The first step in processing nicotine's effects on the brain is the drug's interaction with neuronal nicotinic receptors (nAChR). The diversity of nAChR subtypes, their various modes of response (activation, desensitisation, prolonged inactivation), and the complex pharmacokinetics of nicotine delivery conspire to make this a complex issue that is difficult to unravel. The alpha4beta2 nAChR subtype has the highest affinity for nicotine and is the primary candidate for mediating nicotine's central effects. Chronic nicotine exposure (in both humans, animals and cell culture systems) leads to an increase in numbers of alpha4beta2 nAChR (upregulation), with functional implications for withdrawal. However, there is little evidence presently that nAChR upregulation is pertinent to the induction or maintenance of dependence. However, the particular characteristics of the alpha7 subtype of nAChR suggest that it may participate in long term changes in synaptic efficacy that could be relevant to nicotine dependence.
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INTRODUCTION FOR NICOTINE ADDICTION WORKSHOP BY MARCUS MUNAFO AND MIKE MURPHY: The ICRF General Practice Research Group in Oxford recently hosted a workshop on the theme of "Neuroscience, molecular genetics and nicotine addiction". It was chaired by Professor Neal Benowitz from the University of California at San Francisco, a specialist in the field of nicotine pharmacology spending an invited week in the University to work with the Group, and included about 30 other experts from groups around the UK and Sweden, including the ICRF Molecular Pharmacology Unit in Dundee and the ICRF Health Behaviour unit in London. Their backgrounds included neuroscience, molecular genetics, genetic epidemiology, pharmacology, psychology and clinical medicine, but they were united in a belief about the importance of tobacco control.
It is now recognised that addiction to nicotine lies at the heart of the tobacco control problem. Without the marketing of tobacco its use would dwindle. But while it is so widely promoted smoking research focuses heavily on factors affecting why people continue smoking after trying it and how to get them to stop with psychological support and the use of effective drugs. Although tobacco use is rightly viewed as a social and psychological problem we also need to see it as a pharmacological one with a potential genetic contribution to the addictive process. The need to integrate knowledge about the brain chemistry underlying addiction, the molecular genetics of these cellular processes, and how the drugs which are effective work, was the workshop's subject.
Each of the meeting's 4 sessions kicked off with a brief introduction by speakers who had precirculated draft papers. The sessions considered in turn: (1)how nicotine is metabolised and cleared from the body (2)the brain receptors in which nicotine "docks" to exert its effects (3)the brain pathways involving the neurotransmitter,dopamine,which are thought to contribute to the rewarding properties of nicotine which reinforce a smoker's desire to smoke (4)other non-dopamine pathways that are activated by nicotine and may underly such effects as nicotine withdrawal symptoms.
Understanding how the drugs which are effective (Nicotine Replacement Therapy and some antidepressants) interact with the expression of individual genotypes in the brain and elsewhere to predict success in quitting smoking will help to shed light on which bits of the brain are important in nicotine addiction at both the anatomical and cellular level. The workshop participants emerged pleased to have caught up with advances in fields which were not their speciality and with a strong sense that more enduring closer contact and cooperation will be possible in the future.
1.The contribution that nAChRs might make to the addiction process will depend on factors such as:
-where nAChRs are localised in the brain -their sensitivity to nicotine -nAChR activation versus desensitisation -altered responses after chronic exposure to nicotine
These properties will vary for the different subtypes of nAChR, but as nAChR subtypes have not been fully characterised with respect to their constituent subunits, many aspects of their functional contributions remain poorly defined.
2. nAChR are widely expressed throughout the CNS, albeit at low levels (10-100 times lower than muscarinic receptors), but different subunits show distinct anatomical localisations. The association of nAChR with dopamine pathways is perhaps most relevant to the psychomotor stimulant properties of nicotine. The ventral tegmental area (VTA) and substantia nigra (SN) express mRNAs for a large portfolio of nAChR subunits: alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, (but probably not alpha2 or beta4) (Wada et al., 1989; Le Novere et al., 1996). This suggests that dopaminergic neurones contain multiple nAChR subtypes, and there is pharmacological evidence for both alpha3beta2* and alpha4beta2 subunit combinations (Sharples et al., 2000) (where * denotes the possible presence of other subunits, Lukas et al., 2000). Particularly intriguing is the high expression of alpha6 and beta3 subunits in VTA and SN (Le Novere et al., 1996; Goldner et al., 1997). The expression of these subunits in the brain is very restricted but they are found in high abundance in catacholaminergic nuclei, suggesting a particular relationship with dopamine and noradrenaline systems. The properties conferred by the inclusion of alpha6 and/or beta3 to nAChR function or regulation are generally not known at the present time.
3. Different subtypes of nAChR differ in their sensitivity to agonists, including nicotine. This will determine which nAChR subtypes could be activated during smoking. The alpha4beta2 subunit combination has the greatest sensitivity to nicotine, with an EC50 value of 1-5 microM for the human alpha4beta2 subtype (Gotti et al., 1997). This is the only major nAChR subtype that can be identified by the binding of tritiated nicotine: [3H]nicotine binding is completely absent from the brains of transgenic mice lacking beta2 subunit (Picciotto et al., 1995) and largely absent (except for the interpeduncular nucleus) from those lacking the alpha4 subunit (Marubio et al., 1999). (Although all nAChR recognise nicotine by definition, only the alpha4beta2 subtype has sufficient affinity for nicotine for it to be useful as a radioligand - see below).
4. However, it is important to remember that agonists (including nicotine) can also lead to the desensitisation (short term) or inactivation (longer term) of nAChRs. Agonist binding can result in the transient activation of the nAChR, with a conformational charge causing the central ion channel to open, allowing a flow of cations. But the open state of the channel has a finite lifetime and automatically closes as the nAChR adopts another conformation: the desensitised state (of which there are multiple configurations), in which the channel is closed, the nAChR is refractory to activation but can still bind agonist, now with higher affinity than when in the open state. Subsequently, the nAChR reverts to the resting state, with channel closed but no agonist bound: in this state it is receptive to activation again (Changeux & Edelstein, 1998). For example, for the alpha4beta2 nAChR, nicotine concentrations greater than approx. 0.1 microM will activate the nAChR, converting it to the open state, whereas the desensitised conformation binds nicotine with about 100 times higher affinity (i.e., lower concentration), in the range 1-10 nM. A further complication is that sustained low concentrations of nicotine (1-10nM for alpha4beta2 nAChR) can convert the nAChR to the desensitised state without prior activation. Radiolabelled agonists will stabilise the desensitised (high affinity) state in ligand binding assays (because the tissue is exposed to the agonist for a long time: minutes - hours). Hence equilibrium binding constants reflect the desensitised state of the nAChR (Lippiello et al., 1987).
5. Cigarette smoking is characterised by the repeated inhalation of tobacco smoke which generates boli of nicotine delivered to the brain, superimposed on a relatively stable basal level of plasma nicotine maintained throughout the smoking day. This basal level of nicotine will keep a proportion of nAChR in the desensitised state, while the remaining population of nAChR will be available for activation by nicotine boli, if appropriate concentrations are achieved. Thus we can see how smokers may manipulate their plasma nicotine profiles to achieve a balance of desensitisation versus activation that suits them, c.f. anecdotal smoking for relaxation versus smoking for stimulation. This will also explain why the first cigarette of the day is the most satisfying, as overnight abstinence will have allowed substantial recovery of nAChR from desensitisation.
6. Given that the resting - active - desensitised relationship will differ between nAChR subtypes, and that the concentration of nicotine delivered to the brain is subtly controlled by the smoker, a very complex picture of nAChR responses emerges.
7. It is well documented that the numbers of [3H]nicotine binding sites (alpha4beta2 nAChR) are increased in the brains of smokers examined post mortem (Benwell et al., 1988; Breese et al., 1997), and in the brains of rodents given doses of nicotine daily for a few days (Wonnacott, 1990). This effect can also be produced in cell culture, even when nAChR are expressed heterologously in non-neuronal cells (e.g., Whiteaker et al., 1998). Other nAChR subtypes may also be upregulated but only at higher concentrations of nicotine. The 'upregulation' of receptors was unexpected but is probably a response to desensitisation of the receptor by chronic nicotine exposure (Fenster et al., 1999). There are conflicting reports about the functional status of nAChR after chronic nicotine treatment, with increased, decreased and unchanged levels of responsiveness being reported (Marshall et al., 1997; Hsu et al., 1996). Chronic exposure to nicotine not only transiently desensitises nAChR but can also result in permanent inactivation. alpha4beta2 nAChR are more prone to inactivation than alpha3beta2 * nAChR (Kuryatov et al., 2000). Long lasting inactivation has been seen in native systems, e.g., nicotine-evoked dopamine release from striatal synaptosomes shows partial dose- and time-dependent inactivation (no recovery after one hour; Rowell & Duggan, 1998). Both alpha4beta2 * and alpha3beta2 * nAChR mediate this response (Sharples et al., 2000). Inactivation may reflect the removal of nAChR from the cell surface into an intracellular compartment where the nAChR can still bind hydrophobic ligands.
8. The consensus view is that upregulation of nAChR numbers is an attempt to compensate for the loss of nAChR function through desensitisation, but that compensation may be inadequate (or confounded by removal of nAChR to a functionally irrelevant compartment). However, under circumstances in which nicotine is cleared from the system and desensitisation is reversed, the increased number of receptors could produce an excessive (rebound) response to agonist (including the endogenous agonist acetylcholine). Thus nAChR upregulation may contribute to withdrawal symptoms on quitting smoking.
9. The exact cause and mechanisms of upregulation remain controversial. However, we may ask if this response is relevant to the development of nicotine dependence. Clearly the first step in developing dependence must be the interaction of nicotine with its receptors: indeed, absence of the beta2 subunit in transgenic mice abolishes nicotine self-administration (Picciotto et al., 1998). However, the reversible nature of nAChR upregulation (with a return to normal levels in just a few days in animal models (Wonnacott, 1990), and normal levels are found in ex-smokers (Breese et al., 1997)) is at odds with the long term susceptibility to relapse of smokers after quitting, suggesting that there are other, long term changes in the brain. Rats treated chronically with nicotine by a variety of methods (daily injection, slow release from implanted minipumps, oral consumption) show upregulation of [3H]nicotine binding sites. Dependence, on the other hand, is highly correlated with the rate of access of drug to the brain. Although the nicotine administration routes by injection and minipump are widely used as models of chronic nicotine exposure, neither method adequately models the complex pattern of nicotine delivery achieved by cigarette smoking. It seems probable that peak doses of nicotine delivered to the brain by smokers provoke long-lasting changes downstream from nAChR activation, in dopamine neurones or in brain circuitry.
10. However, there is one nAChR subtype that may be a candidate for long term changes underlying the addiction process. This is the alpha7 nAChR. This subtype is highly calcium permeable (Seguela et al., 1993), has been proposed to participate in synaptic changes akin to long term potentiation (Hunter et al., 1994), is often associated with terminals containing the major excitatory transmitter glutamate (Gray et al., 1996), and is proposed to modulate glutamate synapses onto dopamine neurones in the VTA (Schilstrom et al., 1998). Thus nicotine might influence synaptic remodelling through the alpha7 subtype. It is not very sensitive to nicotine (EC50 = 40 microM; Gotti et al., 1997) and desensitises rapidly, but one study has suggested that 100 nM nicotine may be sufficient to elicit modulation of glutamate transmission via alpha7 nAChR (Gray et al., 1996). Further study of the role of alpha7 nAChR in models of nicotine dependence are warranted, to establish if this nAChR subtype plays a role in the long term changes that underpin drug addiction.
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