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The effects of acute nicotine on the body temperature and striatal dopamine metabolism of mice during chronic nicotine infusion
Neuroscience Letters 284 (2000) 37±40
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The effects of acute nicotine on the body temperature and striatal dopamine metabolism of mice during chronic nicotine infusion Outi Salminen*, Liisa Ahtee Division of Pharmacology and Toxicology, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland Received 11 January 2000; received in revised form 27 February 2000; accepted 27 February 2000
Abstract The effects of acute nicotine administration on body temperature and striatal dopamine metabolism of mice during chronic subcutaneous nicotine infusion were investigated. On the 7th day of nicotine infusion the hypothermic effect of 1 mg/kg nicotine s.c. but not that of 2 mg/kg was weakened suggesting that tolerance developing to nicotine's hypothermic effect during chronic nicotine can be overcome by increasing the dose of nicotine. In saline-infused control mice 1 mg/kg nicotine increased striatal 3,4-dihydroxyphenylacetic acid (DOPAC) but not homovanillic acid (HVA) concentration whereas 2 mg/kg increased both DOPAC and HVA. On the 7th day of nicotine infusion DOPAC and HVA concentrations were similar to control; and acute nicotine did not increase them suggesting that nicotinic acetylcholine receptors (nAChRs) regulating striatal dopamine metabolism were desensitized. The results suggest that the nAChRs mediating nicotine's effects on thermoregulation and brain dopamine metabolism differ. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotine; Constant infusion; Hypothermia; Striatal dopamine metabolism; Desensitization
Nicotine, the psychoactive component of tobacco; creates at least some of its central effects via brain dopaminergic systems. Acute administration of nicotine enhances cerebral dopamine (DA) turnover and metabolism both in mice and in rats [3,4,6,10,14]. However, at large doses nicotine does not alter and was even found to reduce the striatal DA turnover in rats when given i.p. [2] and the striatal concentrations of DA metabolites in mice when given s.c. [4]. This latter phenomenon has been suggested to result from desensitization of the nicotinic acetylcholine receptors (nAChRs) regulating physiologically the striatal dopaminergic neurons. Indeed, nicotinic antagonists blocking the nAChRs reduce the concentrations of striatal DA metabolites in rats and mice [1,4]. Characteristic for the desensitization of the nAChRs is that it both develops and reverses rapidly. Also, tolerance in the classical sense which occurs more gradually, and is longer-lasting; most probably occurs to nicotine's effects such as nausea in smokers [11]. Recently, it has been described a long-lasting inactivation of nAChRs in vitro, which persists long after removal of nicotine and characteristic for which is that after prolonged nicotine * Corresponding author. Tel.: 1358-9-191-59459; fax: 1358-9191-59471. E-mail address: outi.salminen@helsinki.® (O. Salminen).
treatment the nAChRs do not respond to nicotine even after long intervals [15]. Moreover; we reported ®ndings in vivo suggesting occurrence of such long-lasting inactivation of nAChRs regulating cerebral DA metabolism after withdrawal of mice or rats from a prolonged nicotine treatment. Thus, a small nicotine dose did not increase the striatal DA metabolism in mice withdrawn for 24 h from sevenweek oral nicotine administration [12], and in rats acute nicotine did not any more elevate limbic dihydroxyphenylacetic acid (DOPAC) concentration after 24-h withdrawal from 7-day constant nicotine infusion [16]. In the experiment where mice were withdrawn for 24 h from seven-week oral nicotine administration [12], acute nicotine challenge induced signi®cantly less hypothermia in chronically treated mice than in controls suggesting the development of tolerance to the hypothermic effect of nicotine. Nicotine-induced reductions of striatal DA metabolites are in mice closely and inversely correlated with its hypothermic effects and counteracting the hypothermic effect of nicotine enhanced its DA metabolism stimulating effects [4,5]. To study the tolerance and desensitization of nAChRs regulating DA metabolism; we investigated the effects of acute nicotine on rectal temperatures and striatal DA metabolism in mice during constant nicotine infusion. The striatal brain area dissected in the present experiments consists
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 98 3- 6
38
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40
of dorsal as well as ventral striatum and thus includes the nucleus accumbens. An acute dose of (2)-nicotine (1 or 2 mg/kg s.c.) was given to mice on the seventh day of nicotine infusion with the subcutaneous nicotine-releasing reservoirs still in place. Male NMRI-mice weighing 20±27 g at the beginning of the chronic treatment were housed ten to a cage and kept on a standard diet and tap water ad libitum. Lights were on from 06:00 h until 18:00 h and the ambient temperature was kept at 20±228C. Experimental animals were maintained in accordance with the internationally accepted principles; and the experimental set-up was approved by the Committee for Animal Experiments of the Faculty of Science of the University of Helsinki. (2)-Nicotine was administered chronically for 7 days using subcutaneously implanted nicotine releasing reservoirs as described for mice [5]. The nicotine reservoirs containing 40 ml of (2)-nicotine base (Fluka, Buchs, Switzerland) were placed under the skin of mice anaesthetised with pentobarbital (50 mg/kg i.p., Mebunat 60 mg/ml diluted with saline, Orion Pharma, Espoo, Finland). A small sagittal cut of about 1 cm was made behind the ears after local injection of 0.1 ml 0.1% lidocaine solution (Lidocain 5 mg/ml inject., Orion Pharma) s.c., and the reservoir was pushed gently with a longitudinal orientation beneath the skin with the Silastic R (Dow Corning, Midland, Michican, USA) plug most caudally. The incision was closed with surgical suture. Control animals and animals receiving only acute nicotine treatment were sham-operated by placing a reservoir containing 0.9% NaCl-solution (saline) under the skin. The mice received saline (s.c.) or acute nicotine (1 or 2 mg/kg s.c.) 1 h before decapitation on the seventh day of nicotine infusion with the reservoirs still in place. For injections (0.1 ml/100 g) (2)-nicotine base was diluted with saline, and the ®nal solution was adjusted to pH 7.0±7.4 with 0.05 M HCl in saline. After decapitation striata were dissected and frozen on dry ice within 3 min, weighed (mean weight 25 mg) and stored at 2808C until assayed. Rectal temperatures were measured on the seventh day of chronic treatment (either saline- or nicotine-infused mice) before and at 30 and 60 min after acute challenge dose (saline or nicotine). An electric thermocouple (Ellab Instruments; Copenhagen Denmark) was inserted 2.5 cm into the rectum. DT8C is the mean difference of rectal temperatures measured before an acute treatment and 30 or 60 min after acute treatment; the last measurement being immediately before the decapitation. The contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured by HPLC with electrochemical detection after Sephadex G-10 gel chromatographic clean-up of samples [5,16]. When estimating the DA and metabolite samples from all four treatment groups (sham-operated 1 saline, sham-operated 1 acute nicotine; nicotine-infused 1 saline, nicotine-infused 1 acute nicotine) were analysed simulta-
neously. The samples from experiments with 1 and 2 mg/kg of acute nicotine were analysed separately. In brief, tissue samples were homogenized and pH of the homogenates was adjusted to 2.4. After centrifugation supernatants were passed through acidic (0.01 M HCl) Sephadex G-10 columns and a 200 ml of each collected fraction was injected in a C-18 reverse phase (Spherisorb octadecylsilane 2 mm) HPLC column (25 cm; 4.6 mm i.d.) connected to the electrochemical detector (Potentiostat type LC-2A, Bioanalytical Systems with rotating disc working electrode). The plasma concentrations of nicotine and cotinine were measured on the seventh day of chronic infusion using gas chromatography-mass spectrometry (GC-MS). The trunk blood of mice was collected after decapitation. Blood samples were treated and measured as described by Leikola-Pelho et al. [5]. As the metabolite results consisted of several experiments the statistical analyses were carried out by three-way ANOVA (experiment £ chronic nicotine £ acute nicotine). As no signi®cant interactions were found between the experiment and other factors; the randomised block twoway ANOVA was performed using experiments as blocks. If there were signi®cant chronic x acute nicotine interactions (P , 0:1), the analysis was continued by comparing appropriate cell means with linear contrasts. Statistical analysis of changes of rectal temperature was performed by two-way ANOVA of repeated measurements. The results from experiments with 1 and 2 mg/kg of acute nicotine were analysed separately because they were carried out on different days. As signi®cant chronic £ acute £ time interactions were found, the Tukey's post hoc comparisons at timepoints were made. On the 7th day of the chronic nicotine treatment with the nicotine-releasing reservoirs still in place the plasma concentration of nicotine was 207 ^ 32 ng/ml (mean ^ SEM, n 12) and that of cotinine 1366 ^ 258 ng/ml. This agrees well with the previous results of Leikola-Pelho et al. [5]. When challenge doses of 1 and 2 mg/kg were given to nicotineinfused mice, nicotine concentrations in the plasma at 60 min were 301 ^ 56 ng/ml (1 mg/kg, n 12) and 412 ^ 35 ng/ml (2 mg/kg, n 5) and cotinine concentrations 1861 ^ 253 (n 12) and 1529 ^ 284 ng/ml (n 5), respectively. Thus, acute nicotine further elevated plasma nicotine, but not cotinine, concentrations. As shown in Table 1 in the sham-operated mice acute nicotine induced hypothermia dose-dependently (1 mg/kg: F 39:7, P , 0:0001; 2 mg/kg: F 37:8, P , 0:0001). In mice treated chronically with nicotine the hypothermic effect of 1 mg/kg of nicotine was signi®cantly reduced at 30 min; but not at 60 min (acute £ chronic treatment interaction F 6:19, P 0:018). The acute nicotine dose of 2 mg/kg decreased the rectal temperature of chronically treated mice and sham-operated mice about similarly. Thus, increasing the dose of nicotine overcame the reduction of nicotine's hypothermic effect in chronically treated mice. As shown in Fig. 1 the acute 1 mg/kg dose of nicotine
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40 Table 1 The effects of an acute nicotine challenge (1 or 2 mg/kg s.c.) on the body temperature of sham-operated control mice and nicotine-infused mice on the seventh day of chronic treatment with the s.c. implanted reservoirs still in place Treatment
DT8C a 30 min
60 min
1 mg/kg Sham-operated 1 saline Sham-operated 1 nicotine Chronic nicotine 1 saline Chronic nicotine 1 nicotine
2 0.3 ^ 0.1 2 3.3 ^ 0.4** 2 1.0 ^ 0.3 2 1.8 ^ 0.2* oo
2 0.6 ^ 0.2 2 2.8 ^ 0.5** 2 0.6 ^ 0.2 2 1.8 ^ 0.2
2 mg/kg Sham-operated 1 saline Sham-operated 1 nicotine Chronic nicotine 1 saline Chronic nicotine 1 nicotine
2 0.6 ^ 0.2 2 4.9 ^ 0.2** 2 1.8 ^ 0.5 2 4.0 ^ 0.2**
2 0.6 ^ 0.1 2 2.0 ^ 0.4* 2 1.3 ^ 0.4 2 2.5 ^ 0.4**
a Rectal temperatures were measured at 30 or 60 min after saline or nicotine injection. Results are the means ^ SEM of 10 to 19 observations. *P , 0:05, **P , 0:01 as compared with the sham-operated control mice given acutely saline s.c., ooP , 0:01 as compared with the control mice given acutely nicotine (Tukey post-hoc comparison at timepoints after two-way ANOVA of repeated treatments). DT8C mean difference of rectal temperatures measured before and at 30 or 60 min after acute injections.
increased the striatal DOPAC concentration in sham-operated control mice by 35% (F 6:2, P 0:016); but did not affect the HVA concentration. The dose of 2 mg/kg increased the striatal DOPAC by 64% (F 6:13, P 0:019) and HVA by 43% (F 3:47, P 0:071), respectively. On the seventh day of chronic nicotine infusion the striatal DOPAC and HVA concentrations were similar to those of sham-operated mice, although in control mice nicotine at the dose of 1 mg/kg elevated DOPAC and at 2 mg/kg both DOPAC and HVA (acute £ chronic treatment interaction, 1 mg/kg: DOPAC F 3:65, P 0:062; 2 mg/kg: DOPAC F 12:36, P 0:001, HVA F 8:01, P 0:007). Striatal DA concentrations were not altered by any treatment. The hypothermic effect of nicotine is mediated by the central nAChRs [7] and can be prevented by elevating the ambient temperature [4]. In this study we con®rm that chronic nicotine treatment induces tolerance to the nicotine-induced decrease of body temperature [7,8]. Maximum hypothermia induced by acute nicotine doses of 1 and 2 mg/kg occurred at 30 min after administration. Chronic nicotine infused from subcutaneously implanted nicotine-releasing reservoirs for 7 days produced some tolerance to the hypothermia induced by acute nicotine dose of 1 mg/kg but not by the dose of 2 mg/kg. Furthermore; previously we found no tolerance to the hypothermic effect of even larger acute nicotine doses during a similar 7-day chronic nicotine infusion protocol in mice. Thus; nicotine at 3 mg/kg s.c. as well as at 1 mg/kg given four times repeatedly at 30 min intervals decreased the body
39
temperatures of control and nicotine-infused mice to a similar degree [5]. These ®ndings suggest that increasing the nicotine dose overcomes the attenuation of the hypothermic effect of nicotine. This suggests that tolerance in the classical sense, that is characterised by the fact that increasing the dose of the drug overcomes the diminution of its effect following repeated administration; develops to nicotine-hypothermia during chronic treatment. In the present experiments we estimated the striatal concentrations of DA and its metabolites DOPAC and HVA. These two metabolites are hypothesised to be formed at different sites due to the different localizations of the metabolising enzymes, monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). DOPAC is formed by MAO localized inside dopaminergic neurons and thus can be used as an index of intraneuronal synthesis and metabolism. COMT is localized outside dopaminergic neurons and O-methylates DOPAC to HVA which is considered to indicate the sum of DA synthesis, metabolism and release [13,18]. In sham-operated mice both acute nicotine doses increased DOPAC concentration, and the larger acute dose
Fig. 1. The effect of an acute nicotine challenge (1 or 2 mg/kg, 60 min, s.c.) on the striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations in mice on the seventh day of chronic nicotine infusion with the s.c. implanted nicotine-releasing reservoirs still in place. Given are the mean striatal concentrations of DA and the metabolites (columns) ^SEM (vertical bars), n 17±20. **P , 0:01, ***P , 0:001 as compared with the sham-operated control mice given acutely saline s.c., ooP , 0:01, oooP , 0:001 as compared with the sham-operated control mice given acutely nicotine (linear contrasts after randomised block two-way ANOVA). White columns: control mice 1 acute saline, dotted columns: control mice 1 acute nicotine, checkered columns: chronic nicotine 1 acute saline, black columns: chronic nicotine 1 acute nicotine.
40
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40
of 2 mg/kg also increased HVA. These ®ndings agree with those of Haikala et al. [4] that striatal DOPAC concentration is more susceptible to acute nicotine than striatal HVA. Thus, it could be that nAChRs involved in the regulation of the intraneuronal DA metabolism differ in their sensitivity from those involved in impulse-mediated DA release as suggested by Leikola-Pelho et al. [5]. In contrast to the saline-infused mice, acute nicotine treatment induced no changes in the striatal DA metabolism in mice on the seventh day of continuous nicotine infusion. This phenomenon could be mainly caused by the continuous presence of an agonist, in this case nicotine, desensitizing the nAChRs regulating the striatal dopaminergic neurons. It is to be noted that acute nicotine administration during chronic nicotine infusion elevated the plasma nicotine concentration to 300±400 ng/ml. Indeed, the phenomenon of desensitization of nAChRs regulating the DA metabolism seems to be dose-dependent. The results of Leikola-Pelho et al. [5] that 3 mg/kg dose of nicotine did not any more increase DOPAC and HVA and even decreased them during chronic nicotine, supports this suggestion. Similar lack of response to acute nicotine was observed in striatal DA metabolism of rats infused chronically with nicotine for 7 days [16]. Taken together; our results suggest that the responses of striatal DA metabolism and body temperature to acute nicotine are both reduced during constant nicotine infusion. However; the differences in the time courses and the dose-relationships of these effects suggest that they are different phenomena. The tolerance to nicotine's hypothermic effect can be overcome to some degree by increasing the dose of nicotine suggesting that tolerance in the classical sense develops in the nAChRs involved in thermoregulation during chronic nicotine treatment. In contrast, during chronic nicotine treatment acute nicotine did not increase the concentrations of striatal DA metabolites suggesting that desensitization is the main mechanism by which the nAChRs regulating striatal DA metabolism are inactivated under the present experimental conditions. Thus, the nAChRs involved in these two effects differ. Indeed, it is well known that cerebral nAChRs differ in their subunit composition; functional states and regional localization [9,17]. We wish to thank Mrs Eija Labbas and Ms Marjo Vaha for skilful assistance. This research was supported by Finnish Cultural Foundation and by University of Helsinki. [1] Ahtee, L. and Kaakkola, S., Effect of mecamylamine on the fate of dopamine in striatal and mesolimbic areas of rat brain: interaction with morphine and haloperidol, Br. J. Pharmacol., 62 (1978) 213±218. [2] Fuxe, K., Agnati, P., Eneroth, J.-A., Gustafsson, T., HoÈkfelt, A., LoÈfstroÈm, A., Skett, B. and Skett, P., The effect of nicotine
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on central catecholamine neurons and gonadotropin secretion. I. Studies in the male rat, Med. Biol., 55 (1977) 148±157. Grenhoff, J. and Svensson, T.H., Selective stimulation of dopamine activity by nicotine, Acta Physiol. Scand., 133 (1988) 595±596. Haikala, H., Karmalahti, T. and Ahtee, L., The nicotineinduced changes in striatal dopamine metabolism of mice depend on body temperature, Brain Res., 375 (1986) 313± 319. Leikola-Pelho, T., HeinaÈmaÈki, J., Laakso, I. and Ahtee, L., Chronic nicotine treatment changes differentially the effects of acute nicotine on the three main dopamine metabolites in mouse striatum, Naunyn-Schmiedebergs Arch. Pharmacol., 342 (1990) 400±406. Lichtensteiger, W., Hefti, F., Felix, D., Huwyler, T., Melamed, E. and Schlumpf, M., Stimulation of nigrostriatal dopamine neurones by nicotine, Neuropharmacology, 21 (1982) 963± 968. Mansner, R., Alhava, E. and Klinge, E., Nicotine hypothermia and brain nicotine and catecholamine levels in the mouse, Med. Biol, 52 (1974) 390±398. Marks, M.J. and Collins, A.C., Tolerance, cross-tolerance, and receptors after chronic nicotine or oxotremorine, Pharmacol. Biochem. Behav., 22 (1985) 283±291. McGehee, D.S. and Role, L.W., Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons, Annu. Rev. Physiol., 57 (1995) 521±546. Nose, T. and Takemoto, H., Effect of oxotremorine on homovanillic acid concentration in the striatum of the rat, Eur. J. Pharmacol., 25 (1974) 51±55. O'Brien, C.B., Drug Addiction and Drug abuse, In J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon and A. Goodman Gilman (Eds.), Goodman & Gilman's The Pharmacological Basis of Therapeutics, McGraw Hill, New York, 1996, p. 565. PietilaÈ, K., Salminen, O., Leikola-Pelho, T. and Ahtee, L., Tolerance to nicotine's effects on striatal dopamine metabolism in nicotine-withdrawn mice, Eur. J. Pharmacol., 318 (1996) 17±22. Rof¯er-Tarlov, S., Sharman, D. and Tergerdine, P., 3,4-dihydrophenylacetic acid and 4-hydroxy-3-methoxyphenylacetic acid in the mouse striatum: a re¯ection of intra- and extraneuronal metabolism of dopamine? Br. J. Pharmacol., 42 (1971) 343±351. Roth, K., McIntire, S. and Barchas, J., Nicotinic-catecholaminergic interactions in rat brain: evidence for cholinergic nicotinic and muscarinic interactions with hypothalamic epinephrine, J. Pharmacol. Exp. Therap., 221 (1982) 416± 420. Rowell, P.P. and Duggan, D.S., Long-lasting inactivation of nicotinic receptor function in vitro by treatment with high concentrations of nicotine, Neuropharmacology, 37 (1998) 103±111. Salminen, O., SeppaÈ, T., GaÈddnaÈs, H. and Ahtee, L., The effects of acute nicotine on the metabolism of dopamine and the expression of Fos protein in striatal and limbic brain areas of rats during chronic nicotine infusion and its withdrawal, J. Neurosci., 19 (1999) 8145±8151. Sargent, P.B., The diversity of neuronal nicotinic acetylcholine receptors, Annu. Rev. Neurosci., 16 (1993) 403±443. Westerink, B.H.C., Sequence and signi®cance of dopamine metabolism in the rat brain, Neurochem. Int., 7 (1985) 221± 227.
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The effects of acute nicotine on the body temperature and striatal dopamine metabolism of mice during chronic nicotine infusion Outi Salminen*, Liisa Ahtee Division of Pharmacology and Toxicology, Department of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland Received 11 January 2000; received in revised form 27 February 2000; accepted 27 February 2000
Abstract The effects of acute nicotine administration on body temperature and striatal dopamine metabolism of mice during chronic subcutaneous nicotine infusion were investigated. On the 7th day of nicotine infusion the hypothermic effect of 1 mg/kg nicotine s.c. but not that of 2 mg/kg was weakened suggesting that tolerance developing to nicotine's hypothermic effect during chronic nicotine can be overcome by increasing the dose of nicotine. In saline-infused control mice 1 mg/kg nicotine increased striatal 3,4-dihydroxyphenylacetic acid (DOPAC) but not homovanillic acid (HVA) concentration whereas 2 mg/kg increased both DOPAC and HVA. On the 7th day of nicotine infusion DOPAC and HVA concentrations were similar to control; and acute nicotine did not increase them suggesting that nicotinic acetylcholine receptors (nAChRs) regulating striatal dopamine metabolism were desensitized. The results suggest that the nAChRs mediating nicotine's effects on thermoregulation and brain dopamine metabolism differ. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotine; Constant infusion; Hypothermia; Striatal dopamine metabolism; Desensitization
Nicotine, the psychoactive component of tobacco; creates at least some of its central effects via brain dopaminergic systems. Acute administration of nicotine enhances cerebral dopamine (DA) turnover and metabolism both in mice and in rats [3,4,6,10,14]. However, at large doses nicotine does not alter and was even found to reduce the striatal DA turnover in rats when given i.p. [2] and the striatal concentrations of DA metabolites in mice when given s.c. [4]. This latter phenomenon has been suggested to result from desensitization of the nicotinic acetylcholine receptors (nAChRs) regulating physiologically the striatal dopaminergic neurons. Indeed, nicotinic antagonists blocking the nAChRs reduce the concentrations of striatal DA metabolites in rats and mice [1,4]. Characteristic for the desensitization of the nAChRs is that it both develops and reverses rapidly. Also, tolerance in the classical sense which occurs more gradually, and is longer-lasting; most probably occurs to nicotine's effects such as nausea in smokers [11]. Recently, it has been described a long-lasting inactivation of nAChRs in vitro, which persists long after removal of nicotine and characteristic for which is that after prolonged nicotine * Corresponding author. Tel.: 1358-9-191-59459; fax: 1358-9191-59471. E-mail address: outi.salminen@helsinki.® (O. Salminen).
treatment the nAChRs do not respond to nicotine even after long intervals [15]. Moreover; we reported ®ndings in vivo suggesting occurrence of such long-lasting inactivation of nAChRs regulating cerebral DA metabolism after withdrawal of mice or rats from a prolonged nicotine treatment. Thus, a small nicotine dose did not increase the striatal DA metabolism in mice withdrawn for 24 h from sevenweek oral nicotine administration [12], and in rats acute nicotine did not any more elevate limbic dihydroxyphenylacetic acid (DOPAC) concentration after 24-h withdrawal from 7-day constant nicotine infusion [16]. In the experiment where mice were withdrawn for 24 h from seven-week oral nicotine administration [12], acute nicotine challenge induced signi®cantly less hypothermia in chronically treated mice than in controls suggesting the development of tolerance to the hypothermic effect of nicotine. Nicotine-induced reductions of striatal DA metabolites are in mice closely and inversely correlated with its hypothermic effects and counteracting the hypothermic effect of nicotine enhanced its DA metabolism stimulating effects [4,5]. To study the tolerance and desensitization of nAChRs regulating DA metabolism; we investigated the effects of acute nicotine on rectal temperatures and striatal DA metabolism in mice during constant nicotine infusion. The striatal brain area dissected in the present experiments consists
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 98 3- 6
38
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40
of dorsal as well as ventral striatum and thus includes the nucleus accumbens. An acute dose of (2)-nicotine (1 or 2 mg/kg s.c.) was given to mice on the seventh day of nicotine infusion with the subcutaneous nicotine-releasing reservoirs still in place. Male NMRI-mice weighing 20±27 g at the beginning of the chronic treatment were housed ten to a cage and kept on a standard diet and tap water ad libitum. Lights were on from 06:00 h until 18:00 h and the ambient temperature was kept at 20±228C. Experimental animals were maintained in accordance with the internationally accepted principles; and the experimental set-up was approved by the Committee for Animal Experiments of the Faculty of Science of the University of Helsinki. (2)-Nicotine was administered chronically for 7 days using subcutaneously implanted nicotine releasing reservoirs as described for mice [5]. The nicotine reservoirs containing 40 ml of (2)-nicotine base (Fluka, Buchs, Switzerland) were placed under the skin of mice anaesthetised with pentobarbital (50 mg/kg i.p., Mebunat 60 mg/ml diluted with saline, Orion Pharma, Espoo, Finland). A small sagittal cut of about 1 cm was made behind the ears after local injection of 0.1 ml 0.1% lidocaine solution (Lidocain 5 mg/ml inject., Orion Pharma) s.c., and the reservoir was pushed gently with a longitudinal orientation beneath the skin with the Silastic R (Dow Corning, Midland, Michican, USA) plug most caudally. The incision was closed with surgical suture. Control animals and animals receiving only acute nicotine treatment were sham-operated by placing a reservoir containing 0.9% NaCl-solution (saline) under the skin. The mice received saline (s.c.) or acute nicotine (1 or 2 mg/kg s.c.) 1 h before decapitation on the seventh day of nicotine infusion with the reservoirs still in place. For injections (0.1 ml/100 g) (2)-nicotine base was diluted with saline, and the ®nal solution was adjusted to pH 7.0±7.4 with 0.05 M HCl in saline. After decapitation striata were dissected and frozen on dry ice within 3 min, weighed (mean weight 25 mg) and stored at 2808C until assayed. Rectal temperatures were measured on the seventh day of chronic treatment (either saline- or nicotine-infused mice) before and at 30 and 60 min after acute challenge dose (saline or nicotine). An electric thermocouple (Ellab Instruments; Copenhagen Denmark) was inserted 2.5 cm into the rectum. DT8C is the mean difference of rectal temperatures measured before an acute treatment and 30 or 60 min after acute treatment; the last measurement being immediately before the decapitation. The contents of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured by HPLC with electrochemical detection after Sephadex G-10 gel chromatographic clean-up of samples [5,16]. When estimating the DA and metabolite samples from all four treatment groups (sham-operated 1 saline, sham-operated 1 acute nicotine; nicotine-infused 1 saline, nicotine-infused 1 acute nicotine) were analysed simulta-
neously. The samples from experiments with 1 and 2 mg/kg of acute nicotine were analysed separately. In brief, tissue samples were homogenized and pH of the homogenates was adjusted to 2.4. After centrifugation supernatants were passed through acidic (0.01 M HCl) Sephadex G-10 columns and a 200 ml of each collected fraction was injected in a C-18 reverse phase (Spherisorb octadecylsilane 2 mm) HPLC column (25 cm; 4.6 mm i.d.) connected to the electrochemical detector (Potentiostat type LC-2A, Bioanalytical Systems with rotating disc working electrode). The plasma concentrations of nicotine and cotinine were measured on the seventh day of chronic infusion using gas chromatography-mass spectrometry (GC-MS). The trunk blood of mice was collected after decapitation. Blood samples were treated and measured as described by Leikola-Pelho et al. [5]. As the metabolite results consisted of several experiments the statistical analyses were carried out by three-way ANOVA (experiment £ chronic nicotine £ acute nicotine). As no signi®cant interactions were found between the experiment and other factors; the randomised block twoway ANOVA was performed using experiments as blocks. If there were signi®cant chronic x acute nicotine interactions (P , 0:1), the analysis was continued by comparing appropriate cell means with linear contrasts. Statistical analysis of changes of rectal temperature was performed by two-way ANOVA of repeated measurements. The results from experiments with 1 and 2 mg/kg of acute nicotine were analysed separately because they were carried out on different days. As signi®cant chronic £ acute £ time interactions were found, the Tukey's post hoc comparisons at timepoints were made. On the 7th day of the chronic nicotine treatment with the nicotine-releasing reservoirs still in place the plasma concentration of nicotine was 207 ^ 32 ng/ml (mean ^ SEM, n 12) and that of cotinine 1366 ^ 258 ng/ml. This agrees well with the previous results of Leikola-Pelho et al. [5]. When challenge doses of 1 and 2 mg/kg were given to nicotineinfused mice, nicotine concentrations in the plasma at 60 min were 301 ^ 56 ng/ml (1 mg/kg, n 12) and 412 ^ 35 ng/ml (2 mg/kg, n 5) and cotinine concentrations 1861 ^ 253 (n 12) and 1529 ^ 284 ng/ml (n 5), respectively. Thus, acute nicotine further elevated plasma nicotine, but not cotinine, concentrations. As shown in Table 1 in the sham-operated mice acute nicotine induced hypothermia dose-dependently (1 mg/kg: F 39:7, P , 0:0001; 2 mg/kg: F 37:8, P , 0:0001). In mice treated chronically with nicotine the hypothermic effect of 1 mg/kg of nicotine was signi®cantly reduced at 30 min; but not at 60 min (acute £ chronic treatment interaction F 6:19, P 0:018). The acute nicotine dose of 2 mg/kg decreased the rectal temperature of chronically treated mice and sham-operated mice about similarly. Thus, increasing the dose of nicotine overcame the reduction of nicotine's hypothermic effect in chronically treated mice. As shown in Fig. 1 the acute 1 mg/kg dose of nicotine
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40 Table 1 The effects of an acute nicotine challenge (1 or 2 mg/kg s.c.) on the body temperature of sham-operated control mice and nicotine-infused mice on the seventh day of chronic treatment with the s.c. implanted reservoirs still in place Treatment
DT8C a 30 min
60 min
1 mg/kg Sham-operated 1 saline Sham-operated 1 nicotine Chronic nicotine 1 saline Chronic nicotine 1 nicotine
2 0.3 ^ 0.1 2 3.3 ^ 0.4** 2 1.0 ^ 0.3 2 1.8 ^ 0.2* oo
2 0.6 ^ 0.2 2 2.8 ^ 0.5** 2 0.6 ^ 0.2 2 1.8 ^ 0.2
2 mg/kg Sham-operated 1 saline Sham-operated 1 nicotine Chronic nicotine 1 saline Chronic nicotine 1 nicotine
2 0.6 ^ 0.2 2 4.9 ^ 0.2** 2 1.8 ^ 0.5 2 4.0 ^ 0.2**
2 0.6 ^ 0.1 2 2.0 ^ 0.4* 2 1.3 ^ 0.4 2 2.5 ^ 0.4**
a Rectal temperatures were measured at 30 or 60 min after saline or nicotine injection. Results are the means ^ SEM of 10 to 19 observations. *P , 0:05, **P , 0:01 as compared with the sham-operated control mice given acutely saline s.c., ooP , 0:01 as compared with the control mice given acutely nicotine (Tukey post-hoc comparison at timepoints after two-way ANOVA of repeated treatments). DT8C mean difference of rectal temperatures measured before and at 30 or 60 min after acute injections.
increased the striatal DOPAC concentration in sham-operated control mice by 35% (F 6:2, P 0:016); but did not affect the HVA concentration. The dose of 2 mg/kg increased the striatal DOPAC by 64% (F 6:13, P 0:019) and HVA by 43% (F 3:47, P 0:071), respectively. On the seventh day of chronic nicotine infusion the striatal DOPAC and HVA concentrations were similar to those of sham-operated mice, although in control mice nicotine at the dose of 1 mg/kg elevated DOPAC and at 2 mg/kg both DOPAC and HVA (acute £ chronic treatment interaction, 1 mg/kg: DOPAC F 3:65, P 0:062; 2 mg/kg: DOPAC F 12:36, P 0:001, HVA F 8:01, P 0:007). Striatal DA concentrations were not altered by any treatment. The hypothermic effect of nicotine is mediated by the central nAChRs [7] and can be prevented by elevating the ambient temperature [4]. In this study we con®rm that chronic nicotine treatment induces tolerance to the nicotine-induced decrease of body temperature [7,8]. Maximum hypothermia induced by acute nicotine doses of 1 and 2 mg/kg occurred at 30 min after administration. Chronic nicotine infused from subcutaneously implanted nicotine-releasing reservoirs for 7 days produced some tolerance to the hypothermia induced by acute nicotine dose of 1 mg/kg but not by the dose of 2 mg/kg. Furthermore; previously we found no tolerance to the hypothermic effect of even larger acute nicotine doses during a similar 7-day chronic nicotine infusion protocol in mice. Thus; nicotine at 3 mg/kg s.c. as well as at 1 mg/kg given four times repeatedly at 30 min intervals decreased the body
39
temperatures of control and nicotine-infused mice to a similar degree [5]. These ®ndings suggest that increasing the nicotine dose overcomes the attenuation of the hypothermic effect of nicotine. This suggests that tolerance in the classical sense, that is characterised by the fact that increasing the dose of the drug overcomes the diminution of its effect following repeated administration; develops to nicotine-hypothermia during chronic treatment. In the present experiments we estimated the striatal concentrations of DA and its metabolites DOPAC and HVA. These two metabolites are hypothesised to be formed at different sites due to the different localizations of the metabolising enzymes, monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). DOPAC is formed by MAO localized inside dopaminergic neurons and thus can be used as an index of intraneuronal synthesis and metabolism. COMT is localized outside dopaminergic neurons and O-methylates DOPAC to HVA which is considered to indicate the sum of DA synthesis, metabolism and release [13,18]. In sham-operated mice both acute nicotine doses increased DOPAC concentration, and the larger acute dose
Fig. 1. The effect of an acute nicotine challenge (1 or 2 mg/kg, 60 min, s.c.) on the striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations in mice on the seventh day of chronic nicotine infusion with the s.c. implanted nicotine-releasing reservoirs still in place. Given are the mean striatal concentrations of DA and the metabolites (columns) ^SEM (vertical bars), n 17±20. **P , 0:01, ***P , 0:001 as compared with the sham-operated control mice given acutely saline s.c., ooP , 0:01, oooP , 0:001 as compared with the sham-operated control mice given acutely nicotine (linear contrasts after randomised block two-way ANOVA). White columns: control mice 1 acute saline, dotted columns: control mice 1 acute nicotine, checkered columns: chronic nicotine 1 acute saline, black columns: chronic nicotine 1 acute nicotine.
40
O. Salminen, L. Ahtee / Neuroscience Letters 284 (2000) 37±40
of 2 mg/kg also increased HVA. These ®ndings agree with those of Haikala et al. [4] that striatal DOPAC concentration is more susceptible to acute nicotine than striatal HVA. Thus, it could be that nAChRs involved in the regulation of the intraneuronal DA metabolism differ in their sensitivity from those involved in impulse-mediated DA release as suggested by Leikola-Pelho et al. [5]. In contrast to the saline-infused mice, acute nicotine treatment induced no changes in the striatal DA metabolism in mice on the seventh day of continuous nicotine infusion. This phenomenon could be mainly caused by the continuous presence of an agonist, in this case nicotine, desensitizing the nAChRs regulating the striatal dopaminergic neurons. It is to be noted that acute nicotine administration during chronic nicotine infusion elevated the plasma nicotine concentration to 300±400 ng/ml. Indeed, the phenomenon of desensitization of nAChRs regulating the DA metabolism seems to be dose-dependent. The results of Leikola-Pelho et al. [5] that 3 mg/kg dose of nicotine did not any more increase DOPAC and HVA and even decreased them during chronic nicotine, supports this suggestion. Similar lack of response to acute nicotine was observed in striatal DA metabolism of rats infused chronically with nicotine for 7 days [16]. Taken together; our results suggest that the responses of striatal DA metabolism and body temperature to acute nicotine are both reduced during constant nicotine infusion. However; the differences in the time courses and the dose-relationships of these effects suggest that they are different phenomena. The tolerance to nicotine's hypothermic effect can be overcome to some degree by increasing the dose of nicotine suggesting that tolerance in the classical sense develops in the nAChRs involved in thermoregulation during chronic nicotine treatment. In contrast, during chronic nicotine treatment acute nicotine did not increase the concentrations of striatal DA metabolites suggesting that desensitization is the main mechanism by which the nAChRs regulating striatal DA metabolism are inactivated under the present experimental conditions. Thus, the nAChRs involved in these two effects differ. Indeed, it is well known that cerebral nAChRs differ in their subunit composition; functional states and regional localization [9,17]. We wish to thank Mrs Eija Labbas and Ms Marjo Vaha for skilful assistance. This research was supported by Finnish Cultural Foundation and by University of Helsinki. [1] Ahtee, L. and Kaakkola, S., Effect of mecamylamine on the fate of dopamine in striatal and mesolimbic areas of rat brain: interaction with morphine and haloperidol, Br. J. Pharmacol., 62 (1978) 213±218. [2] Fuxe, K., Agnati, P., Eneroth, J.-A., Gustafsson, T., HoÈkfelt, A., LoÈfstroÈm, A., Skett, B. and Skett, P., The effect of nicotine
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