Psychopharmacology (1992) 108:33 39
Psychopharmacology © Springer-Verlag 1992
Tolerance to nicotine following chronic treatment by injections: a potential role for corticosterone James R. Pauly, Elizabeth U. Grun, and Allan C. Collins Instilute for Behavioral Genetics and Department of Psychology, University of Colorado, Campus Box 447, Boulder, CO 80309, USA Received May 28, 1991 / Final version September 9, 1991
Abstract. C57BL/6 male mice were injected intraperitoneally with nicotine (2.0 mg/kg) or saline three times each day (0800 h, 1300 h and 1800 h) for a period of 12 days and then tested for nicotine tolerance using a series of behavioral and physiological tests. For each of these tests, animals that received chronic nicotine treatment were significantly less sensitive to nicotine challenge than were animals that received chronic saline treatment, as indicated by shifts to the right of doseresponse curves. Animals were retested for nicotine sensitivity 2 weeks following cessation of chronic nicotine injections. Tolerance to acute nicotine challenge persisted in nicotine-treated animals. Chronic nicotine treatment by injections did not alter the binding of I.-[3H] nicotine or c~-[125I]-bungarotoxin in any of eight brain regions. Plasma corticosterone (CCS) levels were determined in animals prior to the initiation of the injection series (day 0), and on days 4, 8 and 12 of chronic treatment, immediately before the first injection of the day. CCS levels in nicotine-treated animals were elevated as compared to saline-injected controls by day 12 of treatment. Nicotine-treated animals also had elevated CCS levels 2 weeks after the last chronic injection. Nicotinetreated animals were, however, tolerant to nicotine-induced CCS release. Since previous studies from our laboratory have demonstrated that plasma CCS levels are inversely correlated with sensitivity to nicotine, it is possible that the tolerance to nicotine measured following chronic treatment by injections is due, at least in part, to the elevation in plasma CCS levels. Key words: Nicotine-tolerance- Bungarotoxin-receptors - Corticosterone - Chronic treatment - Nicotinic cholinergic-stress
Drug tolerance can be defined as a reduction in response to a challenge dose following repeated administration of that drug. Both dispositional (metabolic) and cellular changes have been considered to be of primary iraOffprint requests to: J.R. Pauly
portance in regulating the development of tolerance (MacKenzie and Rech 1990). These processes are thought to be regulated primarily by the pharmacological properties and treatment regimen (dose, route of administration, frequency of treatment, duration of treatment, etc.) of the drug in question. It is now apparent, however, that additional factors such as the environmental cues associated with drug delivery also influence the development of tolerance. Many studies have demonstrated that tolerance develops to several of the effects elicited by nicotine. For example, several earlier studies from our laboratory, using mice of the DBA/2 strain, demonstrated that tolerance develops to nicotine's effects on respiration rate, locomotor activity, acoustic startle response, heart rate, body temperature and seizure threshold when mice are chronically treated with nicotine by continuous infusion via indwelling jugular catheters. This tolerance increases with infusion dose (Marks et al. 1983, 1985, 1991) and time of infusion (Marks et al. 1986 a, b) and is associated with an increase in the number of those brain nicotinic receptors that bind L-[3H]-nicotine. Higher infusion doses also elicited increases in the binding of c~-[xzsI]bungarotoxin (BTX), a ligand that binds to lower affinity brain nicotine receptors. Tolerance to nicotine's actions was paralleled most closely by changes in L-[3H]nicotine binding which suggests that up-regulation of this nicotinic receptor may underlie tolerance to nicotine. However, a more recent analysis of the development of tolerance to nicotine in five inbred mouse strains (Marks et al. 1991) suggested that increases in the number of L-[3H]-nicotine binding sites is not inextricably associated with the development of tolerance to nicotine. A significant correlation between tolerance and an increase in the number of brain L-[3H]-nicotine binding sites was observed in some (DBA\2 and C57BL\6) but not in other (C3H and BUB) strains of mice. The development of tolerance to nicotine in the C3H and BUB strains of mice was more highly correlated with an increase in the number of binding sites labeled by ~-[125I]BTX. Rats given nicotine chronically, generally via injections, also develop an increase in the number of CNS
34
high affinity nicotinic receptors as measured by L-[3I'I] nicotine or [3H]-acetylcholine (ACh) binding (Schwartz and Kellar 1983, 1985; Ksir et al. 1985; Martino-Barrows and Kellar 1987; Collins et al. 1988; Hulihan-Giblinet al. 1990; Wonnacott 1990). These two ligands bind to the same subset of nicotinic cholinergic receptors in the mammalian CNS (Clarke et al. 1985; Marks et al. 1986c; Martino-Barrows and Kellar 1987). Tolerance to nicotine-induced decreases in locomotor activity develops following chronic injections (Stolerman et al. 1973; Clarke and Kumar 1983a, b; Collins et al. 1988) and it may be that changes in receptor number explain this reduced sensitivity. Up-regulation of nicotinic receptors has also been suggested to underlie tolerance to nicotineinduced prolactin release (Hulihan-Giblin et al. 1990). However, Ksir et al. (1985) have argued that increases in receptor numbers underlie sensitization to nicotineinduced increases in locomotor activity. Studies examining the time courses of sensitivity and receptor changes have failed to resolve the relationship between changes in receptors and drug sensitivity. For example, in one study rats injected chronically with nicotine developed and lost tolerance to nicotine-induced prolactin release in concert with an elevation and return to normal of brain [3H]-acetylcholine binding (HulihanGiblin et al. 1990). However, studies from our laboratory have yielded different results. Following chronic nicotine injections, tolerance development to the effects of nicotine on locomotor activity and body temperature was significantly correlated with the increase in rat brain L-[3H]-nicotine binding (Collins et al. 1988). This tolerance to nicotine persisted well after receptor binding had returned to control levels. Thus, our results suggest that tolerance persists longer than receptor changes when nicotine is administered using a chronic injection protocol. In contrast, rats treated with nicotine using a chronic infusion method, develop increases in L-[3H]-nicotine as well as increases in ~-[12sI]-bungarotoxin binding, but tolerance to nicotine-included decreases in locomotor activity and body temperature was lost within 4 days following the cessation of chronic treatment, more closely paralleling the return to normal of receptor binding. These findings argue that tolerance to nicotine is regulated by the method of chronic drug treatment and involves more than receptor changes. Chronic injection procedures have also been used to establish whether tolerance to nicotine's effects develop in the mouse (Hatchell and Collins 1977). These early studies detected marked tolerance to nicotine but potential receptor changes were not measured. The goal of the present study was to ascertain whether tolerance development following chronic nicotine injections in the mouse correlates with changes in brain nicotinic cholinergic receptor binding. Because we have shown that altering corticosterone (CCS) levels has marked effects on nicotine sensitivity in the mouse (Pauly et al. 1988, 1990), plasma CCS levels were also monitored. The results obtained indicate that mice chronically injected with nicotine develop profound tolerance to nicotine even though the number and affinity of brain nicotinic receptors is unaffected by this treatment. Preliminary
evidence was obtained which suggests that this tolerance may involve a learning component and may be mediated by an elevation in plasma CCS levels.
Materials and methods Animals. Adult male mice (60-90 days of age) of the C57BL/6/ibg strain were used for these studies. This strain (originally obtained from Jackson Laboratories, Bar Harbor, ME) has been maintained in the breeding colony at the Institute for Behavioral Genetics for more than 20 generations. Mice were group housed (five per cage) under a 12 h light/12 h dark cycle with lights on at 0700 h and provided with pelleted food (Wayne Lab Blox) and water ad libitum. Chronic nicotine treatment. At the beginning of the experiment, animals were transferred from the colony room into a chronic treatment room. Each animal then received chronic treatment with nicotine (2.0 mg/kg) or saline (3 injections per day at 0800, 1300 and 1800 h) for a period of 12 days. (-)-Nicotine base (Sigma Chemical Co., St Louis MO), purified by distillation, was dissolved in physiological saline, neutralized with HCI and injected intraperitoneally in a volume of 0.01 ml/g body weight. The animal's weights were checked every 3 days (following the 0800 h injection) to detect possible changes during the course of chronic treatment. Plasma corticosterone determination. Plasma CCS levels were determined in 8-12 animals from each treatment group on the day prior to the initiation of treatment (day 0), and on days 4, 8 and 12 of chronic treatment. Plasma CCS was also measured in some animals 15 days following the cessation of chronic treatment. Blood samples were collected between the hours of 0700 and 0800 (just prior to the first injection of each day) by retro-orbital sinus puncture. The CCS radioimmunoassay described by Gwosdow-Cohen (1982) was utilized. All samples were processed at the completion of the experiment. Each animal was utilized only once for baseline CCS determinations. CCS antibody was obtained from Dr. G. Niswender (Dept of Physiology and Biophysics, Colorado State University, Fort Collins, CO). Behavioral testing. Following the 1800 h injection on the 12th treatment day, animals were moved from the treatment room to a behavioral testing room where they were tested for behavioral and physiological sensitivity to nicotine on the following day. All testing was performed between the hours of 0800 and 1300. Doses of nicotine (0 2.0 mg/kg) were altered by changing the concentration of nicotine in the injection solution. Nicotine sensitivity- was measured using a battery of tests that has been previously described (Marks et al. 1985 ; Pauly et al. 1988). Nicotine effects on locomotor activity were measured in a symmetrical Y-maze with each arm measuring 26.0 cm x 6.1 cm x 10.2 cm (L x W × H). Each arm was divided into two sections and crosses from one arm/section to another were counted. The number of rears (number of times the animal reared up onto its hind legs) were also counted during the 3 rain test. Y-maze activities were measured 4.5 min after nicotine administration. Heart rate was measured 8.5 rain post-injection using an E and M Physiograph (Narco Biosystems, Houston, TX). Fifteen minutes following nicotine injection, body temperature (rectal) was measured using a Baily Instruments digital thermometer. The time points chosen for these measures reflect the times of maximal nicotine effect based on previously determined time course analyses performed in our laboratory (Marks et al. 1985). For the test battery parameters, five to eight animals were tested at each nicotine dose for each experimental group. Tissue preparation. Sixteen animals from each treatment group were sacrificed by cervical dislocation approximately 2 4 h following behavioral testing. Their brains were removed, washed and
35
Data analysis. Receptor binding data were analyzed by one-way
then dissected into the following eight anatomical regions: cortex, cerebellum, striatum, hypothalamus, hippocampus, midbrain (mostly thalamus), colliculi (inferior and superior) and hindbrain (pons-medulla). Brain regions were placed in 10 volumes of ice-cold buffer (Krebs-Ringers HEPES: NaC1, 118mM; KC1, 4.8 raM; MgSO4, 1.2 mM; CaCL2, 2.5 mM; HEPES, 20 raM; pH adjusted to 7.5 with NaOH). Tissue preparation was essentially that of Romano and Goldstein (1980) as described by Marks et al. (1986c).
analysis of variance (by region). Test battery and CCS release data were analyzed by two-way analysis of variance with Newman-Kuels tests used as post hoc tests, t Tests were used for all other statistical comparisons.
Results
L-[3H]-nicotine binding. The binding of L-[3H]-nicotine (N-methyl-
Nicotine tolerance
[3H], specific activity, 60 Ci/mmol; New England Nuclear) was determined at 4° C using the method of Romano and Goldstein (1980) as modified by Marks and Collins (1982). L-[gH]-nicotine was purified chromatographically as described by Romm et al. (1990) to remove a contaminant that produces what appears to be a low affinity binding site. The final incubation volume was 250 p,1 and each sample contained 150-500 gg tissue protein. Ko and B~,,~values were determined in cortical tissue using six increasing concentrations of radiolabeled nicotine (0.57-18.11 nM) using linear regression analysis of the data transformed to fit the Scatchard equation. Nicotine binding was measured in each of the other brain regions using a single concentration of radiolabeled nicotine (5 nM).
Dose-response curves for the effects of nicotine on the four test battery parameters in chronic nicotine and saline-treated animals are presented in Fig. I (the data in this a n d all o t h e r figures r e p r e s e n t s the g r o u p mean_+ S E M ) . A n i m a l s t h a t received c h r o n i c n i c o t i n e injections were significantly less sensitive ( P < 0 . 0 0 1 ) to the a c u t e n i c o t i n e challenge for e a c h o f the tests. A N O V A results for e a c h o f the test b a t t e r y p a r a m e t e r s were as follows" Y m a z e crosses ( F 1 , 6 a - - 1 9 . 8 5 ) , Y m a z e r e a r s (F1,63 = 24.55), h e a r t r a t e ( F z , 6 3 = 1 9 . 4 3 ) , b o d y t e m p e r a t u r e (Fa,63 = 43.09). T w o weeks f o l l o w i n g c e s s a t i o n o f c h r o n ic n i c o t i n e t r e a t m e n t , a n i m a l s t h a t h a d received c h r o n i c n i c o t i n e injections were still t o l e r a n t to a c u t e n i c o t i n e challenge (Table 1).
c~-[12U]-bungarotoxin (BTX) binding. The binding of e-[azsI]-BTX (Tyr-[~zsI], initial specific activity, 219 Ci/mmol; Amersham) was performed at 37° C using the methods of Marks and Collins (1982). Assays using a single concentration of ligand were performed in each of the eight brain regions using 1.20 nM c~-[~zsI]-BTX. Scatchard analysis of tissue from cortex utilized six concentrations of labeled BTX (0.11-2.06 riM).
Nicotinic cholinergic receptor binding
Protein determination. Protein determinations were performed using the method of Lowry (1951) with bovine serum albumin as the standard.
T h e b i n d i n g o f L-[3H]-nicotine in eight b r a i n r e g i o n s in a n i m a l s t h a t received c h r o n i c n i c o t i n e o r saline t r e a t m e n t for 12 d a y s is p r e s e n t e d in Fig. 2. C h r o n i c n i c o t i n e injections d i d n o t alter L-[3H]-nicotine b i n d i n g in a n y b r a i n region. S c a t c h a r d analysis o f g - [ 3 H ] - n i c o t i n e b i n d ing in c o r t i c a l tissue y i e l d e d similar results. I n salinet r e a t e d a n i m a l s the Bmax for n i c o t i n e b i n d i n g w a s 32.23+_2.40 f m o l / m g p r o t e i n w i t h a KD o f 2.29___ 0.37 n M . I n a n i m a l s c h r o n i c a l l y t r e a t e d w i t h nicotine, similar values were o b t a i n e d (Bma ~ = 35.55 + 3 . 5 0 f m o l / m g p r o t e i n ; K D = l . 7 8 _ + 0 . 4 0 n M ) . T h e b i n d i n g o f c~[a25I]-BTX d e t e r m i n e d a t a single l i g a n d c o n c e n t r a t i o n in all o f the eight b r a i n r e g i o n s e x a m i n e d was also n o t
Nicotine-induced CCS release. Animals that were not used to measure receptor binding were tested for baseline (pre-injection), salineinduced and nicotine-induced CCS release at 0800 h on day 14 of the experiment (n= 8-12 per group). A group of control colony animals that received no prior treatment were also tested. Animals were injected with nicotine (1.0 mg/kg) or saline and blood samples were collected 25 min later. This time point was chosen based on a published time course for nicotine-induced CCS release in C57BL/6 animals (Freund et al. 1988), and provided maximal stimulation of CCS release. A test nicotine dose of 1.0 mg/kg was used to produce a sub-threshold adrenal response, thus avoiding any possible complication of a ceiling effect. Y MAZE CROSSES •
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Fig. 1. Chronic nicotine injections produce profound behavioral and physiological tolerance to acute nicotine challenge. Animals were injected three times a day with nicotine (2.0 mg/kg) or saline for 12 days. Animals were tested for nicotine sensitivity as described in the Methods section. Profound tolerance was present for each measure. ANOVA results for each of the test battery parameters were as follows: Y maze crosses (F1,63 = 19.85), Y maze rears (F1,63 =24.55), heart rate (FI,63 = 19.43), body temperature (F1,63 =43.09)
36 Table 1. The effects of nicotine on several behavioral and physiological measures 2 weeks following the cessation of either chronic nicotine or saline injections. Tolerance persisted in animals that had received chronic nicotine injections for each test battery parameter. Due to the limited number of animals (n=3-4 per g r o u p ) , only one test dose of nicotine was used (1.5 mg/kg). Data represent group means±SEM. * Value significantly different from saline-injected control (ANOVA, P
Psychopharmacology © Springer-Verlag 1992
Tolerance to nicotine following chronic treatment by injections: a potential role for corticosterone James R. Pauly, Elizabeth U. Grun, and Allan C. Collins Instilute for Behavioral Genetics and Department of Psychology, University of Colorado, Campus Box 447, Boulder, CO 80309, USA Received May 28, 1991 / Final version September 9, 1991
Abstract. C57BL/6 male mice were injected intraperitoneally with nicotine (2.0 mg/kg) or saline three times each day (0800 h, 1300 h and 1800 h) for a period of 12 days and then tested for nicotine tolerance using a series of behavioral and physiological tests. For each of these tests, animals that received chronic nicotine treatment were significantly less sensitive to nicotine challenge than were animals that received chronic saline treatment, as indicated by shifts to the right of doseresponse curves. Animals were retested for nicotine sensitivity 2 weeks following cessation of chronic nicotine injections. Tolerance to acute nicotine challenge persisted in nicotine-treated animals. Chronic nicotine treatment by injections did not alter the binding of I.-[3H] nicotine or c~-[125I]-bungarotoxin in any of eight brain regions. Plasma corticosterone (CCS) levels were determined in animals prior to the initiation of the injection series (day 0), and on days 4, 8 and 12 of chronic treatment, immediately before the first injection of the day. CCS levels in nicotine-treated animals were elevated as compared to saline-injected controls by day 12 of treatment. Nicotine-treated animals also had elevated CCS levels 2 weeks after the last chronic injection. Nicotinetreated animals were, however, tolerant to nicotine-induced CCS release. Since previous studies from our laboratory have demonstrated that plasma CCS levels are inversely correlated with sensitivity to nicotine, it is possible that the tolerance to nicotine measured following chronic treatment by injections is due, at least in part, to the elevation in plasma CCS levels. Key words: Nicotine-tolerance- Bungarotoxin-receptors - Corticosterone - Chronic treatment - Nicotinic cholinergic-stress
Drug tolerance can be defined as a reduction in response to a challenge dose following repeated administration of that drug. Both dispositional (metabolic) and cellular changes have been considered to be of primary iraOffprint requests to: J.R. Pauly
portance in regulating the development of tolerance (MacKenzie and Rech 1990). These processes are thought to be regulated primarily by the pharmacological properties and treatment regimen (dose, route of administration, frequency of treatment, duration of treatment, etc.) of the drug in question. It is now apparent, however, that additional factors such as the environmental cues associated with drug delivery also influence the development of tolerance. Many studies have demonstrated that tolerance develops to several of the effects elicited by nicotine. For example, several earlier studies from our laboratory, using mice of the DBA/2 strain, demonstrated that tolerance develops to nicotine's effects on respiration rate, locomotor activity, acoustic startle response, heart rate, body temperature and seizure threshold when mice are chronically treated with nicotine by continuous infusion via indwelling jugular catheters. This tolerance increases with infusion dose (Marks et al. 1983, 1985, 1991) and time of infusion (Marks et al. 1986 a, b) and is associated with an increase in the number of those brain nicotinic receptors that bind L-[3H]-nicotine. Higher infusion doses also elicited increases in the binding of c~-[xzsI]bungarotoxin (BTX), a ligand that binds to lower affinity brain nicotine receptors. Tolerance to nicotine's actions was paralleled most closely by changes in L-[3H]nicotine binding which suggests that up-regulation of this nicotinic receptor may underlie tolerance to nicotine. However, a more recent analysis of the development of tolerance to nicotine in five inbred mouse strains (Marks et al. 1991) suggested that increases in the number of L-[3H]-nicotine binding sites is not inextricably associated with the development of tolerance to nicotine. A significant correlation between tolerance and an increase in the number of brain L-[3H]-nicotine binding sites was observed in some (DBA\2 and C57BL\6) but not in other (C3H and BUB) strains of mice. The development of tolerance to nicotine in the C3H and BUB strains of mice was more highly correlated with an increase in the number of binding sites labeled by ~-[125I]BTX. Rats given nicotine chronically, generally via injections, also develop an increase in the number of CNS
34
high affinity nicotinic receptors as measured by L-[3I'I] nicotine or [3H]-acetylcholine (ACh) binding (Schwartz and Kellar 1983, 1985; Ksir et al. 1985; Martino-Barrows and Kellar 1987; Collins et al. 1988; Hulihan-Giblinet al. 1990; Wonnacott 1990). These two ligands bind to the same subset of nicotinic cholinergic receptors in the mammalian CNS (Clarke et al. 1985; Marks et al. 1986c; Martino-Barrows and Kellar 1987). Tolerance to nicotine-induced decreases in locomotor activity develops following chronic injections (Stolerman et al. 1973; Clarke and Kumar 1983a, b; Collins et al. 1988) and it may be that changes in receptor number explain this reduced sensitivity. Up-regulation of nicotinic receptors has also been suggested to underlie tolerance to nicotineinduced prolactin release (Hulihan-Giblin et al. 1990). However, Ksir et al. (1985) have argued that increases in receptor numbers underlie sensitization to nicotineinduced increases in locomotor activity. Studies examining the time courses of sensitivity and receptor changes have failed to resolve the relationship between changes in receptors and drug sensitivity. For example, in one study rats injected chronically with nicotine developed and lost tolerance to nicotine-induced prolactin release in concert with an elevation and return to normal of brain [3H]-acetylcholine binding (HulihanGiblin et al. 1990). However, studies from our laboratory have yielded different results. Following chronic nicotine injections, tolerance development to the effects of nicotine on locomotor activity and body temperature was significantly correlated with the increase in rat brain L-[3H]-nicotine binding (Collins et al. 1988). This tolerance to nicotine persisted well after receptor binding had returned to control levels. Thus, our results suggest that tolerance persists longer than receptor changes when nicotine is administered using a chronic injection protocol. In contrast, rats treated with nicotine using a chronic infusion method, develop increases in L-[3H]-nicotine as well as increases in ~-[12sI]-bungarotoxin binding, but tolerance to nicotine-included decreases in locomotor activity and body temperature was lost within 4 days following the cessation of chronic treatment, more closely paralleling the return to normal of receptor binding. These findings argue that tolerance to nicotine is regulated by the method of chronic drug treatment and involves more than receptor changes. Chronic injection procedures have also been used to establish whether tolerance to nicotine's effects develop in the mouse (Hatchell and Collins 1977). These early studies detected marked tolerance to nicotine but potential receptor changes were not measured. The goal of the present study was to ascertain whether tolerance development following chronic nicotine injections in the mouse correlates with changes in brain nicotinic cholinergic receptor binding. Because we have shown that altering corticosterone (CCS) levels has marked effects on nicotine sensitivity in the mouse (Pauly et al. 1988, 1990), plasma CCS levels were also monitored. The results obtained indicate that mice chronically injected with nicotine develop profound tolerance to nicotine even though the number and affinity of brain nicotinic receptors is unaffected by this treatment. Preliminary
evidence was obtained which suggests that this tolerance may involve a learning component and may be mediated by an elevation in plasma CCS levels.
Materials and methods Animals. Adult male mice (60-90 days of age) of the C57BL/6/ibg strain were used for these studies. This strain (originally obtained from Jackson Laboratories, Bar Harbor, ME) has been maintained in the breeding colony at the Institute for Behavioral Genetics for more than 20 generations. Mice were group housed (five per cage) under a 12 h light/12 h dark cycle with lights on at 0700 h and provided with pelleted food (Wayne Lab Blox) and water ad libitum. Chronic nicotine treatment. At the beginning of the experiment, animals were transferred from the colony room into a chronic treatment room. Each animal then received chronic treatment with nicotine (2.0 mg/kg) or saline (3 injections per day at 0800, 1300 and 1800 h) for a period of 12 days. (-)-Nicotine base (Sigma Chemical Co., St Louis MO), purified by distillation, was dissolved in physiological saline, neutralized with HCI and injected intraperitoneally in a volume of 0.01 ml/g body weight. The animal's weights were checked every 3 days (following the 0800 h injection) to detect possible changes during the course of chronic treatment. Plasma corticosterone determination. Plasma CCS levels were determined in 8-12 animals from each treatment group on the day prior to the initiation of treatment (day 0), and on days 4, 8 and 12 of chronic treatment. Plasma CCS was also measured in some animals 15 days following the cessation of chronic treatment. Blood samples were collected between the hours of 0700 and 0800 (just prior to the first injection of each day) by retro-orbital sinus puncture. The CCS radioimmunoassay described by Gwosdow-Cohen (1982) was utilized. All samples were processed at the completion of the experiment. Each animal was utilized only once for baseline CCS determinations. CCS antibody was obtained from Dr. G. Niswender (Dept of Physiology and Biophysics, Colorado State University, Fort Collins, CO). Behavioral testing. Following the 1800 h injection on the 12th treatment day, animals were moved from the treatment room to a behavioral testing room where they were tested for behavioral and physiological sensitivity to nicotine on the following day. All testing was performed between the hours of 0800 and 1300. Doses of nicotine (0 2.0 mg/kg) were altered by changing the concentration of nicotine in the injection solution. Nicotine sensitivity- was measured using a battery of tests that has been previously described (Marks et al. 1985 ; Pauly et al. 1988). Nicotine effects on locomotor activity were measured in a symmetrical Y-maze with each arm measuring 26.0 cm x 6.1 cm x 10.2 cm (L x W × H). Each arm was divided into two sections and crosses from one arm/section to another were counted. The number of rears (number of times the animal reared up onto its hind legs) were also counted during the 3 rain test. Y-maze activities were measured 4.5 min after nicotine administration. Heart rate was measured 8.5 rain post-injection using an E and M Physiograph (Narco Biosystems, Houston, TX). Fifteen minutes following nicotine injection, body temperature (rectal) was measured using a Baily Instruments digital thermometer. The time points chosen for these measures reflect the times of maximal nicotine effect based on previously determined time course analyses performed in our laboratory (Marks et al. 1985). For the test battery parameters, five to eight animals were tested at each nicotine dose for each experimental group. Tissue preparation. Sixteen animals from each treatment group were sacrificed by cervical dislocation approximately 2 4 h following behavioral testing. Their brains were removed, washed and
35
Data analysis. Receptor binding data were analyzed by one-way
then dissected into the following eight anatomical regions: cortex, cerebellum, striatum, hypothalamus, hippocampus, midbrain (mostly thalamus), colliculi (inferior and superior) and hindbrain (pons-medulla). Brain regions were placed in 10 volumes of ice-cold buffer (Krebs-Ringers HEPES: NaC1, 118mM; KC1, 4.8 raM; MgSO4, 1.2 mM; CaCL2, 2.5 mM; HEPES, 20 raM; pH adjusted to 7.5 with NaOH). Tissue preparation was essentially that of Romano and Goldstein (1980) as described by Marks et al. (1986c).
analysis of variance (by region). Test battery and CCS release data were analyzed by two-way analysis of variance with Newman-Kuels tests used as post hoc tests, t Tests were used for all other statistical comparisons.
Results
L-[3H]-nicotine binding. The binding of L-[3H]-nicotine (N-methyl-
Nicotine tolerance
[3H], specific activity, 60 Ci/mmol; New England Nuclear) was determined at 4° C using the method of Romano and Goldstein (1980) as modified by Marks and Collins (1982). L-[gH]-nicotine was purified chromatographically as described by Romm et al. (1990) to remove a contaminant that produces what appears to be a low affinity binding site. The final incubation volume was 250 p,1 and each sample contained 150-500 gg tissue protein. Ko and B~,,~values were determined in cortical tissue using six increasing concentrations of radiolabeled nicotine (0.57-18.11 nM) using linear regression analysis of the data transformed to fit the Scatchard equation. Nicotine binding was measured in each of the other brain regions using a single concentration of radiolabeled nicotine (5 nM).
Dose-response curves for the effects of nicotine on the four test battery parameters in chronic nicotine and saline-treated animals are presented in Fig. I (the data in this a n d all o t h e r figures r e p r e s e n t s the g r o u p mean_+ S E M ) . A n i m a l s t h a t received c h r o n i c n i c o t i n e injections were significantly less sensitive ( P < 0 . 0 0 1 ) to the a c u t e n i c o t i n e challenge for e a c h o f the tests. A N O V A results for e a c h o f the test b a t t e r y p a r a m e t e r s were as follows" Y m a z e crosses ( F 1 , 6 a - - 1 9 . 8 5 ) , Y m a z e r e a r s (F1,63 = 24.55), h e a r t r a t e ( F z , 6 3 = 1 9 . 4 3 ) , b o d y t e m p e r a t u r e (Fa,63 = 43.09). T w o weeks f o l l o w i n g c e s s a t i o n o f c h r o n ic n i c o t i n e t r e a t m e n t , a n i m a l s t h a t h a d received c h r o n i c n i c o t i n e injections were still t o l e r a n t to a c u t e n i c o t i n e challenge (Table 1).
c~-[12U]-bungarotoxin (BTX) binding. The binding of e-[azsI]-BTX (Tyr-[~zsI], initial specific activity, 219 Ci/mmol; Amersham) was performed at 37° C using the methods of Marks and Collins (1982). Assays using a single concentration of ligand were performed in each of the eight brain regions using 1.20 nM c~-[~zsI]-BTX. Scatchard analysis of tissue from cortex utilized six concentrations of labeled BTX (0.11-2.06 riM).
Nicotinic cholinergic receptor binding
Protein determination. Protein determinations were performed using the method of Lowry (1951) with bovine serum albumin as the standard.
T h e b i n d i n g o f L-[3H]-nicotine in eight b r a i n r e g i o n s in a n i m a l s t h a t received c h r o n i c n i c o t i n e o r saline t r e a t m e n t for 12 d a y s is p r e s e n t e d in Fig. 2. C h r o n i c n i c o t i n e injections d i d n o t alter L-[3H]-nicotine b i n d i n g in a n y b r a i n region. S c a t c h a r d analysis o f g - [ 3 H ] - n i c o t i n e b i n d ing in c o r t i c a l tissue y i e l d e d similar results. I n salinet r e a t e d a n i m a l s the Bmax for n i c o t i n e b i n d i n g w a s 32.23+_2.40 f m o l / m g p r o t e i n w i t h a KD o f 2.29___ 0.37 n M . I n a n i m a l s c h r o n i c a l l y t r e a t e d w i t h nicotine, similar values were o b t a i n e d (Bma ~ = 35.55 + 3 . 5 0 f m o l / m g p r o t e i n ; K D = l . 7 8 _ + 0 . 4 0 n M ) . T h e b i n d i n g o f c~[a25I]-BTX d e t e r m i n e d a t a single l i g a n d c o n c e n t r a t i o n in all o f the eight b r a i n r e g i o n s e x a m i n e d was also n o t
Nicotine-induced CCS release. Animals that were not used to measure receptor binding were tested for baseline (pre-injection), salineinduced and nicotine-induced CCS release at 0800 h on day 14 of the experiment (n= 8-12 per group). A group of control colony animals that received no prior treatment were also tested. Animals were injected with nicotine (1.0 mg/kg) or saline and blood samples were collected 25 min later. This time point was chosen based on a published time course for nicotine-induced CCS release in C57BL/6 animals (Freund et al. 1988), and provided maximal stimulation of CCS release. A test nicotine dose of 1.0 mg/kg was used to produce a sub-threshold adrenal response, thus avoiding any possible complication of a ceiling effect. Y MAZE CROSSES •
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Fig. 1. Chronic nicotine injections produce profound behavioral and physiological tolerance to acute nicotine challenge. Animals were injected three times a day with nicotine (2.0 mg/kg) or saline for 12 days. Animals were tested for nicotine sensitivity as described in the Methods section. Profound tolerance was present for each measure. ANOVA results for each of the test battery parameters were as follows: Y maze crosses (F1,63 = 19.85), Y maze rears (F1,63 =24.55), heart rate (FI,63 = 19.43), body temperature (F1,63 =43.09)
36 Table 1. The effects of nicotine on several behavioral and physiological measures 2 weeks following the cessation of either chronic nicotine or saline injections. Tolerance persisted in animals that had received chronic nicotine injections for each test battery parameter. Due to the limited number of animals (n=3-4 per g r o u p ) , only one test dose of nicotine was used (1.5 mg/kg). Data represent group means±SEM. * Value significantly different from saline-injected control (ANOVA, P
