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Eery year, ore than fie iion peope ordide diefro the conseqences of soking. These deaths, prin-cipay fro ng cancer, are aoidabe. A foridabeobstace to the preention of these deaths is that tobaccocontains nicotine the aor, if not soe, copondresponsibe for driing the strong addiction to soking1.Of those ho try to qit soking, ony 35% are sccess-f ithot the se of nicotine repaceent therapies, andno ore than one-third are sccessf ith the2. Hocan a sipe cheica sbstance sch as nicotine haeso strong an effect on han behaior? Ansering thisqestion is becoing possibe no that the oecarand cear targets and the physioogica and behaioraeffects of nicotine are being nraeed.
The actions of nicotine are ediated by nicotinicacetychoine (ACh) receptors (nAChRs) (FIG. 1). Toincrease or nderstanding of the contribtion of differ-
ent nAChR oigoers3,4 to nicotine addiction, ne strate-gies hae been deeoped. These incde, first, deetions inice5 of neary a knon AChR sbnit genes6, and tar-geted knock-in gene tations yieding gain-of-fnctionreceptor sbnits7,8; second, the re-expression of adeeted gene either sing indcibe transgenic expres-sion systes9 or by stereotaxic inection of a entiira
ector carrying the issing gene10,11 or the reeant sainterfering RNA11,12; third, the qantitatie anaysis ofthe nerona firing patterns13 and behaiors14 eicitedby nicotine in these ice.
This Reie ais to bridge the gap fro genes tocognition in the nderstanding of nicotine addiction, on
the basis of the recent adances in the oecar bioogyof nAChRs and of ania odes ith odified nAChRgene expression.
Nicotinic receptors
Nicotine interacts ith a broad popation of nAChRhoopentaers and heteropentaers that are distribtedthroghot the PNS and CNS15. nAChRs are aostericebrane proteins that respond to ACh and nicotinicagonists by the fast opening (s to s range) of a cationicchanne that is pereabe to Na+, K+ and, in soe cases,Ca2+ ions4. In the PNS, nAChRs ediate the rapid, phasiceffects of oca, short-asting, high ACh ees. In the brain,fe exapes of fast transission are docented; bthere, nAChRs are aso the target of tonicay reeased AChin oer, odatory concentrations. Of key iportancefor the nderstanding of nicotine addiction, chronic expo-
sre to ACh or nicotinic drgs cases a grada decreasein the rate of this ionic response (100 s to intes),eading to a high-affinity, desensitized, cosed state ofthe receptor3,4,16 and to additiona ong-ter changes inreceptor properties. It aso cases an pregation of thenber of high-affinity receptors in the brain17.
nAChRs are transebrane oigoers consisting offie sbnits (FIG. 1) that rest, in aas, fro thecobinatoria asseby of different nAChR sbnitsencoded by 17 genes. Of these, nine genes encoding-sbnits and three encoding -sbnits are expressedin the brain. The arios nAChR sbnit cobinationsdiffer in their pharacoogica and kinetic properties4
Collge de France and the
Institut Pasteur CNRS URA
2182, 25 rue du Dr Roux,
75015 Paris, France.
e-mail: [email protected]
doi:10.1038/nrn2849
Nicotine addiction and nicotinicreceptors: lessons from geneticallymodified mice
Jean-Pierre Changeux
Abstract | The past decades have seen a revolution in our understanding of brain diseases
and in particular of drug addiction. This has been largely due to the identification of
neurotransmitter receptors and the development of animal models, which together haveenabled the investigation of brain functions from the molecular to the cognitive level.
Tobacco smoking, the principal yet avoidable cause of lung cancer is associated
with nicotine addiction. Recent studies in mice involving deletion and replacement of
nicotinic acetylcholine receptor subunits have begun to identify the molecular
mechanisms underlying nicotine addiction and might offer new therapeutic strategies
to treat this addiction.
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Extracellular
Intracellular
ACh ACh
b
Na+, K+, Ca2+
N
C
a
M1 M2 M3 M4
4
42
2
2
42
7
77
7
7
7c
d
HeteromericHomomeric
and aso in their cear and sbcear ocaizationin the brain15. The to ain types of nAChRs in thebrain are the 7 hoo-oigoer, characterized by a fastactiation, a o affinity and a high Ca2+ pereabiity;and the 42 hetero-oigoer, hich is typified by ahigh affinity and so desensitization3,4. 2 sbnit-containing nAChRs (2*nAChRs) and 7*nAChRs areidey expressed in the brain15, hereas other nAChRsbnits hae a ore restricted expression (FIG. 2). Theexistence of seera fnctiona oigoers ith anysbnit types, different actie site interface profies andbrain distribtions15 has ade it diffict to ecidatethe oecar echanis of nicotine addiction4.
CNS circuits in nicotine addiction
many brain areas are inoed in nicotine addiction(FIG. 2). These incde the dopainergic nerons inthe entra tegenta area (vTA; nces A10) of the
idbrain, hich proect to the prefronta cortex (PFC)and to ibic and striata strctres, in particar thences accbens6,17; and the extended aygdaa,the brain stress syste18, the hippocaps19, thehabeno-interpedncar syste20 and the prefronta(incding insar) cortex21. Fnctiona mRI stdieshae shon that acte nicotine inections in anaesthe-tized ice increase actiation in preibic, anteriorfronta, otor and soatosensory cortices, the ncesaccbens, vTA, sbstantia nigra and the thaas,bt not in the otor cortex and aygdaa22.
Addiction is ieed as the end point of a series oftransitions23,24 that are characterized by a copsionto seek and ontariy take the drg oing to its rein-forcing effects; this eads to a oss of contro of intakesch that it becoes habita and tiatey copsie(BOX 1). when access to the drg is preented, a nega-tie state eerges that anifests in soatic and affectiesigns, refecting a otiationa ithdraa syndroe24.Addiction ths eerges as a chronic reapsing diseaseof the brain.
Nicotine addiction differs fro other drg addictionsin that it has feer obios signs of the bingeintoxicationstage and a ess draatic ithdraa effect than, forexape, opioid or acoho addiction24. Nicotine sef-adinistration does not rest in a progressie escaationanifested by a copsiey increased drg intake24,25bt by a oderate change in a nicotine-rearded earn-ing process26. moreoer, there is itte eidence thatnicotine is absed in its pre for27.
Behavioural effects of nAChR subunits
To nderstand the effects of nicotine exposre thatead to addiction, hich are ediated by nAChRs, est exaine the roe of nAChR sbnits on beha-ior, earning and reard. The endogenos igand ofnAChRs is ACh; ths, deeting a particar nAChRsbnit ay affect the behaior of ice een in theabsence of nicotine.
Contribution of nAChRs to cognition and locomotion.Nicotine and nicotinic agonists enhance cognitieand psychootor behaiors and, conersey, nico-tinic antagonists and oss of nAChRs ipair cognitieperforance2831. Hoeer, these findings hae beendebated and the behaiora conseqences of knock-ing ot specific nAChR sbnits hae therefore beeninestigated.
mice acking the 2 nAChR sbnit (2/ ice)(FIG. 3) sho nora spatia orientation earning inthe ater aze5. By contrast, they do not sho thenicotine-indced eory enhanceent of an aoid-ance response to a id foot shock that is obseredin id-type anias5,9,32, indicating a contribtionof the 2 nAChR sbnit to the retention of aersieeory. In experients in hich to obects arepaced in an open fied, id-type ice progressieyincrease the nber of traectories beteen the toobects, hereas 2/ ice do not, indicating thatcognitie earning strategies inoing spatia e-ory ay aso reqire 2*nAChRs33. moreoer, in a
Figure 1 | stt nAcr. a | Nicotinic acetylcholine receptors (nAChRs) are
transmembrane oligomers consisting of five subunits. b | Each subunit consists of a large
aminoterminal extracellular domain with an immunoglobulinlike sandwich, a
transmembrane domain and a variable cytoplasmic domain. The extracellular domaincarries the acetylcholine (ACh)nicotine binding sites at the boundary between subunits.
The number of binding sites per pentamer ranges, depending on its composition, from
two (in muscle nAChRs or brain 42 nAChRs) to five (in the 7 homopentamer). Sites forallosteric modulators are located in the transmembrane domain. | The two main types
of brain nAChRs are the 7 homooligomer, characterized by a fast activation, alow affinity and a high Ca2+ permeability, and the 42 heterooligomer, typified by ahigh affinity and slow desensitization3,4,146. In addition to the principal subunits (2, 3, 4and6) and complementary subunits (2 and 4) of the nAChR, other subunits (5 and3), the role of which has been recently reevaluated6,37,113, form part of the organizationof various heterooligomers. | Side view of an 7 nAChR pentamer model, showing fivenicotine molecules (dark grey) in the binding sites and the volume of the ion channel
(dark blue). Parts a, and are reproduced, with permission, from ReF. 4 (2009)
Macmillan Publishers Ltd. All rights reserved.
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Prefrontal cortex:orbital, medial, cingulate and insula(executive control)
Amygdala(emotions)
427
242452347
424526236423
4(5)24(5)62623
347
22 (IPN)4234 (HB)3347
HBIPN(withdrawal signs)
Hypothalamus Brain stem(stress symptoms)
NAc shellcore(reinforcement) Dorsal striatum
(habits)
Hippocampus(contextual information)
VTA
SNpc
LDTg PPTg
42 42
Residentintruder test
A tst for social intraction and
aggrssiv bhaviour in
rodnts. An unfamiliar mous
(th intrudr) is introducd
into th cag of a mous that
has bn kpt isolatd in its
rsidnt cag for svral
months.
Navigation
Spontanous locomotor
bhaviour charactrizd by
larg movmnts at fast spd,
aimd at acquiring gnral
information about th
nvironmnt.
Exploration
Spontanous locomotor
bhaviour charactrizd by
small and slow movmnts,
nabling mor prcis
invstigation of thnvironmnt.
Nigrostriatal pathway
Th dopaminrgic pathway
from th substantia nigra to th
striatum, which is associatd
with motor control.
Mesolimbic pathway
Th dopaminrgic pathway
from th vntral tgmntal
ara to th nuclus accumbns
and limbic aras, which is
associatd with rward
procssing.
rsidntintrudr tst, 2/ resident ice sho oreapproach behaior and attept feer escapes to stopthe interaction, sggesting that 2 nAChR sbnitshae a roe in socia interaction33.
mose ocootor behaior as assessed in an openfied can be diided into navigation and xploration14,33.In 2/ ice10,33 the baance beteen these behaiorsis shifted in faor of naigation and their exporatory
behaior is different fro that of id-type ice14,33.This indicates a roe of 2*nAChRs in exporation strat-egies and decision aking 14. Re-expression of the 2sbnit in the vTA by stereotaxic gene expression10,11
restored exporatory behaior in an open fied ithotodifying naigation. Distinct nera echaniss thsnderie exporation and naigation.
This dissociation as frther inestigated by ea-ating the contribtion of endogenos ACh acting on2*nAChRs in the ascending nigrostriatal pathway andmsolimbic pathway11. Re-expression of 2 sbnits inthe sbstantia nigra pars copacta (SNpc) of 2 / icerestored naigation bt not exporation to id-type
ees; the opposite effect occrred hen 2 sbnitsere re-expressed in the vTA. Rests fro RNA sienc-ing experients ere consistent ith these findings 11.These data indicate a dissociation beteen the pstrea,choinergic, nAChR-ediated contro of the nigrostriatapathay and that of the esoibic pathay in regatingexporation and naigation11.
mice acking the gene encoding the 4 sbnit, the
principa partner of the 2 sbnit, hae nora baseineocootor actiity. Hoeer, copared ith id-typeice they sho a sstained increase in cocaine-eicitedocootor actiity and recoer ore qicky fronicotine-eicited ocootor sppression34. nAChRgain-of-fnction tations3,4 that rest in higherreceptor affinity and oss of desensitization of 4* and6*nAChRs case (in the absence of chronic nicotineexposre) increased ocootion and deficits in otorearning8,35. Ths, both the 4 sbnit and the 6 sb-nit contribte to spontaneos ocootor behaior12,hich as anticipated fro their association ith the 2sbnit in high-affinity oigoers3,4,15.
Figure 2 | Nna manm nvv n ntn atn: a m. Many brain areas contain nicotinic
acetylcholine receptor (nAChR) subunits and are involved in nicotine addiction. First, the somata of the dopaminergic
neurons that contribute to nicotine intake and reinforcement are in the ventral tegmental area (VTA) of the midbrain: they
project to the prefrontal cortex and to limbic areas, in particular the hippocampus and nucleus accumbens (NAc) in the
striatum10,147149. These VTA neurons receive cholinergic innervation from the pedunculopontine tegmental nucleus (PPTg)
and the adjacent laterodorsal tegmental nucleus (LDTg)150,151. Second, the emergence of a negative emotional state and
withdrawal syndrome following smoking cessation or nicotine deprivation mobilizes distinct neural circuits that can
include the extended amygdala and brain stress systems18, the hypothalamus, hippocampus19, substantia nigra pars
compacta (SNpc), and/or the habenulainterpeduncular (HBIPN) system20. Third, the switch from voluntary nicotine useto compulsive drug use may represent a global topdown gating transition from control by a prefrontal (cortical and
insular) global neuronal workspace (BOX 1) to subcortical (striatal) control21,82,130.
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Fear conditioning
A form of larning in which an
avrsiv stimulus (for xampl,
an lctric shock) is associatd
with a particular nutral
contxt (for xampl, a room)
or nutral stimulus (for
xampl, a ton), rsulting in
th xprssion of far
rsponss to th originally
nutral stimulus or contxt.
Fractional anisotropy
A paramtr in diffusion tnsor
imaging, which imags brain
structurs by masuring th
diffusion proprtis of watr
molculs. It provids
information about th
microstructural intgrity of
th whit mattr.
The 3 sbnit can assebe ith 6, 4 and 2sbnits in nigrostriata dopainergic pathays. miceacking the gene encoding the 3 sbnit sho ateredocootor actiity and prepse inhibition of the acos-tic starte response (both of hich are partiay regatedby dopainergic transission), spporting a roe for3*nAChRs in odating these behaiors36. Hoeer,behaiora changes eicited by nicotine hae not beentested in these ice.
3/ ice sffer fro high perinata ortaity, hichipairs research on the effects of nicotine in these ice.Copared ith id-type ice, heterozygos 3+/ iceere partiay resistant to nicotine-eicited seizres bthad nora sensitiity to the hypo-ocootor effectsof 0.5 g per kg nicotine (possiby oing to partiaexpression of this sbnit in the heterozygos ice)37.
mice acking 7 nAChRs38 sho nora contextaand far conditioning, spatia eory and anxiety39.Hoeer, in an attentiona task that reies on the integ-rity of the prefronta cortex (at east in rats40), 7/ iceere soer and ess efficient than their id-type it-
terates41. moreoer, ice acking both the 7 and 2sbnits sho greater ipairent in passie aoidanceearning than 2/ ice42.
In concsion, the aaiabe data fro ose std-ies are consistent ith a roe for actiation of 4*, 6*,2* and 7*nAChRs by endogenos ACh in ocootor
behaior and cognitie fnction. The data nderine thecontribtion of these sbnits in ascending dopain-ergic pathays and aso in ore eaborate, top-dongating strategies inoing attentiona contro (BOX 1;FIG. 2). The contribtion of 3 and 3 sbnits to cog-nition and ocootion has been noted bt needs fr-ther inestigation, together ith that of the sti pooryinestigated 5 sbnit.
Role of nAChR subunits in the rewarding effects of nicotine.The reinforcing effects of nicotine can be deonstratedin tests of oitiona nicotine sef-adinistration, hichcan be systeic (by intraenos infsion or introdctioninto the drinking ater43,44) or oca (by nicotine inectioninto the vTA10,45). when ice ere pried ith systeiccocaine sef-adinistration46 and cocaine as sbse-qenty sbstitted ith nicotine, id-type ice con-tined to sef-adinister nicotine, hereas 2/ ice didnot46,47. Drg-naie ice acking the 2, 4 and 6 sb-nits aso did not sef-adinister nicotine systeicay, btthis response as noraized hen these sbnits ere
re-expressed in the vTA44. By contrast, deetion of the7 sbnit did not affect systeic sef-adinistration ofnicotine. when oca, intra-vTA sef-adinistration astested, ice acking the 2 sbnit again did not shonicotine sef-adinistration10. Re-expressing the genein the vTA noraized this behaior. In 4/ ice,
Box 1 | Nicotine addiction and the global neuronal workspace
Addiction is viewed as the end point of a series of transitions from initial voluntary drug use to the loss of conscious
control over this behaviour. The neural correlates of consciousness have been the subject of abundant discussionand
model building. In particular, the global neuronal workspace (GNW) model130 proposes that conscious information
processing recruits networks of neurons with long-range axons that reciprocally connect distinct cortical areas,
including the prefrontal, parietal, temporal and cingulate cortices130133. In the GNW model, the striatum, hippocampus
and amygdala are under the top-down control of the prefrontal cortex (FIG. 2). According to this proposal, the GNW isengaged in decision-making and might therefore have a key role in the loss of control that characterizes the ultimate
stage of nicotine addiction3.
Patients with damage to the ventromedial prefrontal cortex and people who are addicted to drugs of abuse show
similar deficits in cognitive behaviour21. In addition, smokers with brain damage involving the insula undergo a
disruption of smoking addiction and are more likely to quit smoking than smokers with brain damage not involving the
insula134, an observation corroborated by a study in rats135. The insula sends excitatory projections to the nucleus
accumbens and acts in concert with regions that are involved in maintaining specific goals, such as the dorsolateral
prefrontal cortex136. Activation by nicotine might therefore influence the GNW, subverting decision-making to seek and
procure drugs134 and thereby hijacking the cognitive functions exerting inhibitory control to resist drug use21.
Consistent with this possibility, repeated high-frequency transcranial magnetic stimulation of the left dorsolateral
prefrontal cortex combined with either smoking-related or neutral cues reduced cigarette consumption and nicotine
dependence. Furthermore, this treatment blocked the craving induced by daily presentation of smoking-related
pictures137.
Relevant to a possible effect of chronic drug use on the GNW, diffusion tensor imaging studies have shown reduced
frontal white matter integrity in people who show cocaine dependence138 or abuse heroin139. The same method has
revealed that prenatal and adolescent exposure to tobacco smoke alters the development of the microstructure of the
white matter, with increased fractionalanisotropy in right and left frontal regions and in the genu of the corpus
callosum140. These observations suggested that nicotine might directly act on white matter. There is electrophysiological
evidence supporting a direct action of nicotine on axon conduction, possibly at the level of the node of Ranvier80,141,142.
Moreover, low levels of nicotine applied to thalamocortical axons in slice preparations enhance prefrontal cortical
activity80,143. These observations support a direct control of the GNW by nicotine at the white matter level and are
consistent with the expression of functional nicotinic acetylcholine receptors on prefrontal cortical neurons79. The
observations also support the proposal that a gating circuit modulates, in a top-down manner, the nicotine-elicited
activation of the reward system involving the ventral tegmental area101(FIG. 2), which is engaged in the volitional
consumption of nicotine. According to the GNW model, a functional relationship becomes established between nicotine
self-administration and the noradrenaline- and hypocretin-regulated state of awareness144,145, which is a prerequisite for
the self-discipline of conscious behaviour.
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FLAP XhoI NheI SalI BstBI
2 IRES2 EGFP WPRE 3-LTR
U31 kb
5-LTR
Genomic RNApackaging system
Central polypurine tract Central termination sequence(cPPT)
Rev responseelement
Gene ofinterestPromoter
PGK
Gene cluster
A group of nighbouring gns
on a chromosom.
Conditioned place
preference taskA tst for nicotin addiction
potntial in which th mous
rcivs a dos of nicotin in a
distinct nvironmnt, aftr
which th amount of tim th
mous spnds in that
nvironmnt is assssd.
Nicotine discrimination
Th ability of mic to
discriminat nicotin from
salin using a two-bar oprant
procdur with a tandm
schdul of food
rinforcmnt.
intra-vTA sef-adinistration initiay increased andthen decreased, and re-expression of the 4 sbnit in thevTA noraized sef-adinistration. Deetion of the 6sbnit had no significant effect on intracrania nicotinesef-adinistration14. Ths, 42* and 62*nAChRs,bt not7*nAChRs, are necessary and sfficient forsysteic nicotine reinforceent in drg-naie ice44;in intra-vTA sef-adinistration, the 6 sbnit hasno aor roe bt the 4 sbnit is reqired, togetherith the 2 sbnit, for ong-ter sef-adinistrationbehaior.
By contrast, deetion of 5 sbnits or oerexpressionof 4 sbnits enhanced nicotine sef-adinistration48,49,
sggesting a odatory action of these sbnits onnicotine reard pathays. Interestingy, the 5 sbnitis abndanty expressed in dopainergic nerons50.moreoer,the gene encoding this and the 3and 4sbnits for a gn clustr that has been associated ingenoe-ide association stdies ith ng cancer. Thegenes encoding the 3 and 6 sbnits aso for a genecster that has been associated ith nicotine dependence(see Sppeentary inforation S1 (box)).
The rearding effects of nicotine can aso be testedin the conditiond plac prfrnc task, in hich a doseof nicotine is paired ith a distinct enironenta ce.
mice acking the 2 sbnit did not sho nicotine-conditioned pace preference or nicotin discrimination,hereas 7/ ice did51,52. Knock-in ice expressinghypersensitie, high-affinity 4-containing receptors dis-payed conditioned pace preference ith nicotine con-centrations 50-fod oer than id-type ice8. This testaso shoed that the 6 sbnit has a roe in ediatingthe rearding effects of nicotine53.
Oera, these obserations proide eidence thatnAChRs containing 4,2 and to soe extent 5, 6and 4 sbnits ediate the rearding effects of nico-tine8,46, hereas the roe of the 7 sbnit is ess cear(hoeer, see ReF. 54).
Physiological effects of nAChR subunits
Differential effects of nAChR subunits on neural activityin the VTA. Becase the dopainergic syste of the vTAediates the reinforcing effects of nicotine55,56, nicotineis expected to odate firing of these dopainergicnerons. varios nAChR oigoers are indeed expressedon the GABA (-ainobtyric acid)-containinginternerons in the vTA, on excitatory or inhibitoryinpts to the vTA57 and on dopainergic vTA nerons.Dopainergic vTA nerons express nAChR oigoersith arios sbnit copositions (FIG. 4), the distrib-tion of hich ay differ in the soatodendritic andaxona copartents15.
Singe-ce recordings of vTA dopainergic neronsreea to nerona firing rhyths in vivo, hich occrboth spontaneosy and in the presence of nicotine: aso, regar singe-spike firing and a brsting ode58,59
(FIG. 5). The brst-firing ode cases a sbstantiayarger dopaine reease than regar spiking and greateractiation of iediate eary genes in the target areas ofthe dopainergic nerons60,61. The transition fro reg-ar (tonic) firing to brsting (phasic) actiity has beenassociated ith receiing reard-predicting stii andnpredicted reards62. Brst firing is absent in idbrainsice preparations (hich ack afferent fibres)63, indicat-
ing that it depends on the inpt to the dopainergicnerons, incding gtaatergic afferents originatingin part fro the PFC, and choinergic and gtaater-gic inpt fro the tegenta pedncopontine ncesand the aterodorsa tegenta nces, respectiey58,64,65
(FIG. 4). A coser anaysis of the firing patterns of vTAdopainergic nerons13 reeas for odes of spon-taneos firing in id-type anaesthetized ice: first, ao-freqency, regar firing ode ithot brsting(o freqency, o brsting); second, a o-freqencyfiring ode ith abndant brsts (o freqency, highbrsting); third, a high-freqency firing ode ithfe brsting eents (high freqency, o brsting); and
Figure 3 | Mt 2 bnt gn -xpn ng a ntva vt.The lentiviral expression vector contains a
bicistronic cassette that simultaneously expresses the 2 subunit and enhanced green fluorescent protein (EGFP) to enabledetection of transduced cells. Vectors were based on the construct termed pTRIPU3 from which the elongation factor 1(EF1) promoter was removed. The mouse phosphoglycerate kinase (PGK) promoter was amplified by PCR and inserted intothe plasmid, with the restriction sites EcoRI andBamHI upstream and downstream of the PGK promoter, respectively. To
create the 2internal ribosome entry site 2 (IRES2)EGFP construct, a newSalI restriction site was created 3 to the EGFPstop codon in the pIRES2EGFP expression plasmid by mutagenesis, and a newXhoI restriction site was created downstream
of theNheI restriction site. The wildtype mouse 2 subunit was then ligated between theXhoINheI sites of pIRES2EGFP.The2IRES2EGFP construct was then ligated into the pTRIPU3PGK vector usingXhoISalI sites. Finally, the WPRE(Woodchuck hepatitis posttranscriptional regulatory element) sequence was added using SalIBstBI sites. LTR, long terminal
repeat. Figure is modified, with permission, from ReF. 10 (2005) Macmillan Publishers Ltd. All rights reserved.
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NAc
VTA
LDTgPPTg
Glu GABA
GABA
462(3)4(5)262
42
42
42
42
42
7
7
4(5)24(5)6262(3)(34)7
ACh
AChDA
PFCPPTg
forth, a ode ith high-freqency firing and anybrsts (high freqency, high brsting) (FIG. 5). Thecontribtion of the arios nAChR sbnits to thesepatterns hae been inestigated in tant ice.
In vTA dopainergic nerons of 2/ ice, onythe o-freqency, o-brsting ode persisted (FIG. 5),hereas those of 7/ ice shoed ony the high-freqency, high-brsting ode. This sggests thathigh-freqency, high-brst firing reqire the actia-tion of 2* bt not 7*nAChRs. In id-type ice, anintraenos inection of nicotine cased a fast and ss-tained (~10 in) increase in firing freqency in vTAdopainergic nerons. This response as aboishedin 2/ ice46 and restored by entiira re-expressionof 2 sbnits in the vTA10; hoeer, this effect didnot persist for ore than 2 in10. This sggests thatthe sstained firing of dopainergic nerons in thevTA aso depends on the presence of 2*nAChRs inexcitatory strctres proecting to the vTA, sch asgtaate-containing nerons fro the prefronta cor-tex or ponto-tegenta afferents (FIG. 4). In 7/ ice,intraenos nicotine inections cased a arge and rapid
increase in the firing rate of dopainergic nerons13 thatas short-asting and fooed by a patea at a sightyoer firing rate (copared ith the initia increase).This sggests that the absence of 7*nAChR-ediatedgtaatergic excitation nasks an inhibitory effect ofGABAergic nerons (hich do not contain 7*nAChRs)(FIG. 4). The data ipy that a concoitant actiationof 7* and 2*nAChRs ay be necessary for the fexpression of eents eading to nicotine reinforceent,ith 2*nAChRs sitching nerons fro a resting to anexcited state (goba, tonic regation), and 7*nAChRsfiney tning this state after 2*nAChRs hae beenactiated13.
Intraenos nicotine inections in 4/ ice66 casedan increase in the firing rate in the vTA that as oerthan in id-type ice, ithot affecting the eanbrsting actiity. By contrast, intraenos nicotineinections cased a siiar response in vTA neronsof 6/ ice and id-type ice. As the spontaneosactiity of vTA nerons of 4/ and 6/ ice assiiar to that of id-type ice, these findings indicatethat the 4 sbnit, bt not the 6 sbnit, is reqiredfor the transition fro tonic to phasic firing that iscrcia for reinforceent14,66 (in particar, in response toreard-predicting stii and npredicted reards62).
As discssed aboe, the brsting actiity of vTAdopainergic nerons enhances dopaine reease in thestriat (FIG. 4). Tonic ACh reease fro striata choin-ergic (excitatory) internerons is thoght to controdopaine reease in the striat by actiating presyn-aptic nAChRs on the dopainergic terinas6770(FIG. 4).According to one hypothesis71, contined exposre tonicotine desensitizes these nAChRs and so enhances stri-ata dopaine reease fro brsts6770, casing faciitation
of dopaine-dependent reinforceent. In brain sices ofice acking the 6 or the 4 sbnit, dopaine reeasein the nces accbens is not odified by nicotineappication, in contrast to id-type brain sices, shoingthat both 6* and 4*nAChRs hae a key roe in the con-tro of striata dopaine reease by ACh or nicotine inthe nces accbens70. By faoring dopaine reeasefro brsts hie depressing dopaine reease frotonic signas, nicotine acting on 46*nAChRs effec-tiey increases the signa-to-noise ratio of reard-reatedbrst actiity aong dopaine afferents72.
The precise roes of 4 and 6sbnits in nicotinereinforceent ight depend on the differentia ceardistribtion of the fnctiona 4* and 6*nAChRs invTA dopainergic nerons (the 2 sbnit being nec-essary for both 4* and 6*nAChR actiation). Ths,462*nAChRs ight be preferentiay expressed onthe axon terinating in the nces accbens, ith42*nAChRs reaining in the soa. Hoeer, this hassti to be confired experientay.
Athogh nicotine targets nAChRs on both dopain-ergic and GABAergic nerons in the vTA (FIG. 4),it is ncear hich of the to neron types is oreiportant for ediating the effects of nicotine ondopaine reease in the nces accbens. In a stdyon brain sices73, nicotine appication cased a robstincrease in inhibitory postsynaptic crrent (IPSC)
freqency in dopainergic nerons of the vTA,hich as fooed by a decrease to beo baseineafter the reoa of nicotine. This effect as redcedby bocking 7*nAChRs, and copetey aboished bybocking non-7*nAChRs. The athors73 proposedthat the nicotine-eicited increase in IPSC freqencyas de to actiation of 42*nAChRs on GABAergicnerons (FIG. 4) rather than actiation of nAChRs ondopainergic nerons. As nicotine is knon to haean oera stiatory action on dopainergic ne-rons, the athors frther proposed that the transientnicotine-eicited increase in GABA transission todopainergic nerons is fooed by a desensitization
Figure 4 | nAcr bnt nt t ant an nt nntvty
pamng nn m t VTA. Dopaminergic neurons (DA; shown in red) in the
ventral tegmental area (VTA) receive two main types of excitatory inputs. First, from
cholinergic neurons (shown in blue) of the laterodorsal tegmental nucleus (LDTg) and
pedunculopontine nucleus (PPTg); and second, from glutamatergic neurons (shown in
green) of various sources, including the prefrontal cortex (PFC) and the PPTg.
Dopaminergic neurons also receive inhibitory input from GABA (aminobutyric acid)ergicneurons (shown in yellow) and send axonal projections to the nucleus accumbens (NAc),
where they receive inputs from intrinsic cholinergic interneurons (shown in blue).Behavioural and physiological studies with genetically modified mice are revealing the role
of the indicated nicotinic acetylcholine receptor (nAChR) subunits in nicotine reward.
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0
0 2 4 6 8 10
0 2 4 6 8 100 2 4 6 8 10
|
1 mV
1 sec
LFHB
LFHB
HFLBLFLB
100
20
40
60
80
0
100
20
40
60
80
100
0
20
40
60
80
Spikeinburst(%)
Spikeinburst(%)
Spikeinburst(%)
2/
WT
VTA Vec
HFHB
Mean frequency (Hz)
Mean frequency (Hz)Mean frequency (Hz)
LFLB
LFLBHFLB
a b
c d
Spike timing-dependentplasticity
A form of plasticity that rsults
from functional changs in
nurons and/or synapss and
that dpnds on th prcis
timing of action potntials in
connctd nurons.
Giant depolarizing
potentials
An arly typ of lctrical
activity that ariss in th
cours of dvlopmnt and is
probably du to xcitatory
actions of GABA.
of the 42*nAChRs, reoing the tonic choinergicdrie to GABAergic nerons and thereby disinhibitingdopainergic nerons73.
Conersey, in vivo stdies hae ephasized theiportance of 2*nAChRs on vTA dopainergic ne-rons in ediating the effect of nicotine. Intraenosinection of nicotine cased a fast, teporary bt robstincrease in the firing rate and brsting of these nerons13.This effect as aboished in 2/ ice10,46 and redcedin apitde and dration in 7/ ice. It as proposedthat nicotine acting on 2*nAChRs on dopainergicnerons cases a sitch fro a resting to an excitedstate of these ces, ith a ess iportant contribtion
fro 7*nAChRs.The differences of interpretation of the in vitro and
in vivo experients regarding the roe in reinforce-ent of nAChRs on GABAergic erss dopainergicnerons, respectiey, can be addressed sing a en-tiira syste to contro nAChR sbnit expressionspecificay in dopainergic or GABAergic nerons74.Preiinaryin vivo rests sho that 2/ ice inhich the 2 sbnit is excsiey re-expressed indopainergic nerons of the vTA actiey, thoghtransienty, sef-adinister nicotine75; this findingis consistent ith the proposa of the in vivo stdydescribed aboe13.
Physiological effects in other brain areas. Nicotine affectsattention and cognitie perforance in rodents, priatesand hans76,77, sggesting that the edia prefrontacortex (PFC) ight be a target of nicotine. Indeed, 2*and 7*nAChRs are expressed in the ose PFC5,15,78, andiontophoretic appication of nicotinic agonists to pyrai-da ces in ayers IIIII of the rat preibic area od-ates excitatory synaptic transission79. Aso, gtaatereease fro ayer v pyraida nerons in response tonicotine appied to thaaocortica inpt nerons is abo-ished in 2/ice, deonstrating a contribtion of thissbnit to inforation processing in the PFC80.
In sice preparations of ose PFC, spik timing-dpndntplasticity (STDP) can be eicited by pairing stiation ofthe excitatory inpts to pyraida nerons of PFC ayer vith postsynaptic spikes81. Nicotine increases the thresh-od for STDP, eiinating ong-ter potentiation (lTP)and casing synaptic depression of the excitatory inpts tothese ces. This inhibitory effect of nicotine sees to con-tradict the knon cognitie-enhancing effect of nicotine(see ReF. 81for a discssion), bt cod be expained by the
absence of certain featres ofin situ inforation processingin cortica sices13,63.
The PFC, aygdaa and hippocaps proidegtaatergic inpt to nerons in the nces accbens,hich are aso regated by dopainergic nerons inthe vTA (FIG. 2). The conergence of these inpts in thences accbens is thoght to be the nera basis ofa top-don contro, or gating fnction, of attention onnera processes82(BOX 1). Here, I briefy reie the roeof nAChRs in seera coponents of this circit.
Sitaneos extracear recordings of neronaactiity in the PFC, entra hippocaps and ncesaccbens in anaesthetized rats reea that the PFC isreqired for the actiation of the nces accbens bythe entra hippocaps83. Seera nAChR oigoers areexpressed in GABAergic and gtaatergic hippocapanerons. Nicotine enhances seera types of hippoca-ps-dependent earning and odates the indctionof lTP84, the ptatie cear sbstrate of earning andeory. Specificay, nicotine eicits lTP at hippoca-pa synapses throgh 7* and/or 42*nAChRs85 andpregates 4*nAChR ees in gtaate afferentsand GABAergic internerons86. At birth, ost hippo-capa gtaatergic synapses are iatre and fnc-tionay sient, bt they can be conerted presynapticayinto condctie synapses by a brief appication of nico-tine at iatre Schaffer coateraCA1 connections87.
Stdies ith 7/ and 2/ ice shoed that 7* and2*nAChRs are sfficient to odate nicotine-eicitedincreases in the freqency of spontaneosy occrringgiant dpolarizing potntials88. Recordings fro freey o-ing ice frther shoed that nicotine-eicited dopainesignaing in the hippocaps as necessary for lTP atentorhinadentate gyrs synapses89.
GABAergic internerons in the hippocapa stra-t oriensaes express 2 RNA90,91. Interestingy,2*nAChRs do not desensitize in response to nicotineexposre, and continos exposre to nicotine restsin sstained actiation of 2*nAChRs and contin-os discharge of these internerons92. This raises the
Figure 5 | r nAcr bnt n pntan p ng an bt ng n
VTA pamng nn. a | Sample traces showing two types of neuronal firing by
dopaminergic neurons in the ventral tegmental area (VTA): singlespike, regular firing (top)
and burst firing (bottom). A typical burst starts with a short interval (< 80 ms) and ends with
a long interval (> 160 ms). b |Mean firing frequency is plotted against the percentage of
spikes that are within a burst for individual cells of wildtype (WT) mice (b),2/ mice (),and in 2/ mice after reexpression of2 subunits in the VTA using a lentiviral vector (Vec)(). Four main groups of cells can be distinguished: highfiring, highbursting (HFHB) cells;
lowfiring, highbursting (LFHB) cells; highfiring, lowbursting (HFLB) cells; and lowfiring,
lowbursting (LFLB) cells. VTA neurons from2/ mice show only LFLB activity, whereasreexpressing the subunit in the VTA restores HFLB and LFHB activity. Figure is reproduced,
with permission, from ReF. 13 (2006) Cell Press.
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VTA
NAc
IFBNST
Chronic nicotine exposure
Increased glutamate output
Increased dopaminergicneuron bursting activity
Increased dopaminergicneuron activity
Increased dopamineneuron activity
42*nAChR upregulation
Presynaptic 2*nAChRdownregulation
Increased presynaptic7*nAChR transmission
Increaseddopamine releasea
c
b
Intracranial electrical
self-stimulation
A procdur in which rats work
(for xampl, prssing a lvr)
to obtain rwarding lctrical
slf-stimulation through an
lctrod implantd in th
brains rward systm. It is
usd to masur th snsitivity
of th brain rward systm
in vivo.
possibiity that odation of 2*nAChRs throghinhibitory internerons in the hippocaps contribtesto the oera circit. In concsion, 2, 4, 7 and 2nAChR sbnits contribte to the effect of nicotine onhippocapa synaptic pasticity.
Nicotine affects the entra hippocaps-to-accbens pathay. Preiinary findings in ice sg-gest that the pattern of phasic brsting actiity in thences accbens and the degree of hippocapsaccbens coherence are affected by nicotine andthat the atered patterns of actiity can persist ongafter nicotine has been reoed fro the syste93.This sggests a possibe echanis by hich nicotineaffects the concerted action of the nces accbensand the hippocaps throgh the top-don, gatingcontro of the so-caed goba nerona orkspace(BOX 1). Co-ctres of entra hippocaps ne-rons fro 7/ ice and nces accbens neronsfro id-type ice hae recenty deonstrated thecontribtion of the 7*nAChR to the hippocaps-to- accbens pathay. These ongoing experients
reea that 7*nAChRs are reqired to conert thetransient effects of nicotine that are obsered in thispreparation into ong-asting changes in synaptictransission94.
Both 7* and 2*nAChRs are expressed in the basaand atera aygdaa ncei95,96. A singe, o-doseadinistration of nicotine to ose basoatera ayg-daa sices eicits ong-asting faciitation of gtaa-tergic transission at cortico-aygdaa connections97.Pharacoogica stdies in id-type and 7/ icefrther reea that, in the basoatera aygdaa, actia-tion of presynaptic nAChRs containing the 7 sbnit(together ith non-7*nAChRs) faciitates gtaater-gic transission in an actiity-dependent anner 97. Bycontroing nerona actiity in the aygdaa, hich is abrain region knon to hae a roe in contexta earning(for exape, of soking-reated ces), nicotine cododate an iportant aspect of nicotine addiction76.
In sary, gene deetion and re-expression stdieshae reeaed distinct contribtions of 4, 6, 7 and2 sbnits (and possiby the sti argey nexpored 3,5and4sbnits) to the short-ter effects of nico-tine on the physioogy of dopainergic nerons in thevTA erss the SNpc, as e as on dopainergic erssGABAergic nerons in the vTA. moreoer, the stdies
hae distingished the roes of these nAChR sbnits inthe acte behaiora effects of nicotine, as anifestedby cognitie behaiors and nicotine sef-adinistration,for exape. There is aso eidence that acte nicotineexposre ight infence (throgh actiation of 42*and 7*nAChRs) a goba gating circit that incdesthe striat, hippocaps and aygdaa, nder thetop-don contro of the PFC (BOX 1).
Effects of chronic nicotine exposure
Chronic nicotine exposre aters brain reard sys-tes. It increases dopaine reease in the ncesaccbens55,98100, and stdies in rats reeaed that
oitionay consed (bt not pass iey adinis-tered) nicotine oers the threshod ofintracranial lc-trical slf-stimulation for seera eeks25. At the cearee, nicotine sef-adinistration for 2 onths caseshyperactiity of dopainergic nerons in the vTA101.Together, these obserations indicate that sef-adin-istered nicotine resets dopaine reard circits andincreases their sensitiity to sbseqent exposres tonicotine (sensitization). This sees to be niqeto nicotine, as other drgs of abse case decreased sen-sitiity of these circits25,26. Sensitization is thoght to bea key step in the process eading to addiction, and seeraines of research hae expored the oecar echanissnderying this effect (FIG. 6).
Upregulation of VTA nAChRs. A possibe echanisfor sensitization inoes an pregation of high-affinitybrain nAChRs (osty 42-containing oigoers)indced by ong-ter exposre to nicotine17,102,103. Thispregation is de to post-transcriptiona echanissthat incde an increase in receptor sbnit asseby andconforationa atration of the oigoer104,105 acco-panied by strctra changes17. There is soe eidencethat the pregationhas a roe in the increased ocootoractiity and brain reard responses associated ith theeary stages of chronic nicotine exposre98100. BockingnAChRs in the vTA (bt not in the nces accbens)
Figure 6 | Pb manm t- t ng-tm ang a by nntn xp. Chronic nicotine exposure alters the sensitivity of dopaminergic
reward circuits. Several molecular mechanisms have been proposed to underlie this
sensitization.a | Upregulation of highaffinity nicotinic acetylcholine receptors (nAChRs),
mostly 42containing (42*) oligomers, in the ventral tegmental area (VTA) or onmidbrain dopaminergic and GABA (aminobutyric acid)ergic neurons. b | Changes incholinergic transmission (mediated by 2* and 7*nAChRs) in brain circuits at thepresynaptic level. It is possible that chronic nicotine exposure causes a downregulation
of2*nAChRs (possibly following an initial upregulation), followed by an enhanced7*nAChRmediated cholinergic transmission. The brain locus of this process is stilluncertain. | Topdown enhancement of bursting of VTA neurons. Potentiation of activity
in anterior regions of the brain (infralimbic cortex (IF) and bed nucleus of the stria
terminalis (BNST)) induced by chronic nicotine exposure would increase the bursting
activity of dopaminergic neurons by enhancing glutamatergic input to the VTA.
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inhibits ong-ter ocootor hyperactiity in ice, andoderate exposre to the sae nAChR antagonists pro-dces a transient nAChR pregation in the vTA bt notin the nces accbens17,106. Hoeer, there is a co-pex reationship (graphicay represented as an inertedu shape) beteen systeic nicotine concentrationsand effects on lTP and behaior107. This reationshipcan be expained by the initia enhanced actiity (that is,pregation) of nAChRs by nicotine, fooed by desen-sitization, particary at high nicotine concentrations107.upregation of nAChRs ay therefore contribte to aneary step in a seqence of oecar and cear eentsthat tiatey rest in enhanced reard responses tonicotine fooing chronic nicotine exposre17,108.
Chronic nicotine exposre ay aso affect nAChRson both idbrain dopainergic and GABAergic ne-rons, athogh possiby in a different anner86. Directhoe-ce patch-cap recordings sho that passiechronic nicotine adinistration increases the baseinefiring rate and the excitatory effect of actey adinis-tered nicotine on GABAergic nerons in the sbstantia
nigra pars reticata, bt decreases these easres indopainergic nerons86. It is possibe that the preg-ation of nAChRs on GABAergic nerons rests in anenhanced inhibition of idbrain dopainergic ne-rons102. Chronic exposre to nicotine od then restin an enhanced inhibition rather than a sensitization of idbrain dopainergic nerons, ediated bynAChRs on GABAergic nerons (FIG. 4). The potentiacontribtion of sch a process to the effects of chronicnicotine can no be eaated sing differentia expres-sion of genes encoding nAChR sbnits in dopainergic
erss GABAergic nerons74.
Opposing contribution of7* and2*nAChR-mediatedprocesses. A second possibe echanis of sensitizationinoes changes in choinergic transission in braincircits at the presynaptic ee. In 2/ ice, passieong-ter nicotine treatent noraized the ateredfiring patterns of vTA dopainergic nerons and theexporatory deficit obsered in nicotine-naie 2/ice.Hoeer, it did not case an pregation of hetero-eric non-2*nAChRs nor of hooeric 7*nAChRsin the brain, sggesting a presynaptic effect109. moreoer,chronic nicotine treatent of 2/ ice noraized theincreased density of presynaptic, high-affinity choineptake sites that is seen in treatent-naie 2/ ice,particary in the cadate ptaen109. most of these
restoratie effects ere bocked by the 7*nAChRinhibitor ethyycaconitine (mlA), consistent ith apresynaptic ateration at the ee of tonic, 7-ediatedchoinergic transission. Indeed, the phenotype ofid-type ice exposed to chronic nicotine and ith7*nAChRs pharacoogicay bocked as siiar tothat of 2/ ice. Ths, 2* and 7*nAChRs differen-tiay contribte to the ong-ter changes that acco-pany chronic nicotine exposre109: chronic nicotineprobaby cases a donregation of 2*nAChRs (pos-siby fooing an initia pregation), resting in anenhanced 7-ediated choinergic transission, thebrain ocs of hich is sti ncertain.
Recent stdies on the effects of ong-ter actienicotine sef-adinistration (throgh the drinkingater) in 2/ and 7/ ice confired this proposedechanis54. Initiay, 2/ ice, bt not 7/ ice,shoed decreased nicotine consption reatie toid-type ice. Hoeer, after approxiatey 3 eeksof nicotine access, the 2/ ice retrned to id-typeees of nicotine consption, hereas the 7/ icecontined to sho decreased nicotine consption54.These data sggest that, fooing chronic nicotineexposre in id-type ice, there is a fnctiona ba-ance beteen opposing processes ediated by differentnAChR sbtypes, ith the decreased contribtion of2*nAChR-ediated processes being copensated for,in the ong-ter, by 7-ediated synaptic processes atthe ee of ACh reease109,110.
Top-down enhanced bursting of VTA neurons. A thirdpossibe echanis for sensitization has recenty beensggestedon the basis ofin vivorecordings in the rat dringong-ter exposre to nicotine in an actie sef-adin-
istration paradig101. Exposre to sef-adinisterednicotine potentiated the actiity of dopainergic ne-rons in the vTA, anifested by an increase in brst-ing actiity101. This potentiation did not, accordingto the athors, rest fro a direct action of nicotine onthe nAChRs expressed on dopainergic nerons, btfro the potentiation of anterior regions of the brain(infraibic cortex and bed nces of the stria teri-nais), hich are inoed in the oitiona aspect ofnicotine consption. This potentiation od thenincrease, in a top-don anner (BOX 1), the brstingactiity of dopainergic nerons by enhancing gta-atergic inpt to the vTA111(FIG. 4). According to theathors, this echanis, hich inoes enhancedbrsting actiity (discssed aboe), can accont for theeffects of ong-ter sef adinistration of nicotine infreey behaing rats101.
In sary, the key step in nicotine addiction thatrests in the atered response to nicotine associated ithrepeated adinistration reains insfficienty expored,and a singe e-defined echanis is therefore ack-ing. methods and odes26 are aaiabe to distingish or specify the reatie contribtions of the ong-ter, direct effects of nicotine on nAChRs in the reardsystes fro the top-don regation of nicotineconsption by higher brain centres.
nAChRs in nicotine withdrawal symptomsThe ithdraa syndroe that accopanies sokingcessation after chronic tobacco se is one of the factorsthat precdes sccess in qitting112. In hans, thisincdes soatic syptos sch as bradycardia, gas-trointestina discofort and increased appetite acco-panied by eight gain; and affectie syptos sch asirritabiity, anxiety, depressed ood, difficty concen-trating, disrpted cognition and nicotine craing112,113.In rodent odes, rearing, ping, shaking, abdoinaconstrictions, cheing, scratching, facia treor, hyper-agesia and a change in ocootor actiity are consideredsoatic ithdraa syptos, hereas affectie signs
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7 2
2, (4), 5, 4
Thalamus
LC (noradrenaline)6
Raphe nucleus(serotonin)
IPN2, 52
VTA(dopamine)
7 2
Affective withdrawal symptoms 6, 2
Somatic withdrawal symptoms 2, 5, 7, 4
MHbLHb
Limbic system
Basal ganglia
Medial habenula
Contextual fear conditioning
A bhavourial tst in which an
avrsiv stimulus is givn to
an animal in a conditioning
chambr, such that th farrspons can subsquntly b
licitd in th conditioning
chambr in th absnc of th
avrsiv stimulus.
Conditioned place aversion
A form of classical Pavlovian
conditioning in which an animal
larns to avoid a compartmnt
that was prviously paird with
an avrsiv stimulus; for
xampl, th avrsiv stimulus
can b th ngativ affctiv
stat causd by nicotin
withdrawal.
incde anxiety-ike behaiors, eeated reard thresh-ods, ithdraa-indced contxtual far conditioningand ithdraa-indced conditiond plac avrsion113,114
(FIG. 7). The receptor-ediated echaniss ndery-ing nicotine ithdraa can be stdied in anias inhich nicotine ithdraa syptos are eicited bynAChR antagonists115, and in ice acking specificsbnits of the nAChR113,116118. Iportanty, 3, 5,6 and 4 sbnits are predoinanty expressed in thehabenainterpedncar pathays, hereas 2* and7*nAChRs are idey expressed15(FIG. 2).
Soatic ithdraa signs cased by the nAChRantagonist ecayaine fooing chronic nicotineexposre ere siiar in id-type and 2/ ice113,118bt ere ess eident in 2/, 5/, 7/ and 4/ice113,116,117,119. Affectie ithdraa signs are ediatedby different nAChR sbnits, as id-type ice and5/ and 7/ ice shoed ithdraa-eicited anxiety-ike behaior and conditioned pace aersion, bt 2/ice did not113. Frtherore, 7/ ice shoed siiarchanges in fear conditioning to id-type ice ndergo-ing nicotine ithdraa; these changes ere redced in2/ ice120.
6*nAChRs are inoed in the affectieanxiety-ike ithdraa signs, bt not in physica ithdraasigns53. The 6*nAChRs are ocated in the vTA and theocs coeres, here they odate noradrenaine
reease121,122. The contribtion of 6*nAChRs in the vTAto nicotine reard is e estabished, bt it is possibethat, in parae, 6*nAChRs in the ocs coeres ightcontribte to the regation of nicotine ithdraa53.
There is abndant expression of the 4 sbnitin the edia habena and of the 2 and 5 sbnits inthe interpedncar nces20,119, hich is connected to thehabena throgh the fascics retrofexs. In icechronicay treated ith nicotine, ecayaine casedsoatic ithdraa syptos hen icroinected intothe habena or the interpedncar nces, an effect thatas aboished in 2/ and 5/ ice20. Aso, in knock-inice expressing the hypersensitie 4l9A nAChR, bt
not id-type ice, inection of a o dose of nicotineincreased Fos expression (indicating nerona actia-tion) in the entroatera region of the edia habena,sggesting a possibe contribtion to ithdraasyptos by the 4 sbnit in the habena123.
The habena syste has aso been ipicated inithdraa fro other drgs of addiction, incdingopiates, cocaine and acoho124. Ania stdies haeproided eidence that the habena has inhibitoryproections to the vTA and the sbstantia nigra andthat habena esions increase dopaine trnoer inthe nces accbens and the PFC125. Actiity in thehabena and interpedncar nces increases dr-ing nicotine ithdraa possiby throgh redcedactiation of nAChRs thereby controing the acti-ity of dopainergic nerons in the vTA and seroton-ergic nerons in the dorsa raphe nces126, to hichthey proect.
In concsion, the 2, 5, 7 and 4 sbnits regatethe expression of soatic syptos of ithdraa119,hereas 2 and 6 sbnits contribte to affectie
coponents of the nicotine ithdraa syndroe113(FIG. 7). The pattern and distribtion of nAChR sbnitsinoed in the nicotine ithdraa syndroe ight dif-fer fro those engaged in the short-ter, phasic effectsof nicotine, hich od offer noe targets for ong-tersoking cessation therapies.
Conclusions and future directions
There are sbstantia differences beteen the responseof id-type ice and ice acking specific nAChR sb-nits to acte or chronic adinistration of nicotine. Thetant ice share featres ith han genetic ariants,hich egitiizes their se as odes to inestigate theoecar bioogy of nicotine addiction (see ReF. 127and Sppeentary inforation S1 (box)). Inactiationith or ithot re-expression of nAChR genes inthe ose5,10 has identified sets of nAChR sbnitsthat characterize the sccessie steps of nicotine addic-tion, fro the acte effects of nicotine consption toong-ter nicotine addiction.
The sbset of nAChR sbnits engaged in the acteeffects of nicotine on dopainergic reard nerons inthe vTA are being ecidated, as are those inoedin nicotine ithdraa syptos. Hoeer, the nAChRsbnits engaged in the transition fro the short-terto the ong-ter effects of nicotine exposre are stifar fro being nderstood. This is a path that reqires
frther exporation to hep deeop drgs that are oreeffectie than those crrenty aaiabe, hich are pri-ariy designed to target the acte effects of nicotine.
Han genetic stdies on nicotine dependence (seeSppeentary inforation S1 (box)) hae so far onyreeaed eak associations ith the 2 and 7 sbnitgenes, hich pay an iportant part in short-ter nico-tine reard. By contrast, these stdies consistenty shoan association of a 3, 5 and4 sbnit poyorphissand 3 and 6 sbnit poyorphiss ith nicotinedependence128. In ania odes, soe of these sbnitssee to be inoed in nicotine ithdraa rather thanin nicotine reard129; hoeer, ongoing stdies sggest a
Figure 7 | cntbtn nAcr bnt t ntn wtawa ymptm at
t v t ma abnantpna ytm. Subunits shown in green
are implicated in somatic withdrawal symptoms; subunits shown in red are implicated in
affective withdrawal symptoms. IPN, interpeduncular nucleus; MHb, medial habenula;
LC, locus coeruleus; LHb, lateral habenula; VTA, ventral tegmental area.
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possibe contribtion to reard processes as e. moseodes and the han genetics data of nicotine addic-tion ths point to argey nexpored targets in the designof soking-cessation drgs.
An interesting featre of the present discssion isthat, in addition to the ch-stdied effect of nicotineon dopainergic reard systes, there is eidence thata goba gating brain circit82 is inoed in nicotineaddiction, hich incdes the striat, hippoca-ps and aygdaa nder the top-don contro of thePFC101,130. This is consistent ith neroogica and brainiaging data in hans that spport the notion that theoss of contro cased by nicotine addiction ay cor-respond to a disrption of prefronta cortex and insa
fnctions in a goba nerona orkspace (BOX 1). Thisdisrption od sbert attention, reasoning, panningand decision-aking processes that exert inhibitory con-tro to resist drg se21. Nicotine dependence ight thenbe tentatiey ieed as a goba nerona orkspacedisconnection syndroe130, hich od point to noetargets that ediate nicotine action. The conseqencesof these stdies for the nderstanding and treatent ofdiseases sch as depression, Azheiers disease andParkinsons disease reain to be expored. Neertheess,these ose ode experients open a hoe fied ofinestigations, in particar in priates and hans, tonderstand the nero-cognitie processes that are crciato nicotine addiction.
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