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mmmmmmm mmm mmm mmm =Mftm t i Modulation of nicotine receptors by chronic exposure to nicotinic agonists and antagonists Modulation of nicotine receptors 69 of tolerance to nicotine in humans, but tolerance to nicotine has been studied in animals. For example, an actrte injection of nicotine elicits depressed locomotor activity in the rat (Stolerman et al 1973, Clarke & Kumar 1983) and mouse (Hatchell & Collins 1977), but this effect decreases with chronic treatment. Nicotine tolerance and receptor up-regulation Although it is well established that chronic nicotine treatment results in tolerance to the drug, only minimal information is available concerning potential explanations for this tolerance. However, a number of studies have indicated that chronic treatment with nicotine elicits an increase in the number of brain nicotinic receptors. a I Allan C. Collins, Ratan V. Bhat, James R. Pauly and Michael J. Marks Institute for Behavioral Genetics, School of Pharmacy, and Department of Psychology, University of Colorado. Campus Box 447, Boulder, Colorado 80309, USA Abstract. Although numerous studies have demonstrated that chronic nicotine treatment often resufls in tolerance to this drug, the mechanisrns that underlie this tolerance are not well defined. Recent evidence suggests that chronic nicotine treatment results in an up-regutation of brain nicotinic receptors, but the majority of these receptors may be descnsitized or inactivated, thereby explaining tolerance. lhere is evidence that while all mouse strains show increased receptor numbers following chronic nicotine treatment, sonic mouse strains develop maximal changes in [!H ] nicotine binding before any tolerance is detected. Other strains show a high correlation between increase in receptor number and tolerance. Studies with several other nicotinic agonists indicate that up-regulation of nicotinic receptors can occur without changes in drug sensitivity. Similarly, chronic antagonist treatment can also elicit changes in receptors without affecting sensitivity to nicotine. Some of these discrepancies may be due to genetically influenced interactions between the adrenal steroid, corticosterone (CCS), and the nicotinic receptors. The addition of CCS in vitro inhibits binding to nicotinic receptors, and chronic CCS treatment results in decreases in the nurnber of brain nicotinic receptors measured by [12511bungarotoxin binding. Either of these biochemical measures may explain why altering CCS concentrations in vivo results in altered sensitivity to nicotine. It may be that both changes in the number of receptors and altered steroid interactions with the nicotinic receptors explain tolerance to nicotine. 1990 The biology of nicotine dependence. fViley, Chichester (Ciba Foundation Syrnposirnn 152) p 68-86 Chronic drug treatment often results in alterations in the intensity of response to the drug; either reduced (tolerance) or enhanced (supersensitivity) response may be seen. The development of tolerance to nicotine's noxious effects might play a critical role in facilitating the continued use of tobacco. The individuals who develop tolerance to nicotine's noxious effects may be those who progress from experimentation to chronic use. We know very little about the development Studies with the ral Chronic nicotine injection, once or twice daily for seven days, results in an increase in the number of rat brain [ 3H ] acetylcholine (ACh) binding sites (Schwartz & Kellar 1983, 1985). This increase in binding coincides with development of nicotine-induced increases in locomotor activity (Ksir et al 1985, 1987). We examined the time course of tolerance development and loss in Sprague-Dawley rats using a chronic injection procedure (1.6 mg/kg twice daily) (Collins et al 1988). We attempted to correlate alterations in sensitivity to nicotine with alterations in (-)[ 3" ] nicotine binding (note that [3H ] ACh and [ 3H ] nicotine bind to the same site in rat and mouse brain [Marks et al 1986c, Martino-Barrows & Kellar 1987]). Tolerance to nicotine's depressant effects on locomotor activity and body temperature paralleled increases in [3H]- nicotine binding, but tolerance lasted more than the seven days required for nicotine binding to return to control levels in five of the six brain regions. These results suggest that increases in [3H ] ACh/ [3H] nicotine binding may underlie the development of tolerance to nicotine's depressant effects in the rat, but the retention of tolerance may involve factors other than receptor changes. Studies with the DBA/2 inbred ntouse strain Although we have detected the development of tolerance to nicotine in the mouse using chronic injection techniques (Hatchell & Collins 1977), we have been unable to elicit changes in receptor numbers by this means (unpublished data). Consequently, we have chosen to treat mice with nicotine using chronic infusion methodologies. The technique consists of implanting a cannula in the animal's jugular vein, as first described by Barr et al (1979). Chronic nicotine infusion is normally continued for 7-10 days. Two hours after infusion is stopped the animals are injected with either saline or a dose of nicotine ranging from 0.5-2.5 mg/kg; the effects of these injections on a battery of behavioural and 68

.. rs r..r .. r.. .r .. M.. .. a. r= rr .. .. = r. 4.• a I I I I 70 Collins et at physiological measures are determined between 1-15 minutes after injection. The binding of [-1H ] nicotine and 112511 bungarotoxin in membranes prepared from 6-8 brain regions is measured using methods described previously (Marks & Collins 1982). In our first study (Marks et al 1983), we reported that chronic infusion of DBA/2 mice with 0.2, 1.0 or 5.0 mg/kg/h nicotine resulted in tolerance to nicotine's effects on rotarod performance, heart rate and body temperature and in increases in [31i]nicotine binding in nearly all brain regions; significant increases in binding were elicited in some brain regions with the 0.2 mg/kg/h nicotine dose. Increases in 11211 bungarotoxin binding were also observed, but these were restricted to the hippocampus and midbrain and were detected only in animals that had been treated with the 5 mg/kg/h dose. These changes reflected changes in maximal binding (B, ,,), since Kt) values were not altered by chronic nicotine infusion. Thus, chronic nicotine treatment results in an increase in ['H ] nicotine binding at lower doses and in [ 1251 ] bungarotoxin binding at higher doses. Subsequent studies used nicotine infusion doses of 8 mg/kg/h (Marks et al 1985), 3 mg/kg/h (Marks & Collins 1985, Marks et al 1986a), and 2, 4 and 6 mg/kg/h (Marks et al 1986b). These experiments also demonstrated that chronic nicotine Ireatment resulted in tolerance to several actions of nicotine and that increases in [3H I nicotine and 112511 bungarotoxin binding also occurred. Tolerance increased along with infusion dose such that at the highest infusion doses EDsn-like values were twice those obtained in control animals. ['H ] Nicotine binding increased in nearly every brain region studied; maximal changes (50-100Q/o increases in B,„,,„ values) were elicited by the 2 tng/kg/h nicotine dose. [ 1251 ] Bungarotoxin binding increased in only some brain regions (cortex, midbrain and hippocampus), but these effects were relatively small (20-30010) and were detected only at nicotine doses that exceeded 2 mg/kg/h. The time courses of tolerance acquisition and loss have also been examined. In the first of these studies (Marks et al 1985), DBA/2 mice were chronically infused with nicotine (4 mg/kg/h) for 1-12 days. The development and loss of tolerance were measured, as were changes in the binding of [3H ]- nicotine and [ 125I ] bungarotoxin in six brain regions. The development and loss of tolerance to nicotine's effects on Y-maze activities, body temperature and heart rate paralleled the up-regulation and return to control, respec- tively, of [3H]nicotine binding. Significant increases in the binding of [ r2SI ]bungarotoxin were also observed in cortex and hippocampus, but these changes were achieved within two days and did not parallel toler- ance development. In a subsequent study (Miner & Collins 1988), we deter- ntined that acquisition of tolerance to nicotine's seizure-inducing effects closely paralleled the up-regulation of [1251]bungarotoxin binding, but the latter returned to control levels before tolerance to nicotine-induced seizures was lost. i Modulation of nicotine receptors 7~ Genetics of tolerance and receptor up-regulation The studies of nicotine tolerance, described to this point, used the DBA/2 straio. The results obtained with this strain suggested that receptor up-regulation maY play an important role in regulating the development of tolerance to nicotine. However, studies with several other mouse strains (Marks et al 1986a,b, Collins & Marks 1989) have demonstrated that while all mouse strains show increases in [1H I nicotine and [ 12511 bungarotoxin binding after chronic infusion of nicotine, tolerance does not always parallel receptor changes. Some strains, such as the C57BL/6 and DBA/2, develop measurable tolerance to nicotine infused in very low doses, whereas other strains, such as the C3H, fail to develoh measurable tolerance until the nicotine infusion rates exceed 2 ing/kg/h. C3H mice are not tolerant to nicotine at chronic infusion doses that have elicited maximal, or near maximal, changes in [3H j nicotine binding. These results indicate that changes in receptor numbers are not necessarily the cause of tolerance to nicotine. Mechanisms of receptor up-regulation The observation that chronic treatment with the nicotinic agonist, nicotine, results in an increase in nicotinic receptors was unexpected, because many studies have demonstrated that agonist treatment of receptors for other drugs generally results in decreases in receptors, whereas chronic antagonist treatment generally elicits receptor up-regulation. With this in mind, we (Marks et al 1983) and Schwartz & Kellar (1983) argued that nicotinic receptor up-regulation may occur because nicotine treatment results in a short-lived receptor activation followed by a longer-term desensitization. Chronic nicotine administration may cause prolonged desensitization, or even inactivation, of the nicotinic receptors which may result in either an increase in the rate of receptor synthesis or a decrease in the rate of receptor catabolism. Although there seems to be an increase in the absolute number of receptors, it is possible that there is a decrease in the number of activatable receptors, thereby explaining development of tolerance. Cltronic antagonist treatment The desensitization model predicts that chronic treatment with a nicotinic antagonist, such as mecamylamine, should also result in receptor up-regulation. However, Schwartz & Kellar (1985) failed to observe an effect of chronic mecamylamine or dihydro-(3-erythroidine injections on nicotinic receptors. Mecamylamine also did not block the ttp-regulation elicited by chronic nicotine injection. Therefore, Schwartz & Kellar (1985) suggested that agonist activity was required to up-regulate the receptor. .ZVE66E9V09

.. .. r r.r r.. r.. .. .. r... r. r. r.r. rrr ~ r wr. M.. W 72 120 oE ~ 100 ! 80 60 I o 40 v z 20 Cx BRAIN REGION HC Collins et al FIG. I. Effects of chronic nicotine and mecamylamine infusion on ['H]nicotine binding. C57BL/6 mice were infused with saline, nicotine, mecamylamine, or nicotine plus mecamylamine for l0 days. Binding was measured in cortex (CX) and hippocampus (HC) 48 hours after infusion was stopped. Each bar represents the mean ± SEM of six animals. •, P<0.05; •', P<0.01 when compared to control binding. Because chronic antagonist injections do not readily allow the continuous blockade of receptors, we infused C57BL/6 mice chronically with saline (controls), mecamylamine (3 mg/kg/h), nicotine (3 mg/kg/h), or nicotine plus mecamylamine for 10 days. The mice were challenged with saline or I mg/kg nicotine 48 hours after chronic infusion had been stopped; this time was chosen to allow complete elimination of the ntecamylarnine. Animals infused chronically with nicotine were tolerant to the drug, but none of the other treatment groups showed an altered response to it. Thus, chronic mecamylamine infusion did not elicit an altered response to nicotine, but when co-infused with nicotine it blocked the expected tolerance to nicotine. As noted in Fig. l, chronic nicotine infusion resulted in increases in [ 3H J nicotine binding in both the cortex and the hippocampus. However, unlike the results reported by Schwartz & Kellar (1985), chronic tnecamylamine infusion also resulted in increases in [3HJnicotine binding. When the two drugs were infused together, an even greater increase in binding was seen. Scatchard analyses indicated that these treatments resulted in changes in the 8,,,,,, for [ 3111 nicotine binding; Kl) values were unchanged. These results clearly demonstrate that nicotinic receptor antagonists can elicit increases in [ aH ] nicotine binding, but receptor changes need not alter the sensitivity (tolerance) to nicotine. The mecamylamine-nicotine co-infusion yielded a surprising result. The nicotine dose that was used in these studies (3 mg/kg/h) is supramaximal; further increases in nicotine dose do not normally.elicit further increases in binding. However, the addition of inecamylamine resulted in an additive increase in Modulation of nicotine receptors 73 80 20 I I -Sal =-DFP I I . I. I . . I z .I. Cx Cb M H P S T Cot BRAIN REGIONS FIG. 2. Effects of chronic vehicle or diisopropyl fluorophosphate (DFP) injections on ['Hlnicotine binding. C57BL/6 mice were injected every other day with saline or 2 mg/kg DFP for 21 days. Each bar represents mean ± SEM for eight animals. The brain regions studied were cortex (Cx), cerebellum (Cb), midbrain (M), hindbrain (H), hippocampus (P), striatum (S), hypothalamus (T) and colliculi (Col). [3H]nicotine binding. This finding may mean that mecamylamine and nicotine affect different receptor populations. It should also be noted that co-treatment with tnecamytamine blocked the development of tolerance to nicotine. This result also suggests that increases in binding do not necessarily result in tolerance to nicotine. Effects of acetylcholinesterase inhibitors on nicotinic receptors If receptor desensitization is a prerequisite for receptor up-regulation, agonists that vary in affinity for the 13H] nicotine binding site(s) should vary in their relative ability to elicit receptor up-regulation. Chronic treatment with agonists such as ACh, which has very low affinity for the receptor, might be relatively ineffective in eliciting receptor changes, whereas high affinity agonists, such as cytisine, might be very potent in this regard. Schwartz & Kellar (1985) explored this possibility by examining the effects of chronic in jection with the irreversible acetylcholinesterase (AChE) inhibitor, diisopropyl fluorophosphate (DFP), on brain [3H]ACh binding. Chronic DFP treatment induced a decrease in [3HJACh binding. Costa & Murphy (1983) have reported a similar change in UE66EM9

rr r. .. .. ... .. "r r.. s rr. .. mr. rrr r.. rr r. E ~ a 0 I I E 0 z ~ 0 I 74 120 F-1 - Sol = - Phy 90 m 60 U Z 30 I IIIII I * R I * I Collins et al * * Cx Cb M H P S T Col BRAIN REGIONS FIG. 3. Effects of chronic physostigtnine infusion on brain ('HJnicotine binding. C5713E./6 mice were infused continuously with physostigmine (0.1 mg/kg/h) for 10 days. Nicotine binding was measured two hours aftcr infusion was stopped. Each bar rcpresenrs the mcan ±SEM of six animals. The brain regions are designated as for Fig. 2. •,P<0.05 when compared to control. rat brain [41j nicotine binding after chronic disulfoton treatment. These findings might mean that low affinity agonists do not elicit receptor desensi- tization and that the neuron responds to excess stimulation by ACh by decreasing receptor synthesis. In contrast, a recent report by De Sarno & Giacobini (1989) indicated that chronic intracerebroventricular injection of the reversible AChE inhibitor, physostigmine, resulted in an increase in [ 3H ]nicotine binding in rat brain. This result is puzzling, since both DFP and physostigmine should increase synaptic ACh levels. In an attempt to resolve this controversy, we treated C57BL/6 mice chronically with DFP and physostigrnine. Chronic DFP treatment was achieved by injecting the animals with a 2 mg/kg dose of DFP every other day for 21 days, whereas the animals were treated chronically with physostigmine by continuous i.v. infusion (0.1 mg/kg/h) for 10 days. These treatment protocols resulted in comparable inhibition of AChE activity (DFP, 44-66% in eight brain regions; physostigmine, 65% in whole brain). However, different effects on ['Hj- nicotine binding were obtained. Fig. 2 demonstrates that chronic DFP treatment did not alter ['H J nicotine binding in any of (he eight brain regions studied, but this treatment did elicit a reduction in muscarinic receptors (data not shown). Modulation oi nicotine receptors /5 TABLE I Effects of chronic drug treatment on ['11 ] nicotine binding Chronic infusion Cortex Midbrain Hindbrain Saline 29.411.9 82.9± 1.8 41.0± 1.4 Nicotine 47.2±2.9" 106.4±3.4" 57.8±2.0' Anabasine 42.8±2.6" 105.7±4.6' 56.9±3.I` Lobeline 33.3 + 1.8 84.7 ± 2.7 42.6 ± 6.1 Each value represents mean +_ the standard error nicotine binding (fmoles/mg protein), n = 8-12. Animals were Infused with saline (controls) or the indicated drug (18.5 Nmoies/kg/h) for 10 days. 'P<0.0I when compared to the saline-infused control value. In contrast, chronic physostigmine treatment resulted in an increase in [3H]nicotine binding in four of these brain regions (Fig. 3). Because we have failed to detect chronic DFP-induced changes in [3HJnicotine binding using several other treatment protocols and two other inbred mouse strains, we are at a loss to explain our inability to reproduce the results obtained in rats. Nonethe- less, the observation that chronic physostigmine treatment results in receptor up- regulation argues that ACh can, at high levels, produce receptor desensitization. Effects of other cholinergic agonists Schwartz & Kellar (1985) have reported that chronic injection of rats with the high affinity nicotinic agonist, cytisine, results in increases in [3H)ACh binding. Cytisine has greater affinity for the nicotine/ACh binding site than does nicotine (Marks & Collins 1982). In an attempt to determine whether chronic administration of agonists with lower affinity for the receptor also elicits up-regulation, we examined the effects of chronic treatment with the nicotinic agonists, lobeline and anabasine. These compounds effectively compete with nicotine for its binding site (lobeline ICSO=0.075 NM; anabasine IC50=0.35 pM). Table I presents the effects of chronic i.v. infusion (10 days) with equimolar doses (18.5 pM) of nicotine, lobeline and anabasine on [3H] nicotine binding in three brain regions. Consistent with our previously reported results, nicotine infusion resulted in significant increases in binding in all three brain regions. Similarly, anabasine elicited increases in binding in all of the regions, but lobeline was without effect in any of the regions. These results suggest that no clear relationship between affinity for the nicotine binding site and agonist-induced changes in the receptor exists; if affinity predicted ability to up-regulate receptors, lobeline should have been more effective than anabasine. The chronic infusion of anabasine elicited increases in [ 3H ] nicotine binding, but this increase in receptors was not sufficient to change the response to either anabasine or nicotine. This finding also argues that tolerance to nicotine involves more than changes in receptor numbers. VVE66E9V09

MM M M M M =M~Mmmmm mmmm on , ` 1 I I 76 50 40 30 20 10 Collins et al 0 0.5 1 3 Dose of Nicotine infused (mg/kg/hr) FIG. 4. Effects of chronic nicotine infusion on the fast and slow phases of nicotine binding. C57BL/6 mice were infused with saline or nicotine for 10 days. Fast binding (Bf) and slow binding (Bs) were estimated from association curves. Each point represents the mean ± SEM of three determinations. Kinetic analysis of receptor desensitization Lippiello et al (1987) reported that although nicotine appears to bind to a single class of sites, the association kinetics are biphasic. An initial rapid binding process and a slower binding process were detected. The rate and proportion of binding attributable to the slower process were dependent on the nicotine concentration; as the concentration of nicotine added to the incubations was increased, the rate of the slower binding process increased while the fraction of binding attributable to this process decreased. These investigators hypoth- esized that the initial, rapid phase of binding represented binding to a desensitized form of the receptor, and that the slower phase was due to binding to a lower affinity, activatable form of the receptor, which alters its conformation after binding to yield the high affinity, desensitized state. Consistent with this model, the kinetics of dissociation is characterized by a single rate constant. If chronic agonist treatment alters the ratio of activatable:desensitized receptors, this alteration may result in tolerance to nicotine. To determine if chronic nicotine changes this ratio, we infused C57BL/6 mice for 10 days with saline and nicotine (0.5, 1.0, 3.0 and 5.0 mg/kg/h). Two hours after infusion was stopped the animals were sacrificed, their brains removed, the membrane fraction prepared and the association kinetics of [-411 nicotine (20 nM) binding Modulation of nicotine receptors 77 was measured. The association curves were fitted to a two-site model using a least squares method and the amounts of fast phase binding (Bf) and slow phase binding (Bs) were estimated. These values are presented in Fig. 4. Surprisingly, both Bf and Bs increase with chronic infusion, but Bf increases more rapidly at lower doses. Thus, low doses of nicotine increase the number of desensitized receptors, but the number of activatable receptors also seems to change. However, this result must be viewed with some suspicion because we do not know the rate of resensitization of the receptors; i.e. we do not know whether the fast-binding form is converted back to the slow-binding form, and, if so, what the rate of this process might be. If this occurred in less time than was required to remove the brains and prepare the membranes for assay, the results presented in Fig. 4 would not represent a valid estimate of the in vivo Bf:Bs ratio. Clearly, additional studies are required to resolve these issues. In particular, studies of changes in the Bf:Bs ratio must be done in mouse strains that differ in the relationship between tolerance development and receptor tip-regulation. Steroid interaclions with the nicotinic receptors Many smokers increase their tobacco use when placed in a stressful environment (Hall & Morrison 1973, Parkes 1983). In addition, tobacco use may increase the release of adrenal steroids. Because recent studies have demonstrated that certain steroids alter GABA receptor binding and function (Majewska 1987), we are investigating the potential effects of adrenal steroids on response to nicotine and on nicotinic receptor binding. Effects of steroids on sensitivity to nicotine The initial study that suggested that steroids may influence sensitivity to nicotine (Pauly et a) 1988) determined whether adrenalectomy and steroid replacement altered sensitivity to nicotine in C3H mice. Adrenalectomy caused dramatic increases in sensitivity to the effects of nicotine on Y-maze activities, acoustic startle response, heart rate and body temperature. The effects of adrenalectomy could be reversed if the animals were provided with replacement steroid. Furthermore, control animals were made resistant to nicotine if they were treated chronically with corticosterone (CCS). Adrenalectomy did not alter brain 13111 nicotine binding; [ 12511 bungarotoxin binding was elevated, but only in the hippocampus. These results suggest that CCS may have an anti-nicotine effect. In a subsequent study (Pauly et at 1990), we determined whether other mouse strains also exhibit increased sensitivity to nicotine after adrenalectomy. A summary of these results is presented in Table 2. A surprising result was obtained; after adrenalectomy some mouse strains, such as the C57BL/6 and '.~VE66E9V0Z

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ M No I I 78 TABf,E 2 Effects of adrenalectomy on sensitivity to nicotine Collins et al Morrse Startle Y-maze Y-nraze Heart Body strain response rears crosses rate tenrperature A 0 0 0 + 0 BUB + 0 0 0 0 C3H + -F + + + C57 0 0 0 + 0 DBA 0 0 0 4- + LS 0 + + 4 + SS 0 0 0 0 + +indicates that adrenaicctomy resulted in increased sensitivity to nicotine; 0 indicates that it had no effect. DBA/2, showed minimal changes in sensitivity to nicotine; others, such as the C3H and LS, were more sensitive to all of the nicotine effects. The possibility that CCS might exert its anti-nicotine actions by inhibiting binding to the brain nicotinic receptors was examined by measuring binding, in vitro, in the presence of various concentrations of CCS. The effects of CCS on [3H]nicotine binding in four brain regions obtained from C3H mice are presented in Fig. 5. CCS inhibited binding in all of the brain regions, but 100% inhibition was never attained. Similar effects of CCS on [ 12511 bungarotoxin binding were found (data not shown). 120 100 80 60 40 O 0µM W1 t4IiM riS9 701,MFO04 140µ6tM 290µM Q] 700 pM M 1.4 mM I k I CX M13 I-IP 13RAI(v R[;GION Si, FIG. 5. Inhibition of ['H)nicotine binding by corticosterone. Corticosterone (CCS) was added to the standard nicotine binciing assays at the designated concentrations. Inhibition is presented in terms of %± SEM of control binding, Inhibition was measured in cortex (CX), midbrain (MB), hippocampus (HP) and striatum (ST). •, P<0.05 when compared to control. Modulation of nicotine receptors 19 t~p ® 100 ti .--i 20• O 1358 ADX 40% C stiAM ADX ADX 20% CCS ADX 60% C C3H FEMALE BRAIN REGION FIG. 6. Effects of adrenalectomy (ADX) and chronic corticosterone (CCS) treatment on brain [12511 bungarotoxin (BTX) binding. C3H female mice were adrenalectomized and treated chronically with a cholesterol pellet or pellets containing 20016, 40% or 60070 CCS. Binding was measured seven days after ADX. Sham-operated animals were the controls. Each bar represents the mean ±SEM of 6-10 determinations. Binding was measured in cortex (CX), midbrain (M), hippocampus (P), striatum (S) or colliculi (COL). Chronic treatment with corticosterone We have also been examining the effects of chronic CCS treatments on brain nicotinic receptor binding. Chronic CCS treatment has been achieved by preparing pellets containing mixtures of cholesterol and CCS. These pellets are implanted subcutaneously for seven days. This treatment has no effect on [3H]nicotine binding, but chronic CCS treatment has a marked effect on [ 12511 bungarotoxin binding. Fig. 6 presents the effects of adrenalectomy and treatment with 20, 40 and 60% CCS-containing pellets on 112511 bungarotoxin binding in five brain regions. Adrenalectomy elicited an increase in binding only in the hippocampus. CCS treatment reduced binding in all of the brain regions assayed, but a dose-response relationship was not evident. We are currently studying the effects of lower doses and longer treatment times. Scatchard analyses indicated that this reduction in binding is due to a reduction in Bmex; KD values were not altered by chronic CCS treatment. It is important to note that the chronic CCS-treated animals were tolerant to nicotine. This tolerance might be due to CCS-induced inhibition of [ 3H ] nicotine binding, or to the reduction in [t2SI ] bungarotoxin binding. Further studies are required to resolve this issue. 9M6M09

.. r.. .. .. r r .. ..r ~ .. .r. .. .. .r .M s. .. .. tra 1 a 80 Conclttsions Collins et at The studies reported here demonstrate that chronic nicotine treatment results in increases in both [31-1) nicotine and (1251 1bungarotoxin binding. These increases in binding are dose and time dependent. Although studies using a single strain of mouse, the DBA/2, suggest that changes in receptors may explain tolerance to nicotine, studies with other strains indicate that additional factors are involved. One such factor might involve steroid interactions with the nicotinic receptors. Those mouse strains that show a high correlation between receptor changes and tolerance (e.g. the C5713L/6 and DBA/2 strains) also show minimal changes in sensitivity to nicotine after adrenalectomy or CCS adntinistration. On the other hand, the C3H strain is markedly affected by adrenalectomy and shows little correlation between chronic nicotine-induced receptor changes and tolerance development. The observation that chronic mecatnylamine treatment induces an up- regulation of nicotinic receptors supports the notion that nicotine-induced receptor desensitization is responsible for chronic nicotine-induced receptor changes. Since not all nicotinic agonists elicit receptor changes, it does not seem likely that receptor up-regulation can be predicted by the affinity of the agonists. While receptor tip-regulation might be due to desensitization of the receptor, our studies of the effects of chronic nicotine infusion on the association of 13111 nicotine binding failed to provide the expected results: an increase in Bf and a decrease in Bs was expected, but both Bf and Bs increased after chronic nicotine infusion. However, because these assays were done a minimum of two hours after the tissue had been removed from the animal, we do not believe that ttbe results presented here conclusively demonstrate that the expected change in the Bf:Bs ratio was achieved in vivo. Studies of the rate of conversion of the fast-binding form back to the slow-binding form must be done so that the results reported here can be interpreted properly. We suspect that an assay of receptor function might provide results that are less ambiguous. The observation that altering CCS levels alters the sensitivity of the mouse to nicotine may explain why people increase their use of tobacco when stressed. If cortisol; the human equivalent of CCS, also has anti-nicotine actions, it may be that humans increase their tobacco intake when cortisol levels are elevated in an attempt to overcome the partial receptor blockade induced by the steroid hormone. Acknowledgemenls Studies from the authors' laboratory were supported by grants from the National Institute on Drug Abuse (DA-06391 and DA-05131) and from the R. J. Reynolds Tobacco Company. A. C. C. is supported, in part, by a Research Scientist Development Award (DA-00I16). The technical assistance of E. A. Ullman, S. Selvaag and S. Turner is sincerely appreciated. Modulation of nicotine receptors 81 References Barr JE, Holmes DB, Ryan LM, Sharpless SK 1979 Techniques for the chronic cannulation of the jugular vein in mice. Pharmacol Biochem Behav I 1:115-118 Clarke PBS, Kumar R 1983 The effects of nicotine on locomotor activity in nontolerant and tolerant rats. Br J Pharmacol 78:329-337 Collins AC, Marks MJ 1989 Chronic nicotine exposure and brain nicotinic receptors- influence of genetic factors. Prog Brain Res 79:137-146 Collins AC, Romm E, Wehner JM 1988 Nicotine tolerance: an analysis of the time course of its development and loss in the rat. Psychopharmacology 96:7-14 Costa LG, Murphy SD 1983 (3H 1 nicotine binding in rat brain: alteration after chronic acetylcholinesterase inhibition. J Pharmacol Exp Ther 226:392-397 De Sarno P, Giacobini E 1989 Modulation of acetylcholine release by nicotinic receptors in the rat brain. J Neurosci Res 22:194-200 Hall GH, Morrison CF 1973 New evidence for a relationship between tobacco smoking, nicotine dependence and stress. Nature (Lond) 243:199-201 Hatchell PC, Collins AC 1977 Influences of genotype and sex on behavioral tolerance to nicotine in mice. Pharmacol Biochem Behav 6:25-30 Ksir C, Hakan RL, Hall DP, Kellar KJ 1985 Exposure to nicotine enhances the behavioral stimulant effect of nicotine and increases binding of [3H ] acetylcholine to nicotinic receptors. Neuropharmacology 24:527-531 Ksir C, Hakan RL, Kellar KJ 1987 Chronic nicotine and locomotor activity: influences of exposure dose and test dose. Psychopharmacology 92:25-29 Lippiello PM, Sears SB, Fernandes KG 1987 Kinetics and mechanism of L-[1HJnicotine binding to putative high affinity receptor sites in rat brain. Mol Pharmacol 31:392-400 Majewska MD 1987 Steroids and brain activity: essential dialogue between brain and body. Biochem Pharmacol 36:3781-3788 Marks MJ, Collins AC 1982 Characterization of nicotine binding in mouse brain and comparison with the binding of alpha-bungarotoxin and quinuclidinyl benzilate. Mol Pharmacol 22:554-564 Marks MJ, Collins AC 1985 Tolerance, cross tolerance, and receptors after chronic nicotine or oxotremorine. Pharmacol Biochem Behav 22:283-291 Marks MJ, Burch JB, Collins AC 1983 Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226:817-825 Marks MJ, Stitzel JA, Collins AC 1985 Time course study of the effects of chronic nicotine infusion on drug response and brain receptors. J Pharmacol Exp Ther 235:619-628 Marks MJ, Romm E, Gaffney DK, Collins AC 1986a Nicotine-induced tolerance and receptor changes in four mouse strains. J Pharmacol Exp Ther 237:809-819 Marks MJ, Stitzel JA, Collins AC 1986b Dose-response analysis of nicotine tolerance and receptor changes in two inbred mouse strains. J Pharmacol Exp Ther 239:358-364 Marks MJ, Stitzel 1A, Romm E, Wehner JM, Collins AC 1986c Nicotinic binding sites in rat and mouse brain: comparison of acetylcholine, nicotine, and alpha-bungarotoxin. Mol Pharmacol 30:427-436 Martino-Barrows AM, Kellar KJ 1987 [3HJacetylcholine and [3H)(-)nicotine label the same recognition site in rat brain. Mol Pharmacol 31:169-174 Miner LL, Collins AC 1988 The effect of chronic nicotine treatment on nicotine-induced seizures. Psychopharmacology 95:52-55 Parkes KR 1983 Smoking as a moderator of the relationship between affective state and absence from work. J AppI Psychol 68:698-708 Pauly JR, Ullman EA, Collins AC 1988 Adrenocortical hormone regulation of nicotine sensitivity in mice. Physiol Behav 44:109-116 ~ M66EM9

== .rM m.. ... m.. +r r m.. a. M M = mm M 82 I I Discussion Pauly JR, Ulhnan EA, Collins AC 1990 S,rain differences in adrenalectonty-induced alterations in nicotine sensitivity in the mouse. Phartnacol 13iochem Behav 35:171-179 Schwartz RD, Kellar KJ 1983 Nicotinic cholinergic receptor binding sites in the brain: regulation in vivo. Science (Wash DC) 220:214-220 Schwartz RD, Kellar KJ 1985 In vivo regulation of I'l-I lacetylcholine recognition sites in brain by nicotinic cholinergic drugs. J Neurochem 45:427-433 Stolerman LP, Fink R, Jarvik ME 1973 Acute and chronic tolerance to nicotine measured by activity in rats. I'sychopharmacologia 30:329-342 DISCUSSION Changeux: I would like to come back to the muscle nicotinic receptor as a model, although what is true for the muscle receptor may not be exactly correct for the brain nicotine receptor. First, there is a short-term regulation of receptors referred to as desensitization. Another kind of regulation, which has been analysed extensively in the case of the muscle nicotinic receptor, concerns its biosynthesis. During formation of the motor endplate, which lasts 10-20 days, several processes of receptor synthesis occur sequentially or conctirrently. You mentioned the paradox of a decreased response and an increased number of nicotine binding sites. This is also seen in the case of the muscle receptor in extrajunctional areas. At early stages of endplate development, the ACh receptor is present all over the muscle surface. When the junction becomes active, the receptors in the extrajunctional areas disappear as a result of a repression of gene transcription that is dependent on electrical activity. When transmission is blocked, e.g. by curare, the biosynthesis of receptor increases again. I don't know if this is also true for the brain receptor. Infusion of ACh or nicotine is expected to desensitize the postsynaptic membrane and to yield a chronic block. The electrical activity of the postsynaptic cell is expected to decrease as a consequence of this and thereby derepress synthesis of the receptor. Collins: We are addressing those kinds of issues in experiments underway in our laboratory. Rose: Clinically, the two approaches often discussed for nicotine dependence, as well as for other drug dependences, are giving an agonist replacement and giving an antagonist to block reinforcement. Each has problems: giving nicotine alone, for instance, doesn't totally block the reinforcement of smoking a cigarette. On the other hand, if you give mecamylamine, you rnight induce aversive symptoms. Would one get a better clinical effect by combining an agonist with an antagonist? For example, if you combined nicotine with mecamylamine, you could saturate the receptor system but the person would not be intoxicated with nicotine because the level of stimulation would be controlled, nor would they be in a state of withdrawal, because the blockade effect of mecamylamine would be balanced by the nicotine. If the person then smoked a cigarette, there would be no reinforcing effects. Modulation of nicotine receptors 83 Did you ever look at the effects of an acute dose of nicotine on animals that were receiving simultaneously nicotine and mecamylamine? Collins: We have been using minimitters that consist of a tiny radiotransmitter. These give continuous data regarding either the animal's locomotor activity and body temperature, or locomotor activity and heart rate. We have used the locomotor activity and body temperature one to determine the effects of mecamylamine and nicotine. Unfortunately, if we treat an animal with mecamylamine alone, we see profound effects in those two measures, so that experiment didn't work. Stolerman: The findings with adrenalectomy encourage us to look again at stress-related phenomena in connection with nicotine dependence or addiction. I would be interested to see comparable data for a measure of response to nicotine that is more directly linked to drug seeking than is body temperature. Secondly, there's a distinction we need to make with regard to tolerance, if we want to relate tolerance to desensitization phenomena. This is the difference between acute and chronic tolerance to nicotine, both of which have been demonstrated experimentally. Chronic tolerance, the effect produced by repeated administrations of nicotine or by long-term infusions, seems to take quite a long time to develop and to wear off. It seems unlikely that this relates to the desensitization that neurophysiologists talk about, which has a very short duration. Even the acute tolerance produced by single doses of nicotine, where the time course of offset and onset may be in minutes or at the most in hours, seems slow in relation to desensitization. CoAins: It is difficult to interpret experiments designed to relate acute tolerance to receptor desensitization. We need to re-evaluate those experiments in light of the corticosterone date that I presented. The technique involves pretreating the animal with a dose of nicotine, then challenging it with another dose of nicotine later and asking whether or not that pretreatment alters the sensitivity to nicotine. In many cases, you see a reduced response to nicotine. We also know that nicotine injection elicits the release of corticosterone. A potential mechanism for this behavioural desensitization is that nicotine releases the corticosterone and the corticosterone partially blocks the receptor so there is an attenuated response to the challenge dose. Alternatively, it could be due to receptor desensitization. Stolernran: When we reported those results (Stolerman et al 1973), we showed that there was a two hour delay between the administration of the nicotine and the peak of tolerance. We argued against the desensitization hypothesis, and I am encouraged by your results on steroids. Collins: Corticosterone may not be the most important steroid in this regard. We are looking at various corticosterone metabolites; some metabolites may be much more potent than the parent compound. London: Is the inhibition of the nicotinic receptor by corticosterone competitive or non-competitive? . gt£66OV0Z

.. .rr r M ... r.r .. i.. r .. .r .. ... r ..r .r .. .. vi.r 84 Discussion a i I Collins: Non-competitive. London: What concentrations produce inhibition? Collins: We have used concentrations in the bungarotoxin (Bgt) binding assay from as little as I pg/ml, which is equivalent to the concentration in an unstressed animal, all the way up to heroic concentrations. We see inhibition at greater than physiological concentrations. One problem is that all of our binding assays with Bgt binding have had bovine serum albumin added to decrease non-specific binding of the Bgt to glass. BSA has high affinity binding sites for steroids. Consequently, I haven't a clue what the real free concentration in the steroid was that was inhibiting our Bgt binding assay. All I can say is that steroids do inhibit Bgt binding non-competitively. They also non-competitively inhibit binding of ['H ] nicotine to brain membranes. Kellar: The down-regulation of nicotinic binding sites during treatment with cholinesterase inhibitors is seen in rats using at least three different cholinesterase inhibitors. Collins: I think we are seeing a species difference there. Kellar: I don't think you would expect to see up-regulation of nicotinic receptors before tolerance, only after tolerance. There could be no temporal relationship at all. If up-regulation is due to inactivation of receptor function, that may go on for days before you see an up-regulation, so I think it's going to be very hard to see a temporal relationship in that situation. You may see it more easily in the recovery of function. Svensson: We have seen evidence for tolerance development in the mesolimbic dopamine system with repeated nicotine administration. This tolerance appeared with intermittent administration of nicotine but not with continuous adminis- tration of nicotine by an osmotic minipump. It seems like it's neither the total exposure to nicotine nor the duration of the administration but rather a series of high peak concentrations that is important in the development of tolerance. Iversen: What was the measure? Svensson: These were biochemical measures of dopaminergic activity. Collins: That's entirely consistent with a study where we compared pulse infusion of nicotine versus continuous infusion of the same dose. We got much more tolerance with pulse infusion, so we are seeing the same effect on behaviour as you are seeing biochemically. Gray: I am worried about the way the word 'tolerance' has been thrown around, particularly in the context of the overall theme of this meeting, nicotine dependence. There is a general view that dependence on a drug is very closely related to the development of tolerance to the drug. This may be true in some cases. My worry is that it's been taken for granted that nicotine induces general tolerance. There are lots of reasons to think that's not true. I am pretty certain that in nicotine self-administration experiments with animals there is little evidence that the dose that animals choose to self-administer increases during Modulation of nicotine receptors 85 the experiment, except perhaps during a very early period. The human smoker reaches their two pack-a-day stage, or whatever, then stays there for many years. 'Tolerance' is being used, among other things, to refer to the pheno- menon originally described by Ian Stolerman-that an initial effect of locomotor depression changes later to an effect of locomotor increase. That is not tolerance, it is a changed response to the drug. Furthermore, loss of response after a few repeated administrations of the drug may not differ from, for example, the loss of response to an audible tone. It is a characteristic of most behavioural responses that they diminish with repeated elicitation. To go back to the question of the reinforcing effects of the drug, which is probably what dependence is about, this is probably related to dopamine release in the nucleus accumbens. There is clear evidence that that does not change with 15 days of repeated injections of nicotine, as shown by Damsma et al (1989). I am worried that the word tolerance is being used as though this was a general phenomenon relating to nicotine. Collins: You are probably right, but I would like to clear up a point with respect to our work. I don't think many people would question that if you have a shift to the right of the dose-response curve, for biochemical or behavioural responses, you have achieved a reduced response or sensitivity. This, in my opinion, is tolerance. Furthermore, we have strain differences on the degree of that shift. I believe that is tolerance, because it takes a greater challenge dose to elicit the effect seen in a naive animal. However, I think you have an extremely important point that nicotine effects on dopamine release, if that's related to why an animal or a human likes nicotine, may not show development of tolerance. I have argued in other quarters that many of the things that we have been measuring in our behavioural tests are reasons why people would choose not to smoke rather than to smoke. In other words, our results may relate to tolerance to nicotine effects that would deter an individual from smoking. Stolerinan: The answer to Jeffrey Gray's question is straightforward. Dose- response studies show that, acutely, small doses of nicotine can increase locomotor activity and larger doses decrease it. One can find doses of nicotine that acutely decrease activity but after chronic administration they increase it (Stolerman et al 1974, Clarke & Kumar 1983). Other factors are involved, but this shift in the dose-response curve may be the most important. These findings in rats fit well with those of Dr Collins in mice (Marks & Collins 1985, Collins et al, this volume). Overall, there are clear indications of tolerance to the locomotor depressant effect. As with other drugs, the appearance of tolerance depends on the effect measured; Jeffrey Gray is surely right when he says there's no evidence for tolerance to the positive reinforcing effect of nicotine that maintains self-administration.

.r r.. .. .s r ar rr M rmr r.. .. a. .. .. r 86 References Discussion Clarke PBS, Krunar R 1983 The effects of nicotine on locomotor activity in non-tolerant and tolerant rats. Br .1 Pharmacol 78:329-337 Collins AC, Ilhat RV, Pauly JR, Marks MJ 1990 Modulation of nicotine receptors by chronic exposure to nicotinic agonists and antagonists. In: The biology of nicotine dependence. Wiley, Chichester (Ciba Found Symp 152) p 68-86 Damsma G, Day J, Fibiger tIC 1989 I.ack of tolerance to nicotine-induced doparnine release in the nucleus accumbens. Eur J Pharmacol 168:363-368 Marks M1, Collins AC 1985 Tolerance, cross tolerance, and receptors after chronic nicotine or oxolrernorine. Phannacol l3iochem 13ehav 22:283-291 Stolerman IP, Fink R, Jarvik ME 1973 Acute and chronic tolerance to nicotine measured by activity in rats. Psychopharniacologia 30:329-342 Stolerman IP, Bunker P, Jarvik ME 1974 Nicotine tolerance in rats: role of dose and dose interval. Psychopharmacologia 34:317-324 a r Presynaptic nicotinic receptors and the modulation of transmitter release Susan Wonnacott, Alison Drasdo, Elizabeth Sanderson and tPeter Rowell Department of Biochemistry, University of Bath BA2 7AY, UK and tDepartment of Pharmacology and Toxicology, University of Louisville, Kentucky, USA Abstract. Nicotine is increasingly recognized to promote transmitter release in the brain by a direct action on presynaptic terminals. Pharmacological evidence indicates that this action is mediated by nicotinic receptors. From their sensitivity to mecamylamine, neosurugatoxin and neuronal bungarotoxin these presynaptic receptors can be distinguished from a-bungarotoxin-sensitive muscle-type nicotinic receptors, and can be correlated with [3H ] nicotine binding sites in the brain. The release of many transmitters in different brain regions is susceptible to stimulation by nicotine, but this effect is not ubiquitous. However, lesioning and subcellular fractionation studies suggest that the majority of brain nicotinic receptors are located presynaptically, so that a direct influence of nicotine on transmitter release assumes considerable importance. Although the sensitivity of presynaptic receptors is such that they are likely to be partially activated by doses of nicotine obtained by smoking, the desensitization-induced up-regulation of nicotinic binding sites that follows chronic nicotine treatinent raises questions about their functional status during tobacco usage. Chronic administration of the agonist (+)anatoxin-a also up-regulated ['H]nicotine binding sites, and led to increased nicotine-evoked transmitter release in vitro. This could have implications for the involvement of these receptors during withdrawal. 1990 The biology of nicotine dependence. Wiley, Chichester (Ciba Foundation Symposium 152) p 87-105 The early studies of Armitage et at (1969) were among the first to show that nicotine can promote the release of neurotransmitters in the brain, in this case acetylcholine release in the cat cerebral cortex. More recent in vitro studies utilizing brain slices in the presence of tetrodotoxin (Giorguieff-Chesselet et at 1979) or isolated nerve terminals (Rowell & Winkler 1984, Rapier et at 1988) have indicated that nicotine can act directly on the presynaptic tenninal to increase transmitter release, rather than achieving this effect by postsynaptic stimulation and the generation of action potentials. Pharmacological characterization of this.phenomenon suggests that, at least at low nicotine concentrations, this action is mediated by nicotinic acetylcholine receptors. The 87 M66E9fi0g