Symposium Overview: Mechanism of Action of Nicotine on Neuronal Acetylcholine Receptors, from Molecule to Behavior

T. Narahashi*,1, C. P. Fenster{dagger}, M. W. Quick{dagger}, R. A. J. Lester{dagger}, W. Marszalec*, G. L. Aistrup*, D. B. Sattelle{ddagger}, B. R. Martin§ and E. D. Levin

* Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611; {dagger} Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama 35294; {ddagger} MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom; § Department of Pharmacology and Toxicology, Medical College of Virginia, Richmond, Virginia 23298; and Departments of Psychiatry and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

Received April 17, 2000; accepted June 30, 2000

ABSTRACT

Nicotine has long been known to interact with nicotinic acetylcholine (ACh) receptors since Langley used it extensively to chart sympathetic ganglia a century ago. It has also been used as an effective insecticide. However, it was not until the 1990s that the significance of nicotine was increasingly recognized from the toxicological, pharmacological, and environmental points of view. This is partly because studies of neuronal nicotinic ACh receptors are rapidly emerging from orphan status, fueled by several lines of research. Since Alzheimer's disease is known to be associated with down-regulation of cholinergic activity in the brain, a variety of nicotine derivatives are being tested and developed for treatment of the disease. Public awareness of the adverse effects of nicotine has reached the highest level recently. Since insect resistance to insecticides is one of the most serious issues in the pest-control arena, it is an urgent requirement to develop new insecticides that act on target sites not shared by the existing insecticides. The neuronal nicotinic ACh receptor is one of them, and new nicotinoids are being developed. Thus, the time is ripe to discuss the mechanism of action of nicotine from a variety of angles, including the molecular, physiological, and behavioral points of view. This Symposium covered a wide area of nicotine studies: genetic, genomic, and functional aspects of nicotinic ACh receptors were studied, as related to anthelmintics and insecticides; interactions between ethanol and nicotine out the ACh receptor were analyzed, in an attempt to explain the well-known heavy drinker-heavy smoker correlation; the mechanisms that underlie the desensitization of ACh receptors were studied as related to nicotine action; selective pharmacological profiles of nicotine, and descriptions of some derivatives were described; and chronic nicotine infusion effects on memory were examined using animal models.

Key Words: nicotinic acetylcholine receptor (nAChR); nicotine; cholinergic transmission; alcohol; desensitization; anthelmintic; insecticide; memory; hippocampus.

Regulation of Acetylcholine Receptor Desensitization at Low Concentrations of Nicotine (C. P. Fenster, M. W. Quick, and R. A. J. Lester)

Neuronal nicotinic acetylcholine receptors (nAChRs) are localized to pre- and postsynaptic sites in the brain (Role and Berg, 1996Go). In a physiological context, e.g., synaptic stimulation, nAChRs are likely to be exposed to the neurotransmitter acetylcholine (ACh) for very brief periods, during which they will become transiently activated and may even become briefly desensitized. During more prolonged exposure to agonist, such as would occur with chronic nicotine use, these receptors may enter conformational states not normally encountered (Fig. 1Go). These receptor states may directly underlie and/or promote cellular processes that result in tolerance and dependence on nicotine (Dani and Heinemann, 1996Go).



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FIG. 1. Schematic model for receptor upregulation and inactivation. The receptor can exist under normal conditions in several conformations, a low-affinity activatable, R, state and two desensitized states, D1 and D2, with moderate and high affinities for agonist, respectively. At rest, most receptors are in the R conformation. Rapid binding of agonist, A, will promote channel opening. Prolonged exposure to low concentrations of agonist will cause stabilization of the higher affinity desensitized states, with potential consequent long-lasting changes dependent on cellular conditions, e.g., phosphorylation.

 
Any assessment of the consequences of nicotine use at the cellular and molecular levels will require accurate knowledge of the concentration ranges over which nicotine can interact with the various conformational states of nAChRs. In the central nervous system (CNS), an analysis of this type is complicated because an unknown variety of nAChR subtypes exist, and their precise subunit compositions are uncertain. Current research indicates that there are at least 3 major subtypes of nAChRs in the brain, for which partial subunit structure and properties are known: (i) {alpha}-bungarotoxin ({alpha}BTX)-sensitive receptors containing {alpha}7 subunits; (ii) high affinity [3H]nicotine-labeled receptors containing {alpha}4 and ß2 subunits; and (iii) low affinity nAChRs containing {alpha}3 and possibly ß4 subunits (Colquhoun and Patrick, 1997Go; Lindstrom, 1997Go). Several studies have systematically evaluated the specific properties of receptors with these basic compositions. However, in addition, because the steady-state concentrations of nicotine in the CSF after cigarette smoke inhalation have been estimated as in the low to moderate nanomolar range, it is essential to inquire specifically about the long-term effects of these concentrations. Others and we have found that tobacco-related concentrations of nicotine will largely stabilize the high-affinity, desensitized states of the {alpha}4ß2 nAChR subtype (Fenster et al., 1997Go; Hsu et al., 1996Go). Receptors containing {alpha}3 subunits may be relatively unaffected at these doses (see Lindstrom, 1997Go). These data offer direct support for the idea that desensitization could serve as a potential trigger for the increase in the number of high affinity [3H]nicotine-binding sites that occurs following chronic exposure to nicotine (Marks et al., 1983Go; Schwartz and Kellar, 1985Go).

The desensitization-upregulation hypothesis has, however, been difficult to prove, and recent results have suggested that it may be too simple (e.g., Peng et al., 1994Go; Whiteaker et al., 1998Go). The most direct way of testing this theory would be to assess both functional receptor desensitization and the upregulation of receptor number for the same population of receptors, under the same conditions. The most suitable preparation for performing both binding and physiological assays is the Xenopus oocyte, which is capable of expressing millions of functional channels over its large surface area. To estimate the number of nAChRs on the plasma membrane of cells, we modified a [3H]nicotine-binding assay for use on intact oocytes (Chang and Weiss, 1999Go) The half-maximal dose for inducing receptor upregulation was calculated by incubating oocytes for 24 h in various concentrations of nicotine. Long-term desensitization was assessed functionally, rather than indirectly from binding assays (Peng et al., 1994Go; Whiteaker et al., l998), using a protocol similar to the one used for the binding assay. Functional nAChR responsiveness to a fixed test concentration of nicotine was assessed at the start of the assay. Oocytes were then incubated in various concentrations of nicotine for 24 h, and the fractional response remaining was calculated from a second nicotine test application. Changes in receptor function independent of chronic nicotine exposure were determined from changes in parallel control oocytes that did not receive any nicotine exposure. The half-maximal concentrations of nicotine for inducing upregulation and functional desensitization were found to be very similar (~10 nM). Thus, our data support the hypothesis that receptor upregulation may involve the desensitized conformation of the receptor (Fig. 1Go).

Following chronic nicotine exposure in oocytes, {alpha}4ß2 receptor function returns to normal levels very slowly (Hsu et al., 1996Go; Peng et al., 1994Go). Normal recovery from desensitization, slow as it is (Fenster et al., 1997Go), does not appear to account for these apparently "permanently" inactive receptors (Lukas, 1991Go). Since the length of time a receptor spends in any one state is largely related to the rate constant out of that state, we reasoned that if recovery from desensitization became impaired after chronic nicotine exposure, receptors could become "trapped" in seemingly inactive conformations. Inhibition of protein kinase C (PKC) can slowly inactivate {alpha}4ß2 nAChRs (Eilers et al., 1997Go), implying that inactivation of {alpha}4ß2 receptors might be mediated by phosphorylation-dependent effects on the rate of recovery from desensitization. We found that recovery from desensitization could be enhanced by PMA, an activator of PKC, and slowed by calphostin C, a PKC inhibitor. Furthermore, these effects were restricted to a deep state of desensitization (see Boyd, 1987Go), which may only be reached after prolonged exposure to nicotine (Fenster et al., 1999Go). These data alone do not directly demonstrate that the inactivation of {alpha}4ß2 nAChRs during chronic exposure to nicotine results from a loss of recovery from desensitization, but offer a plausible mechanism that may in part explain this phenomenon (Fig. 1Go).

Thus, for {alpha}4ß2 nAChRs expressed in oocytes, receptor desensitization may contribute to the processes that initiate both receptor upregulation and functional inactivation. It need not be the case that these two phenomena are always related, e.g., upregulation could occur independently of inactivation, provided that recovery from desensitization was not dependent on PKC. This could account for the findings that some types of nAChRs, including in some systems those containing {alpha}4 and ß2 subunits, may be upregulated both in number and in function following prolonged nicotine exposure (Rowell and Wonnacott, 1990Go; Gopalakrishnan et al., 1996Go).

This work was supported by PHS grants DA11940 and NS31669.

Interactions of Nicotine and Alcohol at Neuronal Nicotinic Acetylcholine Receptors (nnAChRa) (Toshio Narahashi, William Marszalec, and Gary L. Aistrup)

A positive correlation between alcohol drinking and tobacco smoking is well-established (Bien and Burge, 1990Go; Collins, 1990Go; Zacny, 1990Go). Approximately 70% of alcoholics smoke more than one pack of cigarettes a day, compared with 10% of the general population. Alcohol seems to influence smoking more than smoking influences drinking. However, the mechanism underlying this correlation remains largely to be seen. One possible explanation is that ethanol potentiates the pleasurable or behavior-reinforcing effects associated with nicotine use. Behavioral reinforcement and locomotor stimulation evoked by either nicotine (Balfour et al., 1998Go; Benwell and Balfour, 1992Go; Benwell et al., 1995Go) or ethanol (Koob et al., 1998Go; Phillips and Shen, 1996Go; Samson et al., 1992Go) are both associated with the release of dopamine from mesolimbic dopaminergic terminals located in the nucleus accumbens. Furthermore, stimulation of cholinergic neurons is known to cause release of various neurotransmitters including dopamine, GABA, epinephrine, and glutamate (Wonnacott, 1997Go). Thus, interactions of nicotine and ethanol at neuronal nicotinic acetylcholine receptors (nnAChRs) may explain the linkage between drinking and smoking. We performed patch clamp experiments using rat cortical neurons in long-term primary culture to prove this hypothesis (Marszalec et al., 1999Go).

At least 2 types of currents were generated in response to the application of ACh, a rapidly desensitizing ({alpha}7-like), {alpha}-bungarotoxin ({alpha}-BuTX)-sensitive current, and a slowly desensitizing ({alpha}4ß2-like), {alpha}-BuTX-insensitive current. The {alpha}4ß2-like current was potentiated by the application of 3–300 mM ethanol, while the {alpha}7-like current was slightly inhibited by 30–300 mM ethanol. Nicotine by itself, at a concentration of 300 nM or 1 µM, generated {alpha}4ß2-like currents, albeit in much smaller amplitude than those evoked by ACh. As previously observed with muscle nicotinic AChRs (Wang and Narahashi, 1972Go) and more recently with nnAChRs (Bellwell et al., 1995; Fenster et al., 1997Go; Ochoa et al., 1989Go; Pidoplichko et al., 1997Go), nicotine at low concentrations desensitized the {alpha}4ß2-like receptor of cortical neurons. Nicotine, at concentrations of 30, 100, and 300 nM, reversibly desensitized the ACh-induced current by 38, 54, and 62%, respectively. However, these concentrations of nicotine evoked only small currents by itself, with the EC50 concentration for nicotine-induced desensitization (~100 nM) being about 300-fold less than that required for current activation (~3 µM). It should be noted that blood nicotine levels transiently peak at 30 to 180 nM with each cigarette smoked (Benowitz et al., 1989Go; Henningfield et al., 1993Go; Russell, 1987Go). These peaks, however, are superimposed over continuously rising steady-state nicotine levels of 60 to 300 nM, which tend to persist between each cigarette smoked. Therefore, the nnAChRs are desensitized considerably during repeated cigarette smoking.

Ethanol potentiated {alpha}4ß2-like currents, even in the presence of the desensitizing concentrations of nicotine. In neurons desensitized by 100 to 200 nM nicotine, 100 mM ethanol enhanced residual currents by 30 ± 4% compared with a 24 ± 5% enhancement of control currents (n = 6). Thus, ethanol potentiates the currents to approximately the same extent regardless of the degree of desensitization produced by nicotine perfusion.

An example of a crucial experiment is illustrated in Figure 2Go. Currents evoked by 300 µM ACh pulses were monitored over time (black circles). Perfusion of nicotine at a concentration of 30 nM (solid bar) desensitized ACh-induced currents by 43% (compare traces a with b). Following washout of nicotine, the bath perfusion of 100 mM ethanol (broken bar) potentiated the amplitude of ACh-induced currents by 37% (white circles, compare trace c with d). Now, a co-perfusion of 30 nM nicotine with 100 mM ethanol desensitized ACh-induced currents by 35% (white triangles, compare trace d with e). It should be noted that the nicotine desensitization relative to the initial control response (trace c) is only 6% when ethanol is present (compare traces c with e). Thus, ethanol partially offsets inhibition of the ACh-induced current due to nicotine desensitization.



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FIG. 2. Ethanol opposes the effect of ACh-induced current desensitization due to nicotine perfusion. Current responses were evoked by 300 µM ACh pulses (black circles). A co-application of 30 nM nicotine (solid bar) and ACh (white circles) did not alter the test response until nicotine was introduced into the bath, during which time current desensitization developed (trace a versus b). The test current recovered after nicotine washout (trace c). Next, a co-application of 300 µM ACh and 100 mM ethanol (white squares) potentiated the ACh current above that of the control response (trace c versus d). During this period, 100 mM ethanol was also perfused into the bath (broken bar). When nicotine and ethanol were subsequently perfused together, the response once again underwent desensitization (white triangles, traces d versus e). However, the degree of current reduction observed with nicotine perfusion in the presence of ethanol was less than that observed with nicotine alone (compare traces a/b with traces c/e). From Marszalec et al. (1999).

 
Nicotine has been shown to release dopamine in the nucleus accumbens through activation of {alpha}4ß2-like AChRs at concentrations comparable with those required to evoke currents in cortical neurons (Balfour et al., 1998Go; Benwall and Balfour, 1992; Benwell et al., 1995Go). It has also been demonstrated that ethanol-induced locomotion and dopamine release are blocked by the nicotinic channel blocker mecamylamine (Blomqvist et al., 1992Go, 1993Go, 1997Go). These data, when combined with the present results, suggest that ethanol, together with nicotine, enhances dopamine release, resulting in the behavioral reinforcement mediated by this transmitter to increase the incidence of tobacco smoking. If the smoking urge is prompted by the overall recovery of ACh-induced currents from nicotine desensitization, then the potentiation of these currents by ethanol intake might shorten the interval between each cigarette smoked. Ethanol potentiation and nicotine desensitization of ACh-induced currents may form the basis for the heavy drinking-heavy smoking correlation. It remains to be seen exactly how these in vitro interactions between ethanol and nicotine are reflected in each step of behavioral changes.

Supported by NIH grant AA07836.

Identifying Members of the Nicotinic Acetylcholine Receptor Subunit Family Targeted by Anthelmintics and Insecticides (David B. Sattelle)

The nematode worm Caenorhabditis elegans, and the fruitfly Drosophila melanogaster have proved to be excellent genetic models with which to investigate the nervous system and are now facilitating the identification of particular ionotropic receptor subunits that are targeted by pesticides. Investigations employing genetics, genomics, and functional studies on cloned, heterologously expressed receptor subunits have contributed to these advances. Here we review the recent developments in understanding of the nicotinic receptor (nAChR) subunit gene families of these organisms and the identification of nAChR subunit family members targeted by anthelmintics and insecticides.

The C. elegans nAChR Gene Family
By offering access to a fully sequenced genome, an advanced "genetic toolkit", and a wealth of neurobiological information, the nematode Caenorhabditis elegans is well suited to the functional analysis of genes encoding synaptic proteins. Cholinergic chemical synapses at which acetylcholine (ACh) is the neurotransmitter have been well studied. The cha-1 gene encodes choline acetyltransferase and appears to be the only gene for this ACh synthesizing enzyme (Alfonso et al., 1994Go). The nAChRs (composed of combinations of {alpha} and non-{alpha}-type subunits) are ionotropic receptors, mediating the fast synaptic actions of ACh, yet for no organism is the full complement of nAChR subunits known. In the nematode C. elegans, nAChRs are present on nerve (Treinin and Chalfie, 1995Go) and muscle (Fleming et al., 1997Go), and subunits are encoded by a large gene family. Brenner (1974) first isolated mutants of this nematode resistant to tetramisole (the enantiomeric mixture in which levamisole, an anthelmintic drug, is one stereoisomer). Of the 11 genes found to be associated with resistance to levamisole, 3 encode nAChR subunits: lev-1, unc-29 (non-{alpha}) and unc-38 ({alpha}) (Fleming et al., 1997Go). In the same study, Fleming et al. showed that expression in Xenopus oocytes of combinations of these subunits that include the UNC-38 {alpha} subunit resulted in low-amplitude, inward currents in response to 100 µM levamisole. These currents were suppressed by nicotinic antagonists such as mecamylamine (100 µM). Mutant phenotypes show that UNC-38 and UNC-29 subunits are necessary for nAChR function, whereas the LEV-1 subunit is not. Two dominant mutant alleles of LEV-1, each with a single amino acid change in the second transmembrane (M2) region, which contributes most of the residues that line the channel, were found to be highly resistant to levamisole. A gain of function mutation of the deg-3 {alpha} subunit-encoding gene, which leads to the degeneration of a small set of neurons, also alters a residue in the M2 region (Treinin and Chalfie, 1995Go). The identification of viable nicotinic receptor mutants in C. elegans permits manipulation of receptor expression and synaptic targeting in vivo.

At this time, 13 nicotinic receptor {alpha} and 7 non-{alpha} subunits have been shown to be transcribed in C. elegans, and other candidates are under investigation, making it the largest nAChR {alpha} subunit gene family and also the largest nAChR gene family overall currently known (Mongan et al., 1998Go). Sequence diversity is evident in the M2 channel-lining region and in regions that contribute to the ACh binding site (Mongan et al., 1998Go; Sattelle, 1998Go). At present, 4 genes are known to encode ACh, inactivating enzymes in C. elegans, the acetylcholinesterases (AChEs) (Arpagaus et al., 1994Go; Grauso et al., 1998Go). The genes ace-1, ace-2, ace-x, and ace-y were found to encode 4 pharmacologically distinct classes of AChE. Thus extensive and diverse gene families encode proteins mediating fast synaptic responses to the neurotransmitter ACh and the termination of its actions.

nAChR Subunits Contributing to Levamisole Sensitivity
So how does this wealth of new information assist in identifying the subunits contributing to nAChR subtypes that may be targeted by anthelmintics? In the case of genetic screens based on resistance to levamisole, a drug that activates nAChRs and at higher concentrations shows open channel block. It appears that this approach highlights, from an extensive family, a particular subset of nAChR subunits. We have generated functional heteromeric nAChRs by expressing,in Xenopus oocytes, combinations of {alpha} and non-{alpha} subunits that are products of genes linked to levamisole resistance. This results in functional recombinant nAChRs that mimic several of the properties of native nematode muscle nAChRs, including activation by levamisole (Fleming et al., 1997Go). In this aspect of their pharmacology, they differ strikingly from the only other well characterized C. elegans recombinant nAChRs, which result from the expression of the ACR-16 (= Ce21) nAChR subunit. In the case of ACR-16 homomeric receptors, levamisole has no agonist action but is in fact an nAChR antagonist (Ballivet et al., 1996Go). Thus, pharmacologically distinct recombinant receptors have already been generated using only a small fraction of the known nAChR family members, and different actions of levamisole have been observed. Based on the observed hypercontraction that precedes the cessation of movement in levamisole-treated C. elegans, it appears that the responses of the heteromeric receptors formed from genes linked to levamisole resistance provide the best approximation to date of the native receptors targeted by levamisole. Undoubtedly the current picture may be further refined as more C. elegans nAChR family members are cloned and expressed.

The D. melanogaster nAChR Gene Family
The nAChRs in insects appear to be confined to the nervous system (Breer and Sattelle, 1987Go; Sattelle, 1980Go). Although, to date, the D. melanogaster nAChR gene family is less well-characterized than that of C. elegans, there are detailed studies on 5 nAChR subunits. The cDNAs encoding {alpha} subunits sad (=D{alpha}2), als, d{alpha}3 and the non-{alpha} subunits ard and sbd have all been sequenced (Gundelfinger, 1992Go) but attempts at heterologous expression of a functional nAChR from only Drosophila subunit combinations have so far failed to yield robust functional receptors. This is the case in experiments using either transient expression in Xenopus oocytes (Bertrand et al., 1994Go; Matsuda et al., 1998Go), or stable expression in Drosophila cell lines (Lansdell et al., 1997Go). Thus, it appears that other nAChR subunits remain to be discovered. The only known recombinant insect nAChR composed entirely of insect subunits is the homomer-forming {alpha}L1 subunit of the locust Schistocerca gregaria (Marshall et al., 1990Go).

Nevertheless, robust functional expression can be obtained for nAChR hybrid receptors composed of a Drosophila {alpha} subunit and a vertebrate neuronal non-{alpha} subunit (ß2). Thus SAD/ß2 receptors and ALS/ß2 receptors have been generated and aspects of their pharmacology compared (Bertrand et al., 1994Go). These authors show that, whereas the SAD-containing hybrid nAChR is insensitive to {alpha}-bungarotoxin, the ALS-containing hybrid receptor is highly sensitive to this toxin. It is of interest that earlier studies on native insect nAChRs identified nAChR subtypes that differed in their sensitivity to this snake neurotoxin (Breer and Sattelle, 1987Go; Sattelle, 1980Go; Sattelle et al., 1983Go). Differences in affinity for nicotinic ligands have been observed between hybrid SAD/ß2 and vertebrate brain {alpha}4/ß2-type receptors (Matsuda et al., 1998Go). With the genome sequence of Drosophila advancing rapidly and the pharmacological diversity observed to date in native and hybrid recombinant nAChRs containing different Drosophila subunits, it seems we can anticipate diverse and extensive nAChR gene families in insects as well as nematodes.

nAChR Subunits Contributing to Imidacloprid Sensitivity
The insecticide with the fastest growing sales worldwide is imidacloprid, a potent ligand at insect nAChRs (Bai et al., 1991Go). It is a member of the nitromethylene/nitroguanidine class of insecticides and other members of this class are active on insect nAChRs (Sattelle et al., 1989Go). The nitromethylene nithiazin was found to have agonist actions on native insect nAChRs and on the recombinant locust {alpha}L1 nAChRs expressed in Xenopus oocytes (Leech et al., 1991Go). Of particular interest has been the recent comparison of the SAD/ß2 hybrid receptor and the vertebrate {alpha}4/ß2 heteromer (Matsuda et al., 1998Go). These authors compared the actions of imidacloprid and other nAChR ligands ((+)-epibatidine, (-)-nicotine and ACh) on both expressed recombinant receptors. Imidacloprid, alone of the 4 agonists, behaved as a partial agonist on the {alpha}4/ß2 receptor; (+)-epibatidine, (-)-nicotine, and ACh were all full, or near-full agonists. Imidacloprid was a partial agonist on the hybrid Drosophila SAD/ß2 receptor, as was also the case for (-)-nicotine, whereas (+)-epibatidine and ACh were full agonists. The EC50 for imidacloprid was reduced by replacing the vertebrate {alpha}4 subunit by Drosophila SAD. This substitution resulted in an increase in EC50 for the 3 other ligands tested. Thus the SAD subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nAChRs.

In conclusion, genetic, genomic and functional studies are enhancing our understanding of molecular and functional diversity in nAChR gene families and are providing insights into subunits targeted by anthelmintic drugs and insecticides.

Pharmacological Properties of Central Nicotinic Receptors (Billy R. Martin)

The pharmacological effects of nicotine are extremely complex as a result of several factors. First, nicotine produces a wide range of effects on the central nervous system, including excitation, sedation, analgesia, cognitive alterations, hypothermia, seizures, etc., depending upon the dose. Secondly, tachyphylaxis develops readily to many of nicotine's effects, which complicates efforts to systematically characterize this pharmacological profile (Damaj et al., 1996bGo; Damaj and Martin, 1996Go). The recognition that nicotine is one of the most highly addictive drugs has attracted considerable attention in recent times and has led to increased efforts to elucidate the mechanisms by which nicotine produces central effects. Moreover, understanding the actions of nicotine in the central nervous system provides a strategy for development of smoking cessation therapies devoid of serious nicotine side effects.

Most of nicotine's effects have been attributed to its interaction with the cholinergic ion channels {alpha}4ß2, {alpha}2ß2, and {alpha}7. Nicotine either stimulates or depresses spontaneous activity under some circumstances, reduces body temperature, generates a discriminative stimulus, and produces antinociception, all of which are blocked by the ganglionic antagonist mecamylamine. Nicotine exhibits high affinity for a binding site in brain tissue that is thought to represent primarily the pentameric ion channel {alpha}4ß2. Structure-activity relationship studies have established a reasonable correlation between affinity for 3H-nicotine-labeled binding sites and depression of spontaneous activity and analgesia, particularly when only those compounds that are antagonized by mecamylamine are considered (Damaj et al., 1996aGo). These structure-activity relationship studies have been aided by the discovery of the highly potent epibatidine that greatly extended the potency range under consideration (Damaj et al., 1994Go). More recently, the emphasis has been on development of agonists and antagonists with selective pharmacological profiles, in order to further characterize the effects that are mediated via {alpha}4ß2 receptors. One such compound is metanicotine, which is active in a wide range of analgesic tests but is devoid of depressant properties and seizure activity, even at very high doses (Damaj et al., 1999Go). This profile contrasts that of nicotine, which is equiactive in depressing spontaneous activity and producing analgesia, and producing seizures at slightly higher doses. Metanicotine is almost 10 times more potent at {alpha}4ß2 than {alpha}3ß2 receptors expressed in Xenopus oocytes. The involvement of {alpha}4ß2 receptors in analgesia remains to be fully characterized. However, the findings that nicotine was devoid of activity in the hot-plate test and considerably less potent in the tail-flick procedure in {alpha}4 and ß2 "knockout" mice established that these receptor subunits are critical for nicotine analgesic activity (Marubio et al., 1999Go).

There have been suggestions that nicotine-induced seizures are mediated through the homologous {alpha}7 pentameric receptor subtype. Nicotinic agonists produce seizures to varying degrees, and their potency does not appear to be directly related to affinity for 3H-nicotine-labeled binding sites. Seizures are blocked by both mecamylamine and methyllycaconitine, the latter of which has some preference for {alpha}7 subtypes. However, nicotine-induced seizures are highly susceptible to agents that alter intracellular calcium levels, an observation that underscores the role of receptor and non-receptor mechanisms (Damaj and Martin, unpublished observations).

Presently, it is difficult to assign specific pharmacological effects to nicotine receptor subtypes. However, {alpha}4ß2 receptors are strongly implicated in nicotine-induced analgesia as well as in depression of the central nervous system. The {alpha}7 receptor is undoubtedly involved in nicotine-induced seizures. Presently, it is not possible to establish whether other receptor subtypes may also play a role in some of these actions. Further development of selective agonists and antagonists for all receptor subtypes, coupled with a better characterization of receptor subtypes in the central nervous system, will provide new strategies for elucidating the physiological role of nicotinic receptors and for developing pharmacotherapies for diseases of the cholinergic nervous system.

Chronic Nicotine Infusion Effects on Memory: A Ventral Hippocampal Mechanism (Edward D. Levin)

Nicotinic systems are involved in a wide variety of neurobehavioral functions. Some effects of nicotine such as cognitive enhancement hold promise for the development of therapeutic treatments for cognitive dysfunction (Arneric et al., 1995Go; Levin et al., 1993aGo; Warburton, 1992Go). For the development of nicotinic-based therapeutics, it is important to determine the critical mechanisms for the therapeutic effect in order to guide the development of novel nicotinic drugs that are effective with minimal side effects.

Nicotine has been widely found to improve cognitive performance in experimental animals (Levin and Simon, 1998Go). In an experimental rat model, we have found that chronic 4-week nicotine infusion with osmotic minipumps significantly improves working memory performance in the radial-arm maze (Levin et al., 1997Go) (Fig. 3Go). Important for the potential therapeutic use of nicotine is the finding that the memory enhancing effect does not diminish with chronic administration. With chronic infusion over a period of 4 weeks via osmotic minipumps, the memory enhancement caused by nicotine did not diminish (Levin et al., 1993aGo). In fact, with 3 weeks of high-dose infusion, there was a persistence of the memory improvement for at least 2 weeks after withdrawal (Levin et al., 1990Go). Chronic nicotine infusion selectively improved working memory without significantly affecting reference memory (Levin et al., 1996bGo).



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FIG. 3. Chronic nicotine infusion effects on 8-arm radial maze choice accuracy. Seven studies (mean ± standard error of the mean). The means are for 3 weeks of testing (twice per week) during nicotine infusion (SC) and for 2 weeks after withdrawal. Entries to repeat are the number of correct arm entries before an error is made. Chronic nicotine infusion significantly (p < 0.005) improved choice accuracy during the period of dosing. After withdrawal, the rats given the lower nicotine dose (5 mg/kg/day) were not different from controls, while the rats given the higher dose (12 mg/kg/day) had a persistent improvement in accuracy that was significantly better than controls (p < 0.05).

 
In humans, nicotine administration via skin patch has been found in our studies to significantly improve attentional performance in patients with Alzheimer's disease, schizophrenics, adults with attention deficit/hyperactivity disorder, and normal nonsmoking adults (Jones et al., 1992Go; Levin et al., 1996aGo,bGo, 1998Go; White and Levin, 1999Go). Since nicotine has been shown to have adverse effects in terms of cardiovascular effects and the development of dependence, it is important to determine the critical mechanisms for the potential therapeutic effects, so that the side effects can be avoided and the potential therapeutic effects enhanced.

Work with the experimental rat model has revealed that the ventral hippocampus is critical to nicotinic involvement in memory function. We found local infusions of nicotinic antagonists into the ventral hippocampus to cause significant working memory impairments. Mecamylamine, as well as the specific {alpha}4ß2 antagonist DHßE and the specific {alpha}7 antagonist MLA caused significant impairments in working memory performance on the radial-arm maze (Fig. 4Go) (Felix and Levin, 1997Go; Kim and Levin, 1996Go). In a recent study using the 16-arm maze, we have replicated these effects at lower doses and have found more pronounced effects with working vs. reference memory (Levin et al., 1999bGo). Given these effects, we hypothesized that this area is a critical locus for chronic nicotine infusion-induced memory improvements.



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FIG. 4. Acute ventral hippocampal infusions of the {alpha}7 nicotinic antagonist MLA and the {alpha}4ß2 nicotinic antagonist DHßE caused significant (p < 0.025) dose-related impairments in choice accuracy (entries to repeat) in the 8-arm radial maze (mean ± standard error of the mean).

 
Knife cut lesions of the fimbria-fornix did not impair the memory improvements caused by chronic nicotine administration (Levin et al., 1993bGo). In contrast, small ibotenic acid lesions in the ventral hippocampus blocked the nicotine effect (Levin et al., 1999cGo). Half of the rats with each lesion treatment were given subcutaneous infusions of nicotine with osmotic minipumps (5 mg/kg/day) or saline. As seen previously, chronic nicotine infusion significantly (p < 0.05) improved working memory performance in the sham-lesioned group. The ventral hippocampal lesion blocked this effect even though by itself this small lesion did not impair working memory performance (Fig. 5Go). These data show that the ventral hippocampus is critical for chronic nicotine-induced memory improvements. Recently, we found that the selective {alpha}7 nicotinic agonist AR-R 17779 also reversed working memory deficits caused by fimbria-fornix knife cut lesions in rats (Levin et al., 1999aGo). This mechanism further demonstrates the importance of hippocampal nicotinic systems for cognitive function and specifies the relevance of {alpha}7 receptors.



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FIG. 5. Chronic nicotine infusion (5 mg/kg/day) caused a significant improvement in 8-arm radial maze choice accuracy (entries to repeat) in sham-operated rats. This effect was abolished in the rats with small ibotenic acid lesions to the ventral hippocampus (mean ± standard error of the mean). The small lesions themselves did not impair accuracy.

 
These data show that the ventral hippocampus is critical for nicotinic involvement in memory function and that the memory improvement caused by chronic nicotine infusion is blocked by small lesions within the hippocampus (Levin et al., 1999cGo. Given that lesions of the septohippocampal fiber bundles did not block this effect, it seems that nicotinic receptors located presynaptic to this connection were not critical for the chronic nicotine effect. Intrahippocampal, ibotenic-acid lesions did block the effect, suggesting that nicotinic receptors postsynaptic to the septohippocampal pathway were critical for the chronic nicotine-induced memory improvement. These findings indicate that both {alpha}4ß2 and {alpha}7 nicotinic receptors within the hippocampus are critically important for memory.

NOTES

1 To whom correspondence should be addressed. Fax: (312) 503-1700. E-mail: tna597{at}northwestern.edu. Back

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