Psychopharmacology
Psychopharmacology 63, 7 11 (!979)
~ ' by Springer-Verlag 1979
Effects of Nicotine Administration and Its Withdrawal on Plasma Corticosterone and Brain 5-Hydroxyindales Maureen E. M. Benwell and D. J. K. Balfour Department of Pharmacology and Therapeutics, Dundee University Medical School, Ninewells Hospital, Dundee, Scotland
Abstractso The effects of nicotine administration and its withdrawal on the levels of brain hydroxyindoles and plasma corticosterone have been studied in the rat. Daily injections of nicotine (0.4mg/kg s.c.) rapidly induced tolerance to the increase in plasma corticosterone seen in response to acute nicotine. Withdrawal of the drug from chronically treated animals caused a significant increase in plasma corticosterone. Hippocampal 5-hydroxytryptamine (5-HT) was reduced in nicotine-treated rats, significantly so in those treated for more than 20 days. The 5-hydroxyindole acetic acid (5-HIAA) concentration in the hippocampus was also reduced by nicotine although this was not a consistent observation. Hypothalamic 5-hydroxyindoles were not affected by nicotine administration itself, but, if tee drug was withdrawn, the concentration of 5-HT was increased after 5 days treatment. The changes in the hypothalamus and hippocampus appeared to be reIativeiy specific since they differed from those seen in the rest of the brain. None of the effects could be related direct!y to changes in the plasma corticosterone concentration.
Key words: Nicotine withdrawal - Plasma corticosterone - 5-Hydroxytryptamine - Hypothalamus Hippocampus
concentration of the hippocampus (Balfour et al., ~975), which contains many gIucocorticoid receptors (McEwan, 1973), and has been implicated in the control of adrenocorticotrophic hormone (ACTH) and glucocorticoid secretion (Scapagnini and Preziosi, !972). Recent behavioural studies (Balfour and Morrison, t975) have suggested that performance in a stressfui shock avoidance schedule may be related to pituitaryadrenocortical activity and to the concentration of 5HT in the hippocampus. Rats trained to perform such a schedule under the influence of nicotine not only perform it better than control animals, but also become dependent upon nicotine for the continued successful performance of the task (Hall and Morrison, 1973; Morrison, 1975a). In this behavioural situation, however, no changes in hippocampal 5-HT could be attributed directly to nicotine administration or its withdrawal (Balfour and Morrison, 1975) although dearly, any effects cou!d well have been masked by changes due to the behavioural task itself. Therefore in the study now reported, the effects of chronic nicotine administration and its withdrawal in unstressed behaviourally naive rats have been investigated in order to try to establish more clearly the possible roles of pituitary-adrenocorficN and brain 5-HT systems in the mechanisms involved in nicotine dependence.
Materials and Methods Although the behavioural effects of chronic nicotine administration have been the subject of a number of reports (Morrison, 1974a, b; Morrison and Stephenson, 1972; Stolerman et ai., 1973), little appears to be known of any biochemical changes which may occur in the brains of chronically treated animals. The acute administration of nicotine to unstressed rats causes an increase in plasma corticosterone (Balfour et al., 1975; Turner, 1975). There are also relatively specific changes in the 5-hydroxytryptamine (5-HT)
Male Sprague-Dawleyrats (Charies River), weighing approximately 150g at the beginning of the experiment, were used. The animals, which were housed in pairs, were given free access to food and water and were exposed to light from 8 a.m. to 8 p.m. daily. In each experiment, six rats were treated with nicotine (four rats) or saline (two rats) for periods up to 40 days, each rat receiving one injection of nicotine (0.4 mg/kg) or saline on the five working days of each week. On the last day of the experiment, two of the nicotinetreated rats received their usual nicotine injection while in the remaining two the nicotine was withdrawn and replaced by saline. The controls received saline throughout. These injection schedules are the same as those described by Morrison (I975a) in bebavioural
0033-3158/79/0063/0007/$01.00
8
Psychopharmacology 63 (1979)
experiments used to demonstrate nicotine dependence. In order to reduce any stressful effects of a novel injection on the last day of the experiment, any rats which were to receive less than five injections as part of the treatment schedule were given five additional daily injections of saline prior to the beginning of the schedule (Balfour et al., 1975). The experiments completed on day 40 were repeated twice (n = six rats for each complete treatment) whereas the shorter experiments (lasting 2, 5 or 20 days) were repeated once (n = four rats for each complete treatment). The nicotine, in the form of its hydrogen tartrate (British Drug Houses), was dissolved in saline, adjusted to pH 7 by the addition of a small quantity of NaOH and injected subcutaneously (0.1 ml/100 g), the dose being expressed as free base (Balfour et al., 1975; Morrison, 1975a). On the last day of the treatment schedule the rats were killed by decapitation 30 rain after their last injection. The brains were quickIy removed, chilled and dissected over ice, using the procedure of Glowinski and Iversen (1966) to separate the hypothalamus and hippocampus from the remainder of the brain which was pooled together (residual brain). Blood samples were also taken from the trunk, placed in chilled heparinised tubes, centrifuged and the plasma collected for the measurement of plasma corticosterone. All the animals were killed between 9 a.m. and 12 noon, when the diurnal variation in plasma corticosterone is minimal (Hodges, 1970). Brain 5-HT and 5-hydroxyindole acetic acid (5-HIAA) levels were measured by the method of Curzon and Green (1970). Plasma corticosterone was measured using the method of Mattingly (1962) adapted for small plasma volumes. Statistical comparisons were made using the Student's t-test (Snedecor, 1956).
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0.~ I Lr3
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5
40
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Psychopharmacology 63, 7 11 (!979)
~ ' by Springer-Verlag 1979
Effects of Nicotine Administration and Its Withdrawal on Plasma Corticosterone and Brain 5-Hydroxyindales Maureen E. M. Benwell and D. J. K. Balfour Department of Pharmacology and Therapeutics, Dundee University Medical School, Ninewells Hospital, Dundee, Scotland
Abstractso The effects of nicotine administration and its withdrawal on the levels of brain hydroxyindoles and plasma corticosterone have been studied in the rat. Daily injections of nicotine (0.4mg/kg s.c.) rapidly induced tolerance to the increase in plasma corticosterone seen in response to acute nicotine. Withdrawal of the drug from chronically treated animals caused a significant increase in plasma corticosterone. Hippocampal 5-hydroxytryptamine (5-HT) was reduced in nicotine-treated rats, significantly so in those treated for more than 20 days. The 5-hydroxyindole acetic acid (5-HIAA) concentration in the hippocampus was also reduced by nicotine although this was not a consistent observation. Hypothalamic 5-hydroxyindoles were not affected by nicotine administration itself, but, if tee drug was withdrawn, the concentration of 5-HT was increased after 5 days treatment. The changes in the hypothalamus and hippocampus appeared to be reIativeiy specific since they differed from those seen in the rest of the brain. None of the effects could be related direct!y to changes in the plasma corticosterone concentration.
Key words: Nicotine withdrawal - Plasma corticosterone - 5-Hydroxytryptamine - Hypothalamus Hippocampus
concentration of the hippocampus (Balfour et al., ~975), which contains many gIucocorticoid receptors (McEwan, 1973), and has been implicated in the control of adrenocorticotrophic hormone (ACTH) and glucocorticoid secretion (Scapagnini and Preziosi, !972). Recent behavioural studies (Balfour and Morrison, t975) have suggested that performance in a stressfui shock avoidance schedule may be related to pituitaryadrenocortical activity and to the concentration of 5HT in the hippocampus. Rats trained to perform such a schedule under the influence of nicotine not only perform it better than control animals, but also become dependent upon nicotine for the continued successful performance of the task (Hall and Morrison, 1973; Morrison, 1975a). In this behavioural situation, however, no changes in hippocampal 5-HT could be attributed directly to nicotine administration or its withdrawal (Balfour and Morrison, 1975) although dearly, any effects cou!d well have been masked by changes due to the behavioural task itself. Therefore in the study now reported, the effects of chronic nicotine administration and its withdrawal in unstressed behaviourally naive rats have been investigated in order to try to establish more clearly the possible roles of pituitary-adrenocorficN and brain 5-HT systems in the mechanisms involved in nicotine dependence.
Materials and Methods Although the behavioural effects of chronic nicotine administration have been the subject of a number of reports (Morrison, 1974a, b; Morrison and Stephenson, 1972; Stolerman et ai., 1973), little appears to be known of any biochemical changes which may occur in the brains of chronically treated animals. The acute administration of nicotine to unstressed rats causes an increase in plasma corticosterone (Balfour et al., 1975; Turner, 1975). There are also relatively specific changes in the 5-hydroxytryptamine (5-HT)
Male Sprague-Dawleyrats (Charies River), weighing approximately 150g at the beginning of the experiment, were used. The animals, which were housed in pairs, were given free access to food and water and were exposed to light from 8 a.m. to 8 p.m. daily. In each experiment, six rats were treated with nicotine (four rats) or saline (two rats) for periods up to 40 days, each rat receiving one injection of nicotine (0.4 mg/kg) or saline on the five working days of each week. On the last day of the experiment, two of the nicotinetreated rats received their usual nicotine injection while in the remaining two the nicotine was withdrawn and replaced by saline. The controls received saline throughout. These injection schedules are the same as those described by Morrison (I975a) in bebavioural
0033-3158/79/0063/0007/$01.00
8
Psychopharmacology 63 (1979)
experiments used to demonstrate nicotine dependence. In order to reduce any stressful effects of a novel injection on the last day of the experiment, any rats which were to receive less than five injections as part of the treatment schedule were given five additional daily injections of saline prior to the beginning of the schedule (Balfour et al., 1975). The experiments completed on day 40 were repeated twice (n = six rats for each complete treatment) whereas the shorter experiments (lasting 2, 5 or 20 days) were repeated once (n = four rats for each complete treatment). The nicotine, in the form of its hydrogen tartrate (British Drug Houses), was dissolved in saline, adjusted to pH 7 by the addition of a small quantity of NaOH and injected subcutaneously (0.1 ml/100 g), the dose being expressed as free base (Balfour et al., 1975; Morrison, 1975a). On the last day of the treatment schedule the rats were killed by decapitation 30 rain after their last injection. The brains were quickIy removed, chilled and dissected over ice, using the procedure of Glowinski and Iversen (1966) to separate the hypothalamus and hippocampus from the remainder of the brain which was pooled together (residual brain). Blood samples were also taken from the trunk, placed in chilled heparinised tubes, centrifuged and the plasma collected for the measurement of plasma corticosterone. All the animals were killed between 9 a.m. and 12 noon, when the diurnal variation in plasma corticosterone is minimal (Hodges, 1970). Brain 5-HT and 5-hydroxyindole acetic acid (5-HIAA) levels were measured by the method of Curzon and Green (1970). Plasma corticosterone was measured using the method of Mattingly (1962) adapted for small plasma volumes. Statistical comparisons were made using the Student's t-test (Snedecor, 1956).
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40
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