), as follows: 1) NaiVe, 2) Nicoane: chronic nicotine x15 days, 3) Control #/: chronic, saline x15 days 4) Withdrawal: chronic nicotine x15 days followed by 20 days withdrawal, 5) Control #2: chronic saline xl5 days followed by 20 days withdrawal. Rats in the naive group were given no injections while rats in the other four groups received subcutaneous injections once daily for 15 days between 08:30-10:00 hours. The active drug treatment consisted of (-)-nicotine hydrogen tartrate, 0.6 mg/kg (calculated as flee base), dissolved in 0.9% NaCI, pH 7.0 (adjusted with I N NaOH). The dose of nicotine was selected on the basis of behavioral studies demonstrating that 0.5 mg/kg or less facilitated while 0.8 mg/kg or more impaired memory in rats (3,4,5) and our findings that gender differences in learning performance was most prominent at a dose of 0.6 mg/kg (6). Control animals received an equal volume (1 ml) of 0.9 % NaCI under the same regimen. Rats in the chronic nicotine and control #I groups were decapitated l day af[er the termination of injections while those in the withdrawal and control #2 groups were maintained without injections for another 20 days prior to sacrifice. Tissue Preparation: Brains were removed and dissected on ice. Whole brain minus cerebellum was homogenized in 50 mM Tds HCI buffer containing 120 mM NaCI, 5 mM KCI, 1 mM MgCI2, 2.5 mM CaCI2. The homogenate was centrifuged at 40 000 x g for 10 min and the pellet was resuspended twice in buffer. Binding Assays: Assays of nAChR binding were performed essentially as previously describe~ usin8 [3H]cyfisine, an asonist that binds with high affinity, slow dissociation, and low nonspecific binding. In vitro assays have demonstrated that the regional distribution of [~H]cytisine binding is almost identical to both [~H]acetylcholine and [3H-Jnicotine binding (7); data on tissue distribution and pharmacological characterization in vivo suggest that [3H]nieotine and[3H]cytisine both label nAChRs in the mouse brain (8). In saturation experiments, 20 mg tissue were incubated with [3H]eytisine in a total volume of 250/~1 for 75 min at + 4°C. Six concentrations of the radio/igand, varying between 0.I 8-16.4 nM were used. (-)Nicotine, 1 mM~ was used to define nonspecific binding. The EBDA and LIGAND programs were used to analyze the data from saturation experiments and to obtain Rosenthal-Seatchard plots (Figure I). Statistical Analyses: K~ and B~, values were initially analyzed in two separate 2-way analyses of variance (ANOVA), with sex and treatment as the factors. The treatment variable included the following five levels: naive, chronic nicotine or saline treatment, withdrawal following chronic nicotine or saline treatment. In the ease of B~, where there was a significant interaction (see results), post hoe analysis was performed using protected t-tests. Power analyses were performed to evaluate the level of confidence in the negative results. The software used for all statistical evaluation was Statview II by Abacus Concepts, Inc.

VoL 61, No. 12, 1997 Sex Differencez in Nicotinic Receptor~ Bound (pmol/g tissue) Fig. 1 Scatchard plot of specific 3H-Cytisine binding in the whole brain era female rat (Naive group). Inset: Saturation plot showing nonspecific (open circles) and specific binding (filled circles). Data are from a representative experiment. Ka -- 1,18 nM, Bm~x -- 52,76 fmoles/20 mg tissue Results There was no significant interaction or main effect (sex or treatment) for K~; however, analysis of B~ data revealed both a significant main effect of treatment (F4,4.~=3.40, p=0.017) and a significant interaction (F4.~3-3.17, p--0.023) (Fig.2). As shown in Figure 2, the cause of the interaction was higher Bm~ values in females compared to males in all groups except the chronic nicotine-treated rats, where an up-regulation was observed in males. Post hoc analysis of the data between male and female rats in the five treatment groups revealed a statistically significant difference between the genders only in those groups that were sacrificed one day after the chronic nicotine regimen (t=2.49, p=O.04). This finding indicated that nAChRs were up-regulated by nicotine only in males (112 % increase in males vs. 8.5 % increase in females), and that the up- regulation was transient, as it was no longer apparent after 20 days of withdrawal Since receptor up-regulation was observed only in the chronic nicotine treatment group, we re- analyzed the data after excluding this group. Our results showed a significant main effect of sex, in that females had a higher B~x compared to males (F.~,~.~=4.04, p=0.05). This finding was consistent with a non-significant trend for Bm~ to be higher in naive females than males (t=2.06, p=0.06). This group was comprised of six animals of each sex, and a power analysis indicated a power of 55%, suggesting a low tevet of confidence. In order to achieve a power of 85%, a'sample size of twelve per group would be required.

Sex Difference~ in Nicotinic Receptors Vol. 61, No. 12, L097 Discussion The finding that chronic nicotine causes an up-regulation of nAChRs in male rats supports most previous observations that chronic nicotine increases nAChRs in several brain regions of rodents (9, I0), and that this up-regulation corresponds with sensitization to the locomotor stimulant action of nicothne after chronic administration (I0). In male Sprague Dawley rats, given 4 mg/kg/day nicotine via mini pumps for either 7 or 14 days, [3H]nicotine binding was increased approximately 50% in the cortex and hippocampus but unchanged in the striatum; the changes reflected alterations in B,~x (l I). Up-regulation of the nAChRs may be species-specific since, in male Wistar rats, no up-regulation of [3H]nicotine binding was observed following chronic (0.4 mg/kg/day for 39 days) s.c. administration of nicotine (12). 100 90 80 70 60 50 40 3O 2O 3.0 2.5 2.0 LO 10 KD Female Male Naive Nicotine N.Control Withdrawal W.Control Naive Nicotine N.Coneroi Withdrawal W.Contrel l~ig. 2 B,~ and Kd values were determined by Scatchard analyses and represent mean _+ s.e.m, of 5-8 saturation experiments. Please refer to the "Results" section for statistical evaluation of the data.

VoL 61, No. 12, 1997 Sex Differences in Nicotinic Receptors PL-189 Despite these findings in male rodents, fewer studies have been performed oa brains from female rodents treated chronically with nicotine. Collins and co-workers have used female rats in some studies. In one of them, they injected 1.6 mg/kg nicotine twice dally, s.c. and measured [3H]nicotine binding at 0,1,2,4 and 8 days. B,~, aiter 8 days was 40 % higher compared to 0 days (13). In a second study, saline or nicotine was administered chronically (0.8 mg/kg) via s.c. infusion for 7 days; there was 100% increase in B,~, which gradually returned to control levels by 21 days after withdrawal (14). As compared with the present study, these investigations by Collins et al. (13,14) employed higher doses of nicotine, more frequent injections or constant infusion, and a shorter regimen of treatment; they also used different radioligands and only female rats. Tizabi et al. (15) treated pregnant Sprague Dawley rats with nicotine (6 mg/kg/day) throughout gestation and measured activity levels and neuronal nACb.R binding using ['~H]cytisine in the brains of male and female pups. An up-regulation of nAChRs was observed only in hyperactive male rats. If females are less susceptible to up-regulation, a treatment regimen with.more frequent injections or constant infusion may be necessary to observe the phenomenon. In our study, whole brain was used for receptor assays. It is possible that up-regulation occurs in discrete regions, and therefore the effect may be diluted in whole brain. Nonetheless, up-regulati0n is more pronounced in males than females of the Sprague~Dawley strain. Available evidence suggests that although nAChRs are heterogeneous due to variety in combinations of subunlts, upregulation measured here reflects an effect on nAChRs comprised of ct-4 and [3-2 subunits. When antisera generated against nonhomologous domains comprising the nAChRs were used in conjunction with [3Hlcytisine binding, only antisera generated against the ~, and [3-2 subunits specifically immunoprecipitated the labeled receptors, and these receptors were upregulated in the cortices of rats treated with nicotine (16). Recent findings from another study in which we are testing the effect of chronic nicotine in male and female Sprague-Dawley rats suggest that the effects described here are due to a pharmacokinetic difference (Pogun, Kimes and London, unpublished). Nicotine was administered chronically according o the same regimen used in the present study, and 24h aRer the termination of treatment, serum samples were taken for assay of nicotine and cotinine by a commercial laboratory (Labstat, Inc., Kitchener, Ontario, Canada). The analytical method used was gas chromatography, with a fused silica capillary column and a thermionic specific detector~ N- ethylnornicotine and 5-methylcotinine were the internal standards to quantitate nicotine and cotinine, respectively. The assay revealed that cotinine levels were not different in serum from male and female rats (290.3 _+ 12.6 and 279.0 _+ 8.3 ng/ml, respectively), but nicotine levels were significantly (t~97"3.76, p<0.005) lower in females (5.2 +_ 1.5 ng/ml) than males (20.6 ± 3.5 ng/ml) implying a shorter half-life of nicotine in females compared to males. This observation may also explain why female rats showed up-regulation in previous studies in which higher doses or more frequent treatment with nicotine were used (i.e. 13,14). The present study empioyed a single dose of nicotine, once daily. Since the half-life of nicotine appears to be shorter in females, the regimen used may have allowed for receptor resensitization between nicotine injections in female rats. In addition, females may be less sensitive to up-regulation, and may need a larger dose to manifest this effect. Although it appears that nicotine was metabolized more rapidly in the female rats than in the males, and that this difference likely accounts, at least to some extent, for the lack of effect in the female rats, other contributors to the sex difference observed here cannot be excluded. Steroids may affect nAChR binding and function through interaction with the lipid environment or the receptor protein itself (17,18), and fast and reversible inhibition of the neuronal nAChI~ by progesterone has been demonstrated (19). Therefore. it seems possible that a higher level of

PI~lg0 Sex Differences in Nicot~c Receptor~ VoL 61, No. 12, 1997 circulating progesterone in the female rats might have rendered nAChRs less susceptible to the action of nicotine. In conclusion, as administered in the present study, nicotine caused an up-regulation of nAChR in male but not female Sprague Dawley rats. This effect did not persist 20 days after withdrawal. Furthermore, in groups that showed no up-regulation, nAChR densities were higher in t~male rats than males. The findings underscore the importance of sex differences in neurochemical properties and pharmacological responses in brain. Acknowledgements This study was supported by grant no. SBAG-Cd-15/3 from TUBITAK and the Intramural Research Program, NIDA/NIH. References 1. M.C. FIORE, The Med. Clin. of North America 76 289-303 (1992). 2. M.E.M BENWELL, D.J.K. BALFOUR and J.M. ANDERSON, J. Neurochem. 50 1243-1247 (tgS8). 3 V HAROUTUNIAN, E. BARNES and K.L. DAVIS, Psychopharmacol. 87 266-27l (1985). 4. D.M. GILLIAM and K. SCHLESINGER, Psychopharmacol. 86 291-295 (1985). 5. A. OLIVERIO, J. Pharmacol. Exp. Ther., 154 350-356 (1966). 6. O. YzLMAZ, L. KANIT, B.E. OKUR and $. POCJ~N, Behav. Pharmacol. (In press) (1997) 7. L.A. PABREZA,, S. DHAWAN and K.J. KELLAR, Mol. Pharmacol. 39 9-12 (1991 ). 8. J.E. FLESHER, U. SCHEFFEL, E.D. LONDON. AND J.J. FROST, Life Sol., 54 1883-1890 (1994). 9. C. KSI~ R. HAKAN, D.P. HALL and K.J., KELLAR, Neuropharmacol. 24 527-531 (1985). 10. M.J. MARKS, E. ROMM and A.C. COLLINS, J. Pharrnacol. Exp. Ther. 235 619-628 (1985). 11. E.M. SANDERSON, A.L. DRASDO, K. McCREA and S. WONNACOT, Brain Res. 617 349-352 (1993). 12. M.E.M. BENWELL and D.J.K. BALFOUR, J. Pharm. Pharmacol. 37 405-409 (1985). 13. A.C. COLLINS, E. ROMM and J.M. WEHNER, Psychopharmacol. 96 7-14 (1988). 14. A.C. COLLINS, E. ROMM and J.M. WEHNER~ Brain Res. Bull. 25 373-379 (1990). 15. Y. TIZABI, E.J. POPKE, M.A. RAI-IMAN, S.M. NESPOR and N.E. GRUNBERG, Pharmacol. Biochem. Behav. (In press) 16. C.M. FLORES, S.W. ROGERS, L.A. PABREZA, B.B. WOLFE and K.J.. KELLAR, Mol. Pharmaeol. 41 31-37 (1992). 17. L. KE and R.J. LUKAS, J Neurochem. 67 1100-1112 (1996). 18. F.J. BARRANTES, FASEB J. 7 1460-1467 (1993). 19. S. VALERA, M. BALL/VET, and D. BERTRAND, Proe. Natl. Aead. Sei. USA 89 9949- 9953 (1992). 0