James R. Pauly

An autoradiographic analysis of cholinergic receptors in mouse brain

Autoradiographic techniques were used to localize cholinergic receptors in the central nervous system of female DBA mice. Nicotinic receptors were identified using [3H]-L-nicotine and alpha-[125I]-bungarotoxin (BTX); [3H]-quinuclidinyl benzilate (QNB) was used to examine muscarinic receptor binding. There was little overlap between the regional distribution of binding sites for these ligands. Nicotine binding was highest in thalamic nuclei, the superior colliculus and the interpeduncular nucleus. For BTX binding, high density receptor populations were identified in the hippocampus, caudate putamen, colliculi (superior and inferior) and various nuclei in the hypothalamus and hindbrain. Muscarinic receptors were distributed more uniformly than nicotinic receptors; the colliculi, hippocampus and cerebral cortex had the highest level of QNB binding. Species differences between rats and mice in terms of cholinergic receptor binding are discussed.

Tolerance to nicotine following chronic treatment by injections: a potential role for corticosterone.

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 dose-response 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 ofl-[3H]-nicotine or α-[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. Nicotine-treated 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.

Chronic corticosterone administration modulates nicotine sensitivity and brain nicotinic receptor binding in c3h mice

Glucocorticoid regulation of nicotine sensitivity was investigated in adrenalectomized (ADX) and sham-operated C3H mice administered chronic corticosterone (CCS) replacement therapy. Hormone pellets (60% CCS or pure cholesterol) were implanted at the time of surgery and animals were tested for nicotine sensitivity in a battery of behavioral and physiological tests. ADX-induced increases in nicotine sensitivity were reversed by chronic CCS replacement. Sham-operated animals that received CCS supplementation were subsensitive to the effects of nicotine. In both ADX and sham-operated animals, chronic CCS administration induced a decrease in the number of CNS nicotinic cholinergic receptors labeled by alpha-[125I]-bungarotoxin. Binding was decreased by 30–60% depending on brain region; no changes in affinity (Kd) were detected. The number of brain nicotinic sites labeled by [3H]-nicotine was unaltered following 1 week of chronic CCS administration. These data support the hypothesis that glucocorticoids modulate nicotine sensitivity in the C3H mouse. In animals chronically treated with CCS, nicotine tolerance may be due to CCS-induced changes in nicotinic cholinergic receptor binding or the presence of high CCS titers at the time of testing.

Adrenocortical hormone regulation of nicotine sensitivity in mice

The possibility that nicotine-induced corticosterone (CCS) release regulates nicotine sensitivity was investigated in female mice of the C3H strain. Adrenalectomy (ADX) resulted in an increase in nicotine sensitivity as measured in a number of physiological and behavioral tests. In ADX animals, chronic CCS (100 micrograms/ml) administered in the drinking solution normalized nicotine sensitivity. Dexamethasone (DEX), a potent synthetic glucocorticoid which interacts with a distinct population of CNS steroid receptors, did not reverse the effects of ADX. Unoperated animals administered CCS (200 micrograms/ml) were protected from the effects of nicotine for several test battery parameters. ADX had no effect on the number of brain nicotinic cholinergic receptors and also did not alter nicotine metabolism. These data support the hypothesis that CCS secretion modulates nicotine sensitivity in the mouse; however, the mechanisms by which this regulation occurs are unknown.

An autoradiographic analysis of alterations in nicotinic cholinergic receptors following 1 week of corticosterone supplementation

Previous studies from our laboratory have established an interaction between central nervous system nicotinic cholinergic systems and glucocorticoid hormones. When mice are treated for 1 week with exogenous corticosterone (CORT) they become insensitive to the behavioral and physiological actions of nicotine and also have a reduction in brain alpha-bungarotoxin (BTX) receptor binding. In the present study, mice were treated with high stress levels of CORT and nicotinic receptor binding was measured using quantitative autoradiographic methods. L-[3H]-nicotine and alpha-[125I]-bungarotoxin were used to label both high- and low-affinity nicotinic sites. Chronic CORT administration reduced BTX binding in 77 of 115 (67.0%) brain regions examined. In general, forebrain regions were more sensitive to this regulation than were more posterior brain regions. Hippocampal and hypothalamic regions were particularly susceptible, with binding in treated animals being reduced by up to 80% in some nuclei. L-[3H]-nicotine sites were not as sensitive to CORT regulation as binding was significantly reduced in only 7 of the 83 (8.4%) regions measured. Regions affected were restricted to the thalamus and septum. The mechanism by which CORT reduces brain nicotinic cholinergic receptor binding is unknown and may or may not be dependent on CNS glucocorticoid receptors.

Chronic corticosterone administration enhances behavioral sensitization to amphetamine in mice

The role of corticosterone (CCS) in regulating sensitization to amphetamines locomotor activating effects was measured in female DBA/2 mice that had been sham-operated or adrenalectomized and implanted with CCS-containing or cholesterol pellets. Three days following surgery, the mice were injected with saline and circular open field locomotor activity was measured for a 5-min time period starting 15 min after injection. Over the next 4 days, amphetamine (1.0-10.0 mg/kg) was injected and locomotor response measured. Control animals (sham-operated, cholesterol pellet) showed increased locomotor activity following their first injection of 5.0 mg/kg and 10.0 mg/kg amphetamine, while ADX animals showed increased activity only after treatment with the 10 mg/kg dose. Chronic CCS treatment did not significantly alter initial responsiveness to amphetamine in either sham-operated or ADX animals, but it did alter the dose-dependent sensitization to amphetamine. Both sham-operated and ADX animals implanted with cholesterol pellets showed increased locomotor response to amphetamine (sensitization) following injection with 2.5, 5.0 and 10.0 mg/kg doses of amphetamine. However, the enhancement of locomotor activity was greater in the sham-operated control animals. CCS-treated sham-operated animals exhibited sensitization to the locomotor-activating effects of amphetamine at the lowest dose used (1.0 mg/kg) and increased stereotype following treatment with the higher doses. ADX/CCS animals developed sensitization to the locomotor-activating effects of amphetamine following chronic injection with the 2.5 mg/kg dose, and showed sensitization to amphetamine-induced stereotypy at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain differences in adrenalectomy-induced alterations in nicotine sensitivity in the mouse

Adult mice of four inbred strains (A, BUB, C57BL, DBA) and two selectively bred lines [Long-Sleep (LS) and Short-Sleep (SS)] were tested for differences in glucocorticoid regulation of nicotine sensitivity. One week following adrenalectomy (ADX), animals were tested for nicotine sensitivity in a battery of tests that included acoustic startle response, Y-maze activity (line crosses and rearings), heart rate and body temperature. Although each type of animal tested had increased nicotine sensitivity in at least one of the test battery measurements, there was clear evidence for a genetic influence on the scope of ADX-induced changes in sensitivity. LS animals had the largest increase in sensitivity with altered responses in four of five tests following ADX. The sensitivity of DBA animals was increased in two tests while for A, BUB, C57BL and SS animals, only one test was affected. ADX-induced alterations in nicotine sensitivity could not be explained on the basis of changes in nicotinic receptor number since changes were consistent across strains. The mechanism by which ADX causes increased nicotine sensitivity is not known. However, these data support the hypothesis that nicotine sensitivity is modulated by adrenal glucocorticoid secretion and also suggest that this phenomenon is under strict genetic control.

Corticosterone reversibly alters brain α-bungarotoxin binding and nicotine sensitivity

Previous studies have shown that chronic corticosterone (CCS) treatment via subcutaneous pellets elicits reduced sensitivity to many actions of nicotine in mice as well as decreased brain alpha-bungarotoxin (alpha-BTX) binding. We report here the time courses of altered sensitivity to nicotine, as measured by acoustic startle, Y-maze crossing and rearing activities, heart rate, and body temperature, and alpha-BTX binding during and after CCS treatment. CCS treatment resulted in rapid decreases in sensitivity to nicotine for four of the five responses that were measured, as well as rapid changes in alpha-BTX binding. Sensitivity to nicotine returned to control levels within 3 days following pellet removal, but alpha-BTX binding returned to control levels in most brain regions 9-11 days after pellet removal. Because the restoration of control sensitivity to nicotine occurred long before alpha-BTX binding returned to control levels, it seems likely that factors other than changes in alpha-BTX binding cause chronic CCS-induced changes in sensitivity to nicotine.

Adrenalectomy reverses chronic injection-induced tolerance to nicotine

A recent study from our laboratory has demonstrated that C57BL/6 male mice that are chronically injected with nicotine develop a profound tolerance to nicotine that is not associated with changes in brain nicotinic receptors. We have proposed that alterations in the secretion of corticosterone (CCS) may regulate tolerance development in chronically injected animals. In the present study we have directly tested this hypothesis. Female DBA/2 mice were injected three times each day for 12 days with saline or 2 mg/kg nicotine. Blood samples were collected at various time points during the course of treatment and plasma CCS levels were determined. The animals were divided into two groups following the last injection on day 12. The first group of animals was tested for nicotine-induced release of corticosterone on day 13 of the experiment and then sacrificed. The brains of these animals were subsequently used to measure nicotinic receptor binding. The second group of animals was adrenalectomized (ADX) or sham-operated on day 13 of the experiment and tested for nicotine sensitivity on day 14 of the experiment. Plasma CCS levels were significantly elevated in animals that were chronically injected with nicotine (versus saline controls) by the fourth day of the experiment. Chronic nicotine-injected animals were tolerant to nicotine-induced CCS release. Animals that were chronically injected with nicotine and sham-operated were tolerant to acute nicotine challenge; however, tolerance to nicotine was not detected in ADX animals. These data support the hypothesis that the capacity to release CCS may underscore the expression of tolerance to nicotine in chronically injected animals.

An analysis of response to nicotine infusion using an automated radiotelemetry system

Previous studies from our laboratory have demonstrated that chronic nicotine infusion evokes tolerance to nicotine injected IP several hours after withdrawal from chronic infusion. This method may introduce problems related to withdrawal reactions and to stress associated with handling of the animals. The studies reported here measured tolerance to nicotine in mice using an automated radiotelemetry system. DBA/2 mice were infused intravenously with saline for 4 days followed by infusion of a 4 mg/kg per h dose of nicotine for 7 days. After the nicotine treatment, the mice were infused with saline for 7 days. The nicotine was infused continuously or in four 1 mg/kg pulses, two 2 mg/kg pulses or one 4 mg/kg pulse each hour. Home cage activity and body temperature were measured throughout the treatment periods using a radiotelemetry system. Nicotine infusion produced an abrupt decrease in body temperature and activity, but this effect was totally reversed within 12 h in the continuously infused and four infusions/h treatment groups. Mice that received one or two infusions/h also showed a rapid response to nicotine that was reversed as treatment proceeded, but nicotine continued to produce a measurable effect for several days. After nicotine withdrawal, temperature and activity returned to predrug infusion values in all of the groups except those infused once per hour. This group showed depressed activity for a minimum of 3 days after nicotine treatment stopped. Thus, the kinetics of nicotine administration affected the intensity of response during continued treatment as well as activity after cessation of chronic treatment.