R. JAIN AND K. MUKHERJEE
Correspondence: Raka Jain
E-mail: rakajain2001@yahoo.com ABSTRACT
BIOLOGICAL BASIS OF NICOTINE ADDICTION
R. JAIN, K. MUKHERJEE
National Drug Dependence Treatment Centre, All India Institute of Medical Sciences, New Delhi-110 029.
Manuscript Received: 17.12.2002 Revised: 21.4.2003 Accepted: 4.6.2003
Nicotine, the primary component of tobacco produces reinforcing effects both in humans and animals. The neurochemical, anatomical and molecular basis of nicotine dependence is very similar to the other drugs of abuse, particularly the psychostimulants. Nicotine has specific acetylcholine receptors (nAChRs) in the brain and other organs. The stimulation of presynaptic acetylcholine receptors increases trans-mitter release as well as the metabolism. Nicotine, like other drugs of abuse triggers the dopamine reward system and increases the extracellular level of dopamine in nucleus accumbens (NAc), which is thought to be responsible for the reinforcing behavior, stimulant and dependence properties of nico-tine. Nicotine also exerts its effect in the brain on non-dopaminergic structures that may account for its positive rewarding effects and some of the symptoms of nicotine withdrawal. Moreover, the actions of nicotine on many systems, including brain stem cholinergic, GABAergic, noradrenergic, and seroton-ergic nuclei, may also help to mediate nicotine effects related to addiction. Further, the neurochemical pathway to nicotine reinforcement is likely to be due to binding of nicotine to nAChRs containing the β
2
subunit, followed by activation of the mesolimbic dopamine system and an initiation of common mo-lecular changes that underlie drug dependence. Furthermore, the constituents in tobacco inhibit both forms of the enzyme monoamine oxidase (MAO-A and MAO-B). This enzyme is important in the breakdown of the amine neurotransmitters, including dopamine, which is thought to mediate the rein-forcing effects of nicotine and contribute to tobacco dependence. Several questions remain regarding nicotine addiction and further research is needed in this direction. Molecular genetic techniques, coupled with behavioral analysis, may prove to be very useful tools for addressing these questions in near future.
Dependence dopamine tolerance
Introduction
Nicotine, is derived from the leaves of tobacco be-longing to the family Nicotiana tabacum and has been in use for centuries. It can be smoked, chewed, or sniffed. Nicotine was isolated from tobacco leaves in 1828 by Posselt and Reimanbasic, since then scientists began studying its effects in the brain and body. This research eventually showed that, although tobacco contains thousands of chemicals, the most active ingredient that acts in the brain and produces addiction is nicotine. Nicotine is an alkaloid (1-me-thyl-2-[3-pyridyl] pyrrolidine). It is the primary
com-ponent of tobacco that leads to addiction. More re-cent research has shown that the addiction produced by nicotine is extremely powerful and is atleast as strong as addictions to other drugs such as heroin and cocaine. In recent years, the use of tobacco has taken a great toll on youth and society. In fact, to-bacco is the gateway drug to other drugs of abuse such as marijuana and alcohol. The notable aspect about tobacco use is that it consistently occurs early in the sequence of problem behaviors. When a young person starts smoking or using tobacco, it is a sig-nal, an alarm that he or she may get involved in other risky behaviors.
Some of the effects of nicotine include changes in respiration, blood pressure, constriction of arteries, and increased alertness. Many of these effects are produced through its action on both the central and peripheral nervous system.
How nicotine acts?
Nicotine acts on the brain and other parts of the ner-vous system. When tobacco is smoked, nicotine enters the bloodstream through the lungs and reaches the brain faster than the drugs that enter the body directly through the veins. When it is chewed or sniffed, nicotine passes through the mu-cosal membranes of the mouth and nose to enter the bloodstream. Nicotine can also enter the blood-stream by passing through the skin. Nicotine reaches the brain within seven seconds. This sudden burst of nicotine causes an instant high blood pressure which is caused by the stimulation of the adrenal glands resulting in discharge of epinephrine. The release of adrenaline causes a sudden release of glucose as well as an increase in blood pressure, respiration and heart rate. In addition, nicotine indi-rectly causes release of dopamine in the brain re-gions that control pressure and motivation. Nicotine is distributed through out the body and brain where it activates specific types of receptors known as cho-linergic receptors.
Nicotine and the brain
Recent research has shown how nicotine acts on the brain to produce a number of behavioral effects. Nicotine readily crosses the blood brain barrier stimulating the nicotinic acetylcholine receptors (nAChR). These nicotinic cholinergic receptors are one of the major acetylcholine receptor subtypes. The stimulation of the presynaptic nAChRs on these neurons increases the transmitter release as well as the metabolism. Chronic administration of nico-tine results in desensitization and inactivation of nAChRs1,2 with subsequent up-regulation of nAChRs
sites. Cholinergic receptors are present in many brain regions especially concentrated in the midbrain tegmentum, the striatum, nucleus accumbens and the ventral tegmentum;3 as well as in muscles,
adrenal glands, the heart, and other organs. These receptors are normally activated by the neurotrans-mitter acetylcholine produced in the brain, and neu-rons in the peripheral nervous system.
Acetylcho-line and its receptors are involved in many activi-ties, including respiration, maintenance of heart rate, memory, alertness, and muscle contraction.
Besides binding to AChRs nicotine also binds to the cholinergic receptors in the autonomic ganglia, ad-renal medulla and the neuromuscular junction. The specific sites for binding in the brain are the hypo-thalamus, hippocampus, thalamus midbrain, brain stem and cerebral cortex. Nicotine also binds to re-ceptors in the nigrostriatal and mesolimbic dopam-inergic neurons. As and when the nicotine receptors are stimulated they release acetylcholine, nor-epi-nephrine, dopamine, serotonin, vasopressin, growth hormone and ACTH. Nicotine is one of the most po-tent stimulants of the mid brain dopamine reward pathway4-6. Nicotine acts on the locus ceruleus
regu-lating vigilance, arousal, concentration and stress reactions making the tobacco users more alert. Nicotine also alters the functions of some of the neu-rotransmitters implicated in the pathogenesis of some of the major psychiatric disorders. These in-clude dopamine, nor-epinephrine, serotonin (5-HT), glutamate, γ-amino butyric acid (GABA) and endog-enous opioid peptides7-9. These effects could be
pr-esynaptic, pre terminal or cell body nicotine recep-tors, rather than mediated through neurotransmis-sion wherein pre-synaptically released acetylcholine acts on postsynaptic, junctional nAChRs to cause neuronal firing10.
Biological theories of nicotine addiction
I. Dopamine and reward pathways
There is substantial evidence to suggest that nico-tine plays a vital role in maintaining the tobacco smoking habit and many habitual smokers become dependent on nicotine. Nicotine is a powerful rein-forcing agent in both animals and humans. The means by which nicotine produces addiction remain unclear. Behavior studies in animals do indicate that nicotine is an addictive drug that reinforces self-ad-ministration, place preference and increases locomotion. The effects of nicotine on tests of reinforcement and behavioral sensitization are pri-marily mediated through the mesolimbic dopamine system. The mesolimbocortical dopamine system consists of neurons with cell bodies localized in the ventral tegmental area (VTA) and axons projecting to
the nucleus accumbens (NAc) and the medial pre-frontal cortex (PFC), being referred to as mesolimbic and mesocortical projections, respectively. Nicotinic receptors concentrated in the VTA and NAc activate the mesolimbic dopamine system, which is thought to be responsible for the reinforcing behaviors like stimulant and dependence properties of nicotine. In this way nicotine is similar to other abused drugs such as amphetamine, cocaine, opiate and alcohol11. The
VTA and its projections to NAc are involved in reward and mediate the reinforcing actions of drug abuse12.
Recently, it has been shown, that key brain chemi-cal involved in mediating the desire to consume drugs is the neurotransmitter dopamine. In the brain, nico-tine stimulates the release of the dopamine in the pleasure circuit. Nicotine increases extracellular lev-els of dopamine in NAc13-15. Lesions of mesolimbic
dopamine neurons attenuate nicotine self-adminis-tration in rats16. It also attenuates the locomotor
stimulant effect of systemically administered nico-tine17. Local injections of nicotine or nicotine
ago-nists into the VTA can result in increased locomo-tion18,19. The strongest effects of nicotine appear to
be on the dopamine cell bodies of the VTA. Injection of nicotinic agonists locally into the dopamine termi-nal fields of NAc stimulates dopamine release20 or
locomotor activity21 indicating that nicotine receptors
on the dopamine terminal are involved in mediating the actions of nicotine.
Using microdialysis, a technique that allows minute quantities of neurotransmitters to be measured in precise brain areas, researchers have discovered that nicotine causes an increase in the release of dopamine in the nucleus accumbens. This release of dopamine is similar to that seen for other drugs of abuse, such as heroin and cocaine, and is thought to underlie the pleasurable sensation experienced by many smokers.
II. Other biological theories related to addiction to nicotine
Other research is providing even more clues as to how nicotine may exert its effects on the brain. Nico-tine produces its central effects through the nAChRs. The cholinergic receptors are relatively large struc-tures that consist of several components known as subunits. The nicotinic receptors are composed of 12 subunits (alpha 2-alpha 10 and beta2- beta10) which play the central role in autonomic
transmis-sion22. The different nicotinic receptors present in the
brain are gated-ion channels made of five subunits. Different combinations make different types of re-ceptors, which vary in terms of affinity and localiza-tion within the brain. One of these subunits, the
β subunit, has recently been implicated as having a role in nicotine addiction. The alpha 4-beta 2 sub-unit combination has the greatest sensitivity to nico-tine. Using highly sophisticated bioengineering tech-nologies, scientists were able to produce a new strain of mice in which the gene that produces the beta 2 subunit was missing. Without the gene for the beta subunit, these mice, which are known as "knockout" mice because a particular gene has been knocked out, were unable to produce any beta subunit5. These
knockout mice, in contrast to mice, with an intact beta subunit, would not self- administer nicotine. These studies demonstrate that the beta subunit plays a critical role in mediating the pleasurable ef-fects of nicotine. The results also provide scientists with valuable new information about how nicotine acts in the brain, an information that may eventually lead to better treatments for nicotine addiction. It is also important to mention that repeated inhala-tion of tobacco smoke generates boli of nicotine de-livered into the brain, superimposed on a relatively stable level of plasma nicotine maintained through out the smoking day. This basal level of nicotine will keep a proportion of nAChR in the desensitized state, while the remaining population of nAChR is avail-able for activation by nicotine boli, if appropriate con-centrations are achieved. This explains how smok-ers manipulate their plasma nicotine profiles to achieve balance desensitization versus activation. This also explains why the first cigarette of the day is the most satisfying, as overnight abstinence al-lows a substantial recovery from nAChR desensiti-zation. This further suggests that when a smoker is asleep, plasma level of nicotine decreases and the nicotinic receptors can gradually recover their ac-tive functional state. In the morning the smoker has a greater number of nAChR active receptor sites (up-regulation) and this might contribute to the de-velopment of withdrawal symptoms and craving. Moreover, it is also well documented that the num-ber of [3H] nicotinic binding sites (nAChR) are in-creased in the brain of smokers examined postmor-tem23,24 and in the brain of rodents given nicotine daily
for a few days25. Other nAChR subtypes may also
be upregulated but only at higher concentrations of nicotine. There are conflicting reports about the func-tional status of nAChR after chronic treatment, with increased, decreased and unchanged levels of re-sponsiveness being reported26. Chronic exposure to
nicotine transiently desensitizes nAChR but can also result in permanent inactivation. The alpha4 beta2 nAChR is more prone to inactivation than alpha3 beta2 nAChR27.
The exact cause and mechanisms of up-regulation remain controversial. However, the reversible nature of nAChR up-regulation (with a return to normal lev-els in few days in animal modlev-els25) is at odds with a
long term susceptibility to relapse (to smoking) sug-gesting that there are other, long term changes in the brain.
The interaction between nicotine and GABAergic system has been quite recently discovered. Since then, several electrophysiological studies have dem-onstrated that nicotinic agonists stimulate the release of GABA from rodent brain tissue and this release was Ca2+ dependent28,29. More recently, the actions
of nicotine on ventral tegmental GABAergic inter-neuron, which modulate the mesolimbic dopamine excitability, have been studied30. Using extracellular
recording techniques in rat brain slices, nicotine was found to increase the firing rate of dopamine and non-dopamine neurons, while the former was more vigorous. These results suggest that nicotine stimu-late the firing rate of dopaminergic neurons of VTA and also the GABA-ergic neurons, which may be an important target for the effects of nicotine on central nervous system. The less robust response in the non-dopamine presumptive GABAergic neurons and their more pronounced desensitization eventually leads to disinhibition of dopamine neurons thereby facilitating a more sustained increase in response of mesolimbic dopamine neurons to nicotine. Studies have shown that acute nicotine stimulates the release of NA in different parts of the brain, and nicotine acts primarily at the locus ceruleus level31.
Moreover, it was also found that in the hippocam-pus, maximal desensitization of nicotine- stimulated NA occurs as early as 40 minutes and persists for at least 100 minutes, thereafter, desensitization be-comes the dominant process32. It seems reasonable
to speculate that this could mediate the 'calming' ef-fects of cigarette smoking. More research is needed on nicotinic receptors and their desensitization state(s) to understand better about the role of nico-tine in different aspects of the dependence process. Tobacco smoking and chronic nicotine administra-tion decreases the concentraadministra-tion of 5-HT in the hip-pocampus33. This effect may reflect reduction in the
concentration of 5-HT because smoking is associ-ated with selective increase in the density of 5 HT1A receptors in this area. There is evidence that hip-pocampus receives serotonergic innervation from the median raphe nucleus. Suppression of 5- HT release in this part of hippocampus brings about anxiolytic response to nicotine when given locally by microin-jection into the dorsal hippocampus34. The effects
of nicotine on 5-HT are difficult to dissociate from those on dopamine neurons. Increased exposure to stressful stimuli is likely to increase the desire to smoke as reported by smokers35. The effects of
nico-tine withdrawal on dopamine release in the brain may exacerbate by the exposure to stressful stimuli and may underlie the role of stress as a factor in tobacco smoking, as well as the role of nicotine on reducing the effects by acting on 5-HT neurons within the hip-pocampus. Currently, there is little evidence for the involvement of the serotonergic system in the posi-tive reinforcing effects of nicotine, but there is some evidence that this system might be involved in the negative reinforcing effects of nicotine withdrawal. Recently, animal models have demonstrated com-monalties between nicotine withdrawal and opiate abstinence syndrome. These studies suggest that nicotine stimulation induce the release of endog-enous opioid peptides in various brain regions re-sulting in overactivation of opiate receptors. The con-sequence would be a state resembling opiate de-pendence. Abrupt termination of the sequence of nicotine stimulated endogenous opioids release and opiate receptor stimulation might then precipitate an opiate abstinence -like state36. Attempts to
demon-strate opioid modulation of smoking reinforcement (cigarette consumption and nicotine self-administra-tion) have been fraught with difficulty37. Further, it
has also been reported that acute treatment with nicotine produced a significant increase in preproenkephalin A mRNA levels in striatum and hippocampus, whereas the chronic treatment
decreases the same in these brain areas38.
Pre-treat-ing rats with mecamylamine blocked these effects of nicotine. This suggests that brain opioid system(s) might be involved in mediating nicotinic responses and its withdrawal, but again further research is needed.
There are some informations regarding the psycho-pharmacological effects of other tobacco alkaloids, or cotinine, the major nicotine metabolite. A recent study suggests that cotinine stimulates nicotinic re-ceptors to evoke the release of DA in a calcium-de-pendent manner from super fused rat straital slices39.
Using advanced neuro- imaging technology, it is pos-sible to actually see the dramatic effect of tobacco smoking on the brain of an awake and behaving hu-man being. Using positron emission tomography (PET), scientists discovered that cigarette smoking causes a marked decrease in the levels of an impor-tant enzyme, monoamineoxidase (MAO), that is re-sponsible for breaking down dopamine. The decrease in two forms of this enzyme MAO- A and B, results in an increase in dopamine levels. Research has shown that although nicotine causes increase in brain dopam-ine, nicotine itself does not alter MAO levels. It affects dopamine through other mechanisms. These leads to the possibility of another component of cigarette smoke other than nicotine may be inhibiting the MAO. Thus, there may be multiple ways by which smoking alters the neurotransmitter dopamine to ultimately produce feelings of pleasure and reward40-42.
In recent years research on the genetic component of cigarette smoking has increased our understand-ing of nicotine dependence. CYP2A6 is the enzyme responsible for the majority of the inactivation of nico-tine in humans. This enzyme is also responsible for activating tobacco-related procarcinogens such as the nitrosamines. A common genetic defect in nico-tine metabolism decreases smoking. Genetic varia-tion in the CYP2A6 gene may protect individuals from becoming nicotine- dependent smokers. Recent find-ings suggest that mimicking this gene defect by in-hibiting CYP2A6 decreases nicotine metabolism43.
Further research is needed in order to improve our understanding of how genetic variation in CYP2A6 alters the risk for nicotine dependence and lowers nicotine consumption. Furthermore, cigarette smok-ing, like other behaviors, shows evidence of genetic heterogeneity. Dopamine transporter (DAT) gene
(SLC6A3) encodes proteins that regulates synaptic levels of dopamine in the brain and is a candidate gene for addictive behaviors44. It is hoped that
re-cent advances in molecular biology, including the completion of the draft sequence of the human ge-nome may help in identifying gene markers that pre-dict a heightened risk of using tobacco and will in-crease our understanding of nicotine dependence45.
Preclinical studies
I. Learning and Memory
The potential role of nicotine has been a cognition enhancer46. Earlier studies reported that nicotine
im-proves learning or performance in several proce-dures of complex mazes, shock avoidance and attentional tasks. Recent studies have been more systematic in their approach and have been reas-sessing the effects of the nicotine in the light of cur-rent concepts of learning and memory. Effects of nico-tine can be defined more specifically in terms of bio-logical processes (attention tasks, spatial tasks, non -spatial tasks)47,48. Details of these are beyond the
scope of this review article. In brief, nicotine has been shown to produce a sort of place preference in rats and mice49,50. The self-administration of nicotine has
also been demonstrated in rats51,52 and mice53. In
rodents nicotine has anxiolytic-like actions in differ-ent behavioral tests namely the mirror chambered54,
the elevated plus maze55, the two compartment
light-dark transition test56 and fear potentiated startle57.
Nicotine and nicotinic agonists improve performance in a variety of cognitive tasks by animals with basal forebrain lesion58. Chronic nicotine intake affects
at-tention and working memory in rodents55. Nicotine
enhances acquisition of spatial radial maze and water maze tasks in normal animals. Rats treated with chronic nicotine for a week and then stopping it also showed enhanced acquisition of tasks as compared to the controls59.
II. Tolerance and dependence
Research on tolerance to nicotine in non-human animals is extensive and research on dependence in animals is expanding that includes studies of withdrawal signs upon cessation of chronic nicotine treatment60 and studies of the onset and persistence
of nicotine self administration61. However, to the best
of our knowledge, no animal study has directly related chronic nicotine tolerance to a measure of
nicotine dependence, such as withdrawal severity or persistence of nicotine self-administration. Wonnacott (1990) suggested that chronic exposure to nicotine increases high affinity binding of nicotinic agonists to brain tissue and induces chronic toler-ance to many of the drug's behavioral and physi-ological effects. The increase in receptor number (up-regulation) has been interpreted as a compensation for agonist-induced desensitization of nAChRs and this prolonged desensitization has been proposed as a mechanism to chronic tolerance to nicotine25,62.
Singer et al have shown the effects of 6-hydroxy-dopamine infusion in the nucleus accumbens on the acquisition of nicotine self administration. Similar attenuation lesions have also shown to produce in the locomotor activity by nicotine self-administration in a procedure that generates robust baseline59. The
effects of acute and chronic administration of nico-tine on locomotor activity have been studied. Admin-istration of nicotine to experimentally naive rats can depress locomotor activity, an effect to which acute and chronic tolerance can develop. In a conditioned taste aversion paradigm, rats learn to avoid consum-ing distinctively flavoured solutions that have previ-ously been paired with nicotine solutions. In rats exposed to test apparatus, nicotine produces mod-erate increase in activity and with repeated expo-sure to the drug, sensitization occurs. Effect of se-lective dopaminergic drugs in nicotine tolerance has also been studied. The result suggests that toler-ance to nicotine may be mediated through dopam-inergic system63.
Commonalties between nicotine and other drugs of abuse
The brain feels the effect of nicotine faster than it feels the effect of a shot of heroin in the arm. A com-mon feature that nicotine shares with non-psychostimulant drugs such as narcotic analgesic, delta-9-tetrahydrocannabinol and ethanol, is the ability of stimulating dopamine transmission prefer-entially in the shell of the nucleus accumbens by activating dopamine neurons that project to this area11,13,14,64. Psychostimulant drugs like
amphe-tamine, cocaine and phencyclidine preferentially stimulate dopamine transmission in the shell, but reduce the firing activity of dopamine neurons as a result of an interference with the dopamine reuptake carrier, leading to accumulation of dopamine extra-cellularly and stimulation of dopamine autoreceptors.
Nicotine dependence resembles non-psychos-timulant drugs with regard to its dopamine-stimulat-ing property upon an endogenous tone on µ-opioid receptors and on 5HT3-receptors.
Evidence also suggests that nicotine addiction, like addiction to other drugs of abuse, is likely to be the result of molecular changes in the brain areas that mediate reinforcement. Nicotine withdrawal has been shown to result in changes in the firing pattern of VTA neurons, implying that adaptation takes place in the mesolimbic dopamine neurons following chronic treatment65. Many drugs of abuse such as
cocaine, morphine and ethanol alter the mRNA lev-els, proteins or activity of tyrosine hydroxylase (TH) in the mesolimbic dopamine system. Like acute co-caine administration, acute administration of nico-tine increases TH activity in the NAc66. In addition,
chronic administration of nicotine can increase TH, mRNA levels and activity in the locus ceruleus, that has been reported following chronic morphine treat-ment67. These changes in the activity and levels of
the rate-limiting biosynthetic enzyme for catechola-mines are likely to be common markers for the de-velopment of dependence for these drugs of abuse. Furthermore, recently it has been reported that self-administration of either nicotine or cocaine results in increased expression of chronic fos-related antigens (fras) in the NAc, the prefrontal cortex and the me-dial caudate68. The common biochemical response
to nicotine and cocaine suggests that similar mecha-nisms may be involved in the development of de-pendence to these different drugs of abuse.
Finally, repeated exposure to nicotine induces adap-tive changes such as tolerance, and sensitization, in the level of the dopamine in the shell and in the core of the nucleus accumbens that resemble those of other drugs of abuse.
Conclusion
In conclusion, nicotine serves as a major reinforcer both in humans and animals. It is a complex behav-ioral phenomenon comprising effects on several neural systems. Recent studies have expanded ini-tial observations that the actions of nicotine on dopaminergic systems increase dopaminergic ac-tivity and release, leading to nicotine-induced rein-forcement. Further, the actions of nicotine on many
systems, including brain stem cholinergic, GABAergic, noradrenergic, and serotonergic nuclei, may also help to mediate nicotine effects related to addiction. The addiction to nicotine is not fully un-derstood as yet and several questions remain. Thus, further research is needed in this direction. The use of molecular genetic techniques, coupled with be-havioral analysis, will be very useful tools for ad-dressing these questions.
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