Division of Pharmacology and ToxicologyDepartment of Pharmacy

University of Helsinki

EFFECT OF NICOTINE ON DOPAMINERGIC NEUROTRANSMISSION

AND EXPRESSION OF FOS PROTEIN

Outi Salminen

ACADEMIC DISSERTATION

To be presented with the permission of the Faculty of Science of the University of

Helsinki, for public critisism in Auditorium 1041 of Biocentre Viikki,

on August 17th , 2000 at 12 o’clock noon

Helsinki 2000

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Supervisor: Professor Liisa Ahtee, M.D.Division of Pharmacology and ToxicologyDepartment of PharmacyUniversity of Helsinki

Reviewers: Docent Jouni Sirviö, Ph.D.OrionPharmaPreclinical ResearchCNS Pharmacology Laboratory, Turku

Docent Sampsa Vanhatalo, M.D.Department of AnatomyInstitute of BiomedicineUniversity of Helsinki

Opponent: Dr. David Balfour, Ph.D.Department of Pharmacology & NeuroscienceNinewells Hospital and Medical SchoolUniversity of Dundee

ISBN 951-45-9458-4 (print)ISBN 952-91-2324-8 (PDF)ISSN 1239-9469

Gummerus Oy, Saarijärvi

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To Arto, Laura and Riikka

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CONTENTS

ABSTRACT...............................................................................................................................6

LIST OF ABBREVIATIONS ..................................................................................................7

LIST OF ORIGINAL PUBLICATIONS ...............................................................................8

1 INTRODUCTION .................................................................................................................9

2 REVIEW OF THE LITERATURE ...................................................................................11

2.1 Neuronal nicotinic acetylcholine receptors (nAChRs) ..............................................112.1.1 nAChR subunits and their distribution.................................................................112.1.2 Presynaptic nAChRs.............................................................................................142.1.4 Desensitization of neuronal nAChRs ...................................................................162.1.5 Upregulation of nAChRs ......................................................................................202.1.6 Nicotine-induced development of tolerance.........................................................22

2.2 Nicotine and dopamine .................................................................................................232.2.1 Dopaminergic pathways in the brain....................................................................232.2.2 Synthesis and metabolism of dopamine ...............................................................252.1.6 Nicotine and brain dopaminergic systems............................................................27

2.3 Nicotine and c-fos ..........................................................................................................302.3.1 Immediate-early gene c-fos ..................................................................................302.3.2 Acute nicotine and c-fos .......................................................................................352.3.3 Intermittent nicotine administration, Fos and Fos-related antigens .....................36

2.4 Brain areas in which nicotine activates c-fos..............................................................372.4.1 Dopaminergic target areas ....................................................................................372.4.2 Other brain areas ...................................................................................................39

3 AIMS OF THE STUDY......................................................................................................42

4 MATERIALS AND METHODS ........................................................................................43

4.1 Animals ..........................................................................................................................43

4.2 Experiments ...................................................................................................................434.2.1 Chronic administration of nicotine .......................................................................434.2.2 Withdrawal from nicotine treatment ....................................................................444.2.3 Nicotine challenge experiments ...........................................................................444.2.4 Measurement of rectal temperatures ....................................................................444.2.5 Dissection of brain................................................................................................454.2.6 Preparation of plasma samples .............................................................................454.2.7 Preparation of brain sections for immunohistochemistry.....................................454.2.8 Drugs ....................................................................................................................46

4.3 Analytical methods ........................................................................................................464.3.1 Estimation of plasma concentrations of nicotine and cotinine using GC-MS (II,

IV)……………….................................................................................................464.3.2 Measurement of tissue contents of dopamine and its metabolites using HPLC-EC

(II, IV) ...................................................................................................................474.3.3 Fos-immunohistochemistry (I, II, III) ...................................................................474.3.4 Statistical analysis ................................................................................................49

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5 RESULTS .............................................................................................................................50

5.1 The plasma concentrations of nicotine and cotinine in rats and in mice (II, IV) ....50

5.2 Nicotine-induced hypothermia in mice (IV) ...............................................................51

5.3 The tissue concentrations o f dopamine and it metabolites, DOPAC and HVA ......525.3.1 The effect of nicotine challenge during chronic nicotine administration in mice

and in rats (II, IV) .................................................................................................525.3.2 The effect of nicotine challenge on rats withdrawn from chronic nicotine

administration (II).................................................................................................54

5.4 The expression of Fos protein in rat brain areas .......................................................555.4.1 Naïve and saline-injected rats (unpublished results)............................................555.4.2 Acute nicotine (I and unpublished results) ...........................................................555.4.3 Acute nicotine and diazepam (I) ...........................................................................575.4.4 Chronic nicotine-infusion and withdrawal (II, III) ...............................................58

6 DISCUSSION.......................................................................................................................60

6.1 The plasma concentrations of nicotine and cotinine ..................................................60

6.2 Nicotine-induced elevation of striatal and limbic DOPAC and HVA concentrations.........................................................................................................................................61

6.3 The effect of chronic nicotine and its withdrawal on the nicotine-induced changesin cerebral dopamine metabolism ...............................................................................62

6.3.1 The desensitization of nAChRs regulating cerebral dopamine metabolism ........626.3.2 The long-lasting inactivation of nAChRs regulating cerebral dopamine

metabolism in rats.............…................................................................................63

6.4 Fos protein expression: brain areas activated by acute nicotine administration....64

6.5 The interaction of nicotine and diazepam in the induction of Fos expression........65

6.6 The effect of chronic nicotine treatment and its withdrawal on nicotine-inducedFos protein expression..................................................................................................67

6.6.1 Dopaminergic target areas ....................................................................................676.6.2 The relationship of nicotine-induced changes between dopamine metabolism and

Fos protein expression in dopaminergic target areas ...........................................686.6.3 Other brain areas ...................................................................................................69

6.7 The development of tolerance in nicotine-infused mice ............................................70

7 SUMMARY AND CONCLUSIONS..................................................................................72

ACKNOWLEDGEMENTS ...................................................................................................74

REFERENCES .......................................................................................................................76

APPENDIX: ORIGINAL PUBLICATIONS I-IV

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ABSTRACT

Nicotine, the psychoactive component of tobacco, acts in the brain via nicotinic ace-tylcholine receptors (nAChRs). Nicotinic receptors are ligand-gated ion channels composed ofdifferent subunits forming either homo- or heteromeric receptors. The common property fornAChRs is that the ion channel desensitizes during exposure to an agonist. Desensitization isa process where repeated or prolonged exposure of a receptor to an agonist leads to areduction in the magnitude of response to a subsequent exposure to an agonist. The rate ofdesensitization and recovery from it depends mostly on the subunit composition of the ionchannel. Chronic nicotine exposure inactivates some brain nAChRs for a longer time period.These long-lived inactive receptor states are likely to underly the development of tolerance tonicotine. They may also be responsible for attenuating the behavioural and physiologicaleffects of the drug and, therefore, be implicated in the process of nicotine dependence.

The present study was conducted in order to examine the effect of sustained chronicnicotine administration and withdrawal on nicotine-induced striatal and limbic dopamine me-tabolism in the rodent brain. The presynaptic effects of nicotine were studied by the determi-nation of concentrations of dopamine and its metabolites. Further, the expression of Fos pro-tein was investigated in rat brain to reveal the postsynaptic effects of acute and chronic nico-tine administration and nicotine withdrawal. The main brain areas studied were the dopami-nergic target areas, the stress-related hypothalamic areas, visual areas as well as interpedun-cular nucleus which is rich in nAChRs. These brain areas most probably contribute tonicotine’s dependence-producing effects.

Nicotine was administered to rats s.c. via osmotic minipumps and to mice via s.c. im-planted nicotine-releasing reservoirs for 7 days. The effects of chronic nicotine administrationand withdrawal on nicotine-induced striatal and limbic dopamine metabolism were analysedin post mortem brain samples of rats and mice by HPLC-EC. Nicotine-induced hypothermiawas studied in mice. Further, the Fos protein immunostaining was studied in the rat brain sec-tions after administration and withdrawal of nicotine.

In both rats and mice the effects of acute nicotine on striatal dopamine metabolismwere attenuated during constant nicotine administration suggesting that the nAChRs regu-lating the dopaminergic neurons were desensitized. In rat studies we found that the nAChRsin the striatal areas appeared to be more easily desensitized than those in the limbic areas.Interestingly, similar desensitization phenomenon was also demonstrated in nicotine’s post-synaptic effects as measured by Fos protein expression in striatal brain areas whereas in lim-bic areas the desensitization of the Fos protein response was not induced as easily or not at all.In the hypothalamic brain areas the attenuation of nicotine-induced Fos expression duringconstant nicotine infusion was also observed. Furthermore, evidence for a long-lasting inacti-vation of nAChRs mediating nicotine’s effects on limbic dopamine metabolism and on Fosexpression in striatal and limbic areas in vivo was found. This indicates the development oftolerance to these effects of nicotine. In mice, the responses of striatal DA metabolism andbody temperature to acute nicotine were both reduced during constant nicotine infusion, al-though the differences in the time courses and the dose-relationships of these effects suggestthat they are separate phenomena. The tolerance to nicotine’s hypothermic effect could beovercome by increasing the dose of nicotine, suggesting that tolerance, in the classical sense,develops in the nAChRs involved in thermoregulation during chronic nicotine treatment.

In these studies the desensitization and long-lasting inactivation of nAChRs weredemonstrated in vivo in rodent brain during chronic nicotine administration and its with-drawal. Further, the levels of desensitization and long-lasting inactivation of nAChRs in vari-ous brain areas seemed to differ. Also, the nAChRs mediating nicotine’s effects on body tem-perature and dopamine metabolism differ. Thus, there seems to be variations in the functionalstates and/or in the subunit combinations of nAChRs mediating nicotine’s effects in brain.

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LIST OF ABBREVIATIONS

ACh acetylcholineACTH adrenocorticotropic hormoneαBgt α-bungarotoxin3-MT 3-methoxythyramineACe central nucleus of amygdalaANOVA analysis of varianceATP adenosine 5’-triphosphateCg cingulate cortexChAT choline acetyltransferaseCOMT catechol-O-methyltransferaseCPU caudate-putamenCRH corticotropin-releasing hormoneDA dopamineDG dentate gyrusDMPP 1,1-dimethyl-4-phenylpiperazinumDOPA dihydroxyphenylalanineDOPAC 3,4-dihydroxyphenylacetic acidFOS-IS Fos-immunostainingGC-MS gas chromatography-mass spectrometryHVA homovanillic acidi.p. intraperitoneallyIPN interpeduncular nucleusLC locus coeruleusMAO monoamineoxidasemRNA messenger ribonucleic acidMH medial habenulaMT medial terminal nucleus of the accessory optic tractNA noradrenalineNAcc nucleus accumbensnAChR nicotinic acetylcholine receptorNMDA N-methyl-D-aspartateNRS normal rabbit serumPaC parietal cortexPB sodium phosphate bufferPBS phosphate buffered salinePFC prefrontal cortexPVN hypothalamic paraventricular nucleuss.c. subcutaneouslyS.E.M. standard error of the meanSC superior colliculusSIM selected ion monitoringSNc substantia nigra pars compactaSON supraoptic nucleusTTX tetrodotoxinVTA ventral tegmental area

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LIST OF ORIGINAL PUBLICATIONS

This dissertation is based on the following publications, herein referred to by their

Roman numerals (I-IV):

I Salminen O, Lahtinen S, Ahtee L (1996) Expression of Fos protein in various

rat brain areas following acute nicotine and diazepam. Pharmacol Biochem

Behav 54: 241-248.

II Salminen O, Seppä T, Gäddnäs H, Ahtee L (1999) The effects of acute nicotine

on the metabolism of dopamine and the expression Fos protein in striatal and

limbic brain areas of rats during chronic nicotine infusion and its withdrawal.

J Neurosci 19: 8145 – 8151.

III Salminen O, Seppä T, Gäddnäs H, Ahtee L (2000) Effect of acute nicotine on

Fos protein expression in rat brain during chronic nicotine and its withdrawal.

Pharmacol Biochem Behav 66: 87-93.

IV Salminen O, Ahtee L (2000) The effects of acute nicotine on the body

temperature and striatal dopamine metabolism of mice during chronic nicotine

infusion. Neurosci Lett 284: 37-40.

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1 INTRODUCTION

Nicotine, acting at neuronal nicotinic acetylcholine receptors (nAChRs), is the primary

component of tobacco that drives its habitual use (Benowitz 1996). Smoking a

cigarette results in a rapid bolus of nicotine that activates the mesolimbic

dopaminergic system in particular, producing pleasure and reward. Further, in

smokers, nicotine levels accumulate over 6-8 h during smoking (Benowitz 1996) to

reach a low steady state concentration which has been suggested to cause both

reversible desensitization and long-term inactivation of nAChRs. In smokers the

increases in the amounts of some nAChR subtypes has been demonstrated.

Additionally, smokers seem to adjust their nicotine intake to regulate their nAChR

response (Collins and Marks 1996; Dani and Heinemann 1996; Wonnacott et al.

1996).

Nicotine and nicotinic drugs could be important in some neurological diseases because

it has been shown that a substantial decrease in nAChRs is characteristic of both

Alzheimer’s and Parkinson’s diseases (Lange et al. 1993; Whitehouse et al. 1988).

Epidemiological studies also indicate that smoking may have a protective association

in Parkinson’s disease and, to a lesser extent, Alzheimer’s disease (Morens et al.

1995). In Parkinson’s disease, there is a reduction of dopamine due to the degeneration

of the substantia nigra. It is known that presynaptic nAChRs can modulate the release

of striatal and limbic dopamine (Wonnacott 1997) and that nicotine can be

neuroprotective against glutamate-induced excitotoxicity (Akaike et al. 1994). Thus,

nicotine might be protective against Parkinson’s disease and nicotinic drugs might be

useful in therapy of Parkinson’s disease. The effects of nicotine on several other

diseases suggest that nAChRs may be involved to some extent in their pathology

and/or therapy. For example, nicotine potentiates the therapeutic properties of

neuroleptics in treating Tourette’s syndrome (Sanberg et al. 1997). It has also been

reported that the α7 subunit of nAChRs may be responsible for the attentional deficit

that may be a predisposing genetic factor for schizophrenia (Adler et al. 1998). It has

been found that α7 nAChRs are reduced in brains of schizophrenic patients (Guan et

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al. 1999). Further, it has been observed that schizophrenic patients smoke heavily to

self-medicate with nicotine (Dalack et al. 1998).

Along with activation of nAChRs by the rapid bolus of nicotine, long-term application

of the drug can lead to inactive states of these nAChRs, some of which are readily

reversible and others which are not (Collins and Marks 1996; Dani and Heinemann

1996; Hsu et al. 1996; Lester and Dani 1994; Lukas et al. 1996). An understanding of

the effects of chronic nicotine exposure on various nAChR subtypes, on

neurotransmitter release, especially dopamine, and on postsynaptic target areas might

provide better insights into mechanisms of nicotine dependence, tolerance and

withdrawal and into the effects of medication with nicotine or nicotinic drugs.

In this study the effects of constant chronic nicotine infusion and its withdrawal on

nicotine-induced striatal and limbic dopamine metabolism were investigated. The

presynaptic effects of nicotine were studied by determination of cerebral

concentrations of dopamine and its metabolites. Moreover, the expression of Fos

protein was investigated to reveal the postsynaptic effects of acute and chronic

nicotine and the withdrawal. The main brain areas studied were the dopaminergic

target areas. Additionally areas rich in nAChRs were examined, such as the stress-

related hypothalamic areas, visual areas and interpeduncular nucleus. These brain

areas are in all probability involved in the dependence producing effects of nicotine as

well as in its beneficial effects in Parkinson’s disease and schizophrenia, and also in its

ability to relieve anxiety.

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2 REVIEW OF THE LITERATURE

2.1 Neuronal nicotinic acetylcholine receptors (nAChRs)

2.1.1 nAChR subunits and their distribution

Genes that are similar in sequence to the genes encoding the subunits of nicotinic

acetylcholine receptors (nAChRs) at the neuromuscular junction encode the subunits

of neuronal nAChRs. To date, eight neuronal nAChR α genes (α2 − α9) and three

nAChR β genes (β2 − β4) have been cloned (Deneris et al. 1989b; Duvoisin et al.

1989; Elgoyhen et al. 1994; Lamar et al. 1990; Schoepfer et al. 1990; Séguéla et al.

1993; Wada et al. 1990b; 1989). Neuronal nAChR α genes encode the ligand binding

subunit with the β subunits appearing to regulate the rate at which agonists and

antagonists dissociate from the receptors, as well as contributing to the rate at which

agonist-bound receptors open. The β subunits do not function on their own, suggesting

that they form part of a hetero-oligomer receptor (Boyd 1997; Luetje and Patrick

1991). Homo-oligomeric receptors can be assembled from α7, α8 or α9 subunits, with

α7 and α8 also combining to form hetero-oligomeric receptors (Boyd 1997).

Neuronal nAChRs can be divided on the basis of snake venom toxin α-bungarotoxin

(αBgt) -binding to those receptors that do not bind αBgt and those that do bind αBgt

(Lindstrom et al. 1995). Neuronal nAChRs that do not bind αBgt include

combinations of α2, α3, α4 and α6 with β2 and β4 subunits; sometimes additionally

including α5 or β3 subunits. α4β2 nAChRs account for > 90% of the high affinity

nicotine-binding sites in rat brain (Flores et al. 1992). α3 or α6 containing nAChRs are

thought to be present in smaller amounts in more limited regions of the brain (Le

Novère et al. 1996). Another snake venom toxin distinct from αBgt is neuronal

bungarotoxin (nBgT), which has a selectivity for neuronal vs. muscle nAChRs. There

seems to be considerable parallelism between nBgT-binding sites and α3 subunit

mRNA in rodent brain (Luetje et al. 1990; Schulz et al. 1991). As a result this receptor

population is sometimes recognised as a third neuronal nAChR subclass (Decker et al.

1995).

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Neuronal nAChRs that bind αBgt include α7, α8, α9 subunits, which can function as

homomeric nAChRs. The characteristics of the homomeric subtypes include a very

rapid rate of desensitization and a high level of permeability to calcium resembling

that of the NMDA type of glutamate receptor (Bertrand et al. 1993; McGehee and

Role 1995; Revah et al. 1991; Séguéla et al. 1993). Rapid desensitization limits the

ability of homomeric nAChRs to respond to sustained intense stimulation. High

calcium permeability permits calcium ions to act as second messengers to affect many

cellular processes (Lindstrom 1997). In the brain α7 nAChRs represent > 90% of the

high-affinity αBgt binding sites, and they are present in brain at about equal amounts

with α4β2 nAChRs in a similarly wide ranging and overlapping but distinct

distribution (Séguéla et al. 1993). α8 has been found only in chickens (Schoepfer et al.

1990) and α9 is found only in limited areas of the rat nervous system (Elgoyhen et al.

1994).

Several nAChR ligands have been used to study the distribution of nAChRs, including3H-acetylcholine (ACh), 3H-nicotine and 125I-αBgt (Clarke et al. 1985b; Marks et al.

1986). The distribution of labeling seen with 3H-ACh coincides well with 3H-nicotine

(Martino-Barrows and Kellar 1987). Table 2.1. shows the distribution of nicotinic

receptor binding of the aforementioned ligands and subunit mRNA in the rat brain.

More recently, 3H-cytisine and 3H-epibatidine have also been used to localize

nAChRs. Distribution of 3H-cytisine binding is generally consistent with that of 3H-

nicotine (Pabreza et al. 1991). Epibatidine can detect nAChRs also labeled with 3H-

nicotine or 3H-ACh. In addition, epibatidine also detects nAChRs with lower affinity

for nicotine, which are not readily detected using 3H-ACh or 3H-nicotine (Marks et al.

1998; Perry and Kellar 1995; Zoli et al. 1998).

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Table 2.1 Distribution of nicotinic receptor binding and subunit mRNA in several ratbrain regions (modified from Shacka and Robinson 1996).

Brain region* Subunit mRNA 3H-NIC and3H-ACh binding

ααBgt-binding

Cerebral cortex α3, α4, α5, α7,β2, β4

+ +

Septum α4, α7, β2 - +Hypothalamus α3, α4, α7, β2, β4 - +Thalamus α3, α4, α7,

β2, β3, β4+ -

Hippocampalformation

α2, α3, α4, α5, α7,β2, β4

+ +

Medial habenula α3, α4, α7,β2, β3, β4

+ -

Amygdala α3, α4, α7, β2 - +Substantia nigraand/or VTA

α3, α4, α5, β2, β3 + -

Interpeduncularnucleus

α2, α4, α5, α7,β2, β4

+ +

Superiorcolliculus

α4, α7, β2 + +

*Only areas exhibiting moderate to strong intensity of hybridization signal or ligandbinding (+) are included. Expression of multiple mRNAs in a structure does not implyidentity of receptor site within that structure. Also, distribution of receptor ligandbinding or mRNA expression is not restricted to structures listed above. – indicates notdetected.

As shown in Table 2.1, nAChRs are widely distributed in the brain. There is

abundance of evidence that neurons located in many sites of the brain express

nAChRs, which can be activated by exogenous acetylcholine and nicotinic agonists

(Clarke 1993). There is also a rich literature describing the anatomical organization of

brain cholinergic neurons (see for review Butcher 1995). In contrast, there are very

few documented sites of nicotinic cholinergic transmission in the brain. Anatomical

mapping studies show that nAChRs are present in brain areas that receive cholinergic

innervation, as shown by choline acetyltransferase (ChAT) immunoreactivity

(Armstrong et al. 1983; Wada et al. 1990b; 1989). This apparent match is consistent

with the possibility of nicotinic cholinergic transmission, but according to Clarke

(1993) it provides no more than weak evidence. At peripheral sites (e.g. autonomic

ganglia, neuromuscular junction), selective stimulation of cholinergic nerves is

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possible, while in the brain, this is difficult or impossible (Clarke 1993). Despite this,

cholinergic transmission mediated by nAChRs has been identified in cerebral cortex,

medial habenula, interpeduncular nucleus and in substantia nigra/ventral tegmental

area (Clarke 1993). Of these, the interpeduncular nucleus (IPN) is the most

convincingly identified site. In addition to the different nAChR subunits, IPN contains

amongst the brain nuclei the highest level of ChAT, acetylcholine esterase and high

affinity choline uptake activity. ChAT-like immunoreactive fibres and synaptic

boutons have also been observed in IPN (Clarke 1993).

2.1.2 Presynaptic nAChRs

Neuronal nAChRs can be categorised based on their location in the neuron. Although

this kind of classification is not clear-cut, nAChRs have mainly been divided into pre-

and postsynaptic receptors (Colquhoun and Patrick 1997). Recently, neuronal nAChRs

were subdivided even further into somatodendritic, preterminal and presynaptic

receptors (Wonnacott 1997). Preterminal receptors are axonal receptors modulating

transmitter release which is sensitive to tetrodotoxin (TTX), while in contrast,

presynaptic nAChRs elicit neurotransmitter release through a TTX-insensitive manner.

In the case of nAChRs, most receptors studied seem to have a presynaptic function in

the CNS, which does not preclude a postsynaptic role. There are undoubtedly

postsynaptic nAChRs on neurons in many areas of the brain (Colquhoun and Patrick

1997), but it is unclear whether the postsynaptic nAChRs mediating responses to

exogenously applied agonists are involved in synaptic transmission (Sorenson et al.

1998).

Activation of neuronal nAChRs on innervating axons can alter the release of a number

of neurotransmitters. It is now known that presynaptic nAChRs are present in the

nigrostriatal dopaminergic pathway and in the hippocampus. Most of the evidence in

support of a presynaptic location for nAChRs is based on lesion studies. Lesioning of

specific inputs to a nucleus leads to a loss of nicotinic-binding sites in that nucleus.

This implies that the binding sites are on the innervating axons and their loss is due to

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degeneration of these innervating axons (Colquhoun and Patrick 1997). In many cases,

however, it is difficult to rule out that the receptors are postsynaptic and their

maintenance requires the presence of the innervating axons. This is unlikely to be the

case in the striatum, where lesioning of nigrostriatal neurons with 6-hydroxy-

dopamine caused a significant decrease in the number of 3H-acetylcholine binding

sites (Schwartz et al. 1984). Because striatal neurons do not express nicotinic genes at

detectable levels (Deneris et al. 1989a; Séguéla et al. 1993; Wada et al. 1990b; Wada

et al. 1989), it is unlikely that the decrease in nicotine-binding sites is due to the loss of

receptors on these striatal neurons. The disappearance is more likely due to the loss of

presynaptic nAChRs on innervating nigral axons. These lesioning studies are

consistent with the idea that activation of nAChRs on nigrostriatal neurons modulates

transmitter release (Colquhoun and Patrick 1997). In striatal slices and synaptosomes

acetylcholine, nicotine or nicotinic agonists release dopamine in a dose- and calcium-

dependent manner (Giorguieff et al. 1977; Giorguieff-Chesselet et al. 1979; Grady et

al. 1992; Rapier et al. 1988; 1990; Soliakov et al. 1995). These nAChRs that are

activated by nicotinic agonists are likely to locate to the innervating nigral axons and

are, thus, termed “presynaptic nAChRs” (Wonnacott 1997). Distinct subtypes of

presynaptic receptors have also been shown to regulate the release of other

neurotransmitters, such as noradrenaline from hippocampus (Clarke and Reuben 1996;

Sacaan et al. 1995), glutamate from prefrontal cortex, medial habenula and

hippocampus (Gray et al. 1996; McGehee et al. 1995; McGehee and Role 1995; Vidal

and Changeux 1993), GABA from the interpeduncular nucleus (Léna et al. 1993) and

hippocampus (Alkondon et al. 1997), acetylcholine from cortex and hippocampus

(Lapchak et al. 1989; Rowell and Winkler 1984) and 5-hydroxytryptamine from

striatum (Reuben and Clarke 2000).

2.1.3 Pharmacology of brain nAChRs

Nicotinic receptors in central nervous system, ganglions and at the neuromuscular

junction differ in their affinity and response to various cholinergic agonists and

antagonists. Each receptor subtype has a specific pattern of sensitivity to the agonists

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acetylcholine, nicotine, DMPP or cytisine (see Table 2.2). Both α and β subunits

contribute to the pharmacological properties of each subtype (Luetje and Patrick

1991).

Table 2.2 Sensitivity of rat nAChRs expressed in oocytes to various agonists(modified from Boyd 1997).

Subtype

combination

Agonists relative

potency

References

α2β2 N > D = A > C Luetje and Patrick 1991

α3β2 D > A > N >C Cachelin and Jaggi 1991,

Luetje and Patrick 1991

α4β2 A = N > D > C Luetje and Patrick 1991

α2β4 C >N > A > D Luetje and Patrick 1991

α3β4 C > N = A = D Cachelin and Jaggi 1991,

Luetje and Patrick 1991

α4β4 C > N > A > D Luetje and Patrick 1991

α7 N = C > D > A Séguéla et al. 1993

α9 A >> D Elgoyhen et al. 1994

Note: N = nicotine; A = acetylcholine; C = cytisine; D = 1,1-dimethyl-4-phenylpiperazinum.

2.1.4 Desensitization of neuronal nAChRs

Desensitization is a process where repeated or prolonged exposure of a receptor to an

agonist leads to a reduction in the magnitude of response to subsequent exposure to the

agonist. The desensitization of the nAChR response is an intrinsic property of the

isolated nAChR protein which does not require any obligatory covalent modification

(Changeux et al. 1987). In 1957, Katz and Thesleff reported that when acetylcholine

was applied iontophoretically to frog skeletal muscle, the tissue showed a typical

response; but when the agonist was allowed to act for a prolonged time, the tissue no

17

longer responded. A cyclical model of desensitization was then suggested for nAChRs

(Katz and Thesleff 1957; Rang and Ritter 1970; Figure 2.1).

Figure 2.1 A cyclical model for desensitization (Katz and Thesleff 1957): S = conditioningdrug concentration; A = free receptors; SA = active drug-receptor complex (=open ionchannel); SB = ’refractory’ drug-receptor complex (= desensitized state), a and b = affinityconstants; k1 being the forward, k2 the reverse rate constant of the desensitizing step.

This model allows for two paths to desensitization. One route consists of the receptor

in a resting state which rapidly binds acetylcholine to form an active complex (= open

ion channel) which then slowly converts to a desensitized state (= acetylcholine still

bound but the channel is non-conducting). When acetylcholine is no longer present the

agonist dissociates from the receptor and the receptor returns to the resting state. The

conversion from the active state to the desensitized state involves a change in the

binding site for the agonist molecule from a low affinity state to a high affinity state

(Sine and Taylor 1979; Weiland and Taylor 1979). Thus, the continued presence of an

agonist molecule will eventually shift nAChR from a low affinity resting state through

an active state and then to a high affinity desensitized state. A second route to the

liganded desensitized form of the receptor can occur at low agonist concentrations at

which binding to the unliganded, desensitized form of the receptor occurs. In other

words, nAChRs can be desensitized by activating and subactivating concentrations of

agonists (Fenster et al. 1997; Grady et al. 1994; 1997; Lester and Dani 1995; Marks et

al. 1994; 1996). Both routes to desensitization are possible due to the desensitized

conformation of the receptor having a higher affinity for the agonist than the resting

receptor and that the unliganded resting state and desensitized state of the receptor are

interconvertible (Katz and Thesleff 1957; Marks 1998). Thus, following exposure to

either activating or subactivating concentrations of agonist for sufficient time, a

18

significant population of receptors are refractory to activation because of the

accumulation of the liganded desensitized nonfunctional receptor.

Most of the experimental data dealing with various aspects of nAChR desensitization

can be accounted for by the general model (Changeux et al. 1984; Heidmann et al.

1983; Neubig and Cohen 1980) which is a modified version of the cyclic model

proposed by Katz and Thesleff. Formally, this model is within the general framework

of the concerted model for allosteric transitions in multimeric proteins (Monod et al.

1965) as applied to the nAChR (Karlin 1967). This general model consists of four

states for the nAChR molecule: (1) resting (closed) or (2) active (open) state or (3)

slowly or (4) rapidly desensitized, refractory, high affinity states of the receptor

(Changeux et al. 1984; Edelstein et al. 1996). Therefore, desensitization consists of

two distinct kinetic processes, a fast and a slow component (Felzt and Trautmann

1982; Sakmann et al. 1980). After brief desensitization, most receptors only have time

to reach a fast desensitized state from which recovery is rapid. During prolonged

desensitization, more receptors are converted to the slowly reached desensitizated

state, from which recovery is slow (Fenster et al. 1999a). In addition, it has been noted

that prolonged exposure to acetylcholine causes an incomplete recovery of receptor

function which could be termed as long-lasting inactivation and which may represent

yet another phase of desensitization (Simasko et al. 1986) or more likely, a mechanism

that can be distinguished from reversible nAChR desensitization (Ke et al. 1998;

Rowell and Duggan 1998).

There are different factors which appear to influence the desensitization of neuronal

nAChRs. The intracellular domains of transmembrane receptors, including muscle-

type nAChRs, are readily phosphorylated by various intracellular kinases, which have

been shown to both promote desensitization and increase the rate of it (Huganir et al.

1986; Huganir and Miles 1989). However, Cachelin and Colquhoun (1989) cast doubt

to the idea that phosphorylation could be a normal mechanism to produce

desensitization, because the process was observed either in reconstituted receptor or in

artificial intracellular medium which contains no ATP. Later, it has been shown that in

the case of neuronal nAChRs, their phosphorylation state appeared important in

19

determining recovery from desensitization (Fenster et al. 1999a; Khiroug et al. 1998).

Furthermore, when nAChRs are desensitized, they become susceptible to modulation

by calcium, via intracellular second messenger mechanisms such as serine/threonine

kinases and calcineurin (Khiroug et al. 1998).

For heteromeric nAChRs the α subunit determines the apparent affinity of nicotine to

the active and desensitized states of a nAChR; the β subunit contributes by

determining the overall time course of the development of desensitization (Cachelin

and Jaggi 1991; Fenster et al. 1997). Generally it has been found that desensitization

develops rapidly in β2-containing nAChRs and slowly in β4-containing nAChRs; α4-

containing receptors seem to be more sensitive to desensitizing levels of nicotine than

α3-containing receptors (Fenster et al. 1997; Vibat et al. 1995). The homomeric α7

receptor has the highest sensitivity to nicotine as regards both activation and

desensitization; α7 nAChRs also demonstrate the fastest desensitization kinetics

(Couturier et al. 1990; Fenster et al. 1997; Revah et al. 1991; Séguéla et al. 1993). The

desensitization of homomeric α7 receptors suggests that the β subunit plays a

modulatory, rather than a permissive role, in desensitization (Fenster et al. 1997).

The process of desensitization has recently attracted increasing attention because of its

ability to influence synaptic transmission over extended periods, thus controlling the

time course of synaptic events and enabling sustained changes in the efficacy of

synaptic transmission (Jones and Westbrook 1996). Also, desensitization has been

suggested to play a role in nicotine dependence in smokers (Lukas et al. 1996; Marks

1998). Desensitization of nAChRs on prolonged exposure to nicotine has been

implicated in cognitive enhancement, relief of depression and anxiolysis but also in

withdrawal and substance dependence in smokers (Lindstrom 1997; Lukas et al.

1996). Many smokers report that the first cigarette of the day is the most pleasurable

(Benowitz 1996; Dani and Heinemann 1996) and so the effect of subsequent cigarettes

may depend on the interplay between activation and desensitization of multiple

nAChRs (Pidoplichko et al. 1997). Pidoplichko et al. (1997) noted that even the low

concentration of nicotine (0.1 µM) applied in vitro, which may be maintained during

much of a smoker’s day (usually 0.1-0.5 µM), can cause significant desensitization of

20

nAChRs. The rate and recovery from desensitization varies depending on which types

of nAChRs are expressed on a particular neuron or in different areas of the brain.

Thus, multiple phases of nAChR desensitization and recovery could underlie aspects

of tolerance such that a second dose of nicotine does not elicit the same effects as the

first. The sustained stay of nicotine in the circulation and slow recovery from deep

levels of desensitization may explain the pleasurable effect of the first cigarette of the

day. This could be explained by the hypothesis that nAChRs only completely recover

from long-term desensitization after a night-time abstinence from smoking

(Pidoplichko et al. 1997).

2.1.5 Upregulation of nAChRs

Usually, chronic agonist treatment results in a decrease, or downregulation of receptor

number, which is accompanied by tolerance to the agonistic actions of the drug in

question. Further, chronic antagonist treatment seems to elicit an upregulation of

receptor number and an accompanying supersensitivity to agonist actions.

Surprisingly, chronic nicotine exposure increases the amount of brain high affinity 3H-

ACh or 3H-nicotine binding sites (primarily α4β2 nAChRs) (Flores et al. 1992; Marks

et al. 1983; 1985; Marks and Collins 1985; Schwartz and Kellar 1983; 1985) while

producing a smaller increase in the amount of αBgt binding sites (α7 nAChRs)

(Collins and Marks 1987; Marks et al. 1983) in both rats and mice. Nicotine and

acetylcholine bind to the same sites in mouse and rat brain whereas αBgt binding

shows different distribution (Clarke et al. 1985b; Marks et al. 1986). The increase of

high-affinity binding sites is not because of an increase in transcription of α4 and β2

subunits (Marks et al. 1992). Instead, it may be the result of a decrease in the rate of

turnover of α4β2 nAChRs on the cell surface (Peng et al. 1994). Rowell and

Wonnacott (1990) found a strong correlation between the Bmax of nicotine-binding and

the nicotine-stimulated release of dopamine in the same tissue, indicating that the

increased binding sites are functional receptors. Contrary to this, Marks et al. (1993)

observed reduced nicotinic receptor function after chronic nicotine infusion at doses

which result in upregulation of nicotinic receptors. Therefore, upregulation is thought

21

to result from a nicotine-induced conformational change, perhaps to a desensitized or

inactivated conformation (Marks et al. 1983; Schwartz and Kellar 1983) rather than a

result of signals mediated by ion flow through the α4β2 nAChR channel. The former

suggestion is probably the case because the nicotinic antagonist mecamylamine can

also induce upregulation of both cloned α4β2 nAChRs and brain nAChRs (Collins et

al. 1994; Pauly et al. 1996; Peng et al. 1994). The mechanisms of upregulation of α3

and α7 nAChRs appear to differ from those of α4β2 nAChRs not only in nicotine

sensitivity, extent and surface expression, but also pharmacologically, in that

mecamylamine was unable to induce upregulation of α3 and α7 subunits (Peng et al.

1997). Differences in regulation among nAChRs could be explained by subtype-

specific nicotine sensitivities of both the desensitized and active receptor states

(Fenster et al. 1997).

Later, conflicting results have been obtained when the correlation of receptor

desensitization and upregulation have been thoroughly studied in vitro. Fenster et al.

(1999c) demonstrated that the nicotine concentration required to induce both

desensitization and upregulation is the same, while some others (Peng et al. 1994;

Whiteaker et al. 1998) have shown that the concentrations necessary to induce

desensitization and upregulation can differ by several orders of magnitude, specifically

that upregulation needs a greater nicotine concentration than desensitization. This

discrepancy may be explained by differences in methodological details; whether cell

homogenates (Whiteaker et al. 1998) or intact oocytes (Fenster et al. 1999c) have been

utilised and whether the nAChRs studied were expressed on the surface or

intracellularly. Clearly, some form of interaction of nAChRs with nicotine is likely to

precipitate both desensitization and upregulation, meaning that there might be some

common steps in the mechanisms of both processes (Ke et al. 1998).

The possible relevance of the nicotine-induced changes in receptor numbers was

emphasized by the findings that nicotinic receptors labeled by 3H-nicotine, 3H-

epibatidine and 3H-cytisine are increased in homogenates of autopsied brain samples

from smokers (Benwell et al. 1988; Breese et al. 1997; Perry et al. 1999). The increase

in 3H-nicotine binding levels in humans was dose-dependent and mostly affected by

22

the daily nicotine intake before death, rather than the overall amount smoked during a

lifetime (Breese et al. 1997). Smokers who had quit smoking at least 2 months before

death had levels of 3H-nicotine binding which were comparable to levels found in non-

smoking subjects (Breese et al. 1997). This suggests that nicotine-induced up-

regulation of receptor numbers is a temporary effect, similar to that found in rodents

(Collins et al. 1990a; Marks et al. 1985). The study of Breese et al. (1997) along with

studies in rodents clearly implies that the increased nicotinic receptor numbers in

smokers’ brains are a consequence of smoking, rather than smoking behaviour being a

consequence of an intrisincally higher number of nicotinic receptors (Perry et al.

1999).

2.1.6 Nicotine-induced development of tolerance

Drug tolerance is a state of decreased responsiveness to the pharmacological effect of

a drug as a result of prior exposure to that drug or to a related drug. The degree of

tolerance, in general, may vary within very wide limits. Usually in the tolerant

organism, although the ordinarily effective dose is less effective or is even entirely

ineffective, an increased dosage will again elicit the typical drug response. Thus, as a

rule, tolerance is a quantitative change in sensitivity to a drug. Sometimes, however,

the drug effect cannot be obtained at any dosage in the tolerant organism (Cox 1990).

Chronic nicotine exposure renders some brain nAChRs inactivated for a long time

period (see section 2.1.4). These long-lived inactive receptor states are likely to

underlie the development of tolerance to nicotine (Fenster et al. 1999b) and may be

responsible for attenuation of the behavioural and physiological effects of nicotine

such as locomotor depressant actions (Marks et al. 1983; Marks and Collins 1985;

Stolerman et al. 1973) and decrease in body temperature (Collins et al. 1988b;

Mansner et al. 1974; Marks et al. 1983; Marks and Collins 1985).

Early studies performed in mice suggested that the upregulation of brain nicotinic

receptors might be associated with tolerance to nicotine (Marks et al. 1983; 1985)

23

whereas later studies indicate that upregulation can occur before tolerance

development (Marks et al. 1991) and tolerance can develop without receptor changes

(Pauly et al. 1992). Similarly, studies with the rat have shown that tolerance to

nicotine’s locomotor depressant effects is not necessarily associated with receptor

changes (Collins et al. 1990b). It seems that tolerance to the effects of nicotine in vivo

cannot be fully explained by either the changes in the number of binding sites or in the

functional state of the nAChRs (Marks et al. 1993).

2.2 Nicotine and dopamine

2.2.1 Dopaminergic pathways in the brain

Dopaminergic neurons can be divided into 3 major classes depending on the length of

the dopaminergic tract: ultrashort, intermediate-length and long-length systems

(Cooper et al. 1996). The long projections consist of three separate tracts: nigrostriatal,

mesolimbic and mesocortical projections (Björklund and Lindvall 1984; Figure 2.2)

Cell bodies of the neurons forming these major ascending dopaminergic pathways are

located in the brainstem, in the substantia nigra pars compacta (A9) and ventral

tegmental area (A10) (Dahlström and Fuxe 1964; Ungerstedt 1971). The A9 neurons

project mainly to the caudate-putamen or to the dorsal striatum forming the

nigrostriatal dopamine system (Fuxe et al. 1985) A minor component of the

nigrostriatal system projects from the A8 area (dopamine-rich neurons dorsal to A10

and caudal to A9) to the ventral putamen (Björklund and Lindvall 1984; Ungerstedt

1971). The mesolimbic dopaminergic pathway is formed by neurons that project from

the A10 area to the nucleus accumbens (i.e. the ventral striatum), olfactory tubercle

and other limbic regions such as amygdala, hippocampus and septum. In addition, the

A10 area sends axons to the cortical areas, e.g. to the medial, prefrontal, entorhinal and

cingulate cortex, and this system is known as the mesocortical dopaminergic pathway

(Björklund and Lindvall 1984).

24

Figure 2.2. The nigrostriatal, mesolimbic and mesocortical dopaminergic projections in ratbrain. Adapted from Wiley et al. (1996), with permission. TO = the olfactory tubercle.

The nigrostriatal system is involved in the regulation of movement and also subserves

some cognitive processes whereas the mesolimbic pathway is involved in arousal,

locomotor activity, reward, motivational and affective states and euphoria induced by

drugs (Fuxe et al. 1985). Behavioural studies have shown that mesolimbic and

nigrostriatal dopaminergic systems are involved in different aspects of activation,

probably linked with incentive-motivational or reward processes (including those

engaged by drugs of abuse), in the case of the mesolimbic (ventral striatal) system, and

response preparatory mechanisms, in the case of the mesostriatal (dorsal striatal)

system (Robbins et al. 1998). These differential effects on behaviour may be explained

by the inputs to these regions. The ventral striatum receives a strong allocortical input

from the prefrontal cortex and amygdala, which may be important for the convergence

of information about reward, whereas the neocortical inputs of the dorsal striatum may

be implicated in cognitive and response-related functions, such as visuospatial

processing and attention to action (Dunnett and Robbins 1992). The activity of dorsal

25

and ventral striatum may be co-ordinated as a general response to activating stimuli

which leads to behavioral activation (Dunnett and Robbins 1992). The mesocortical

pathway is important in higher cortical functions (Fuxe et al. 1985) and finally the

frontal cortical dopamine projections may have important role in the regulation of

subcortical dopamine activity (Dunnett and Robbins 1992).

2.2.2 Synthesis and metabolism of dopamine

Dopamine synthesis originates from the amino acid precursor tyrosine, which is

transported across the blood-brain barrier and is actively concentrated by

dopaminergic neurons. The hydroxylation of L-tyrosine by tyrosine hydroxylase to L-

dihydroxyphenylalanine (L-DOPA) is the rate-limiting step of dopamine synthesis. L-

DOPA is subsequently converted to dopamine by L-aromatic amino acid

decarboxylase in the cytoplasm of cells (Cooper et al. 1996). Active transporters then

carry dopamine to synaptic vesicles, where the molecules are protected from

catabolizing enzymes. Amongst other factors, presynaptic dopamine autoreceptors

modulate the rate of tyrosine hydroxylation. Autoreceptors are activated by dopamine

released from the nerve terminal, resulting in feedback inhibition of dopamine

synthesis. Autoreceptors can modulate both synthesis and release of dopamine.

Dopamine synthesis also depends on the rate of impulse flow in the dopaminergic

pathway (Cooper et al. 1996). Dopamine is released in a calcium-dependent manner

when an action potential invades the terminal of the neuron. The extent of dopamine

release appears to depend on the rate and pattern of firing (Cooper et al. 1996). The

burst-firing pattern leads to enhanced release of dopamine from dopaminergic neuron

compared with single-spike pattern (Grace and Bunney 1984a; 1984b). Most of the

released dopamine is then transported back into the terminal by specific dopamine

transporter molecules (Reith et al. 1997).

The metabolism of dopamine can take place in the synaptic cleft, in the cytoplasm of

nerve terminal and inside glial cells. The main enzymes, which metabolize dopamine,

are catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) (Sharman

26

1973). COMT is found both in membrane-bound and in cytoplasmic soluble forms

(Rivett et al. 1983). The soluble COMT is localised in glial cells and the membrane-

bound COMT in postsynaptic neurons (Kaakkola et al. 1987; Rivett et al. 1983). MAO

is located both intra- and extraneuronally (Agid et al. 1973).

Figure 2.3. A simplified diagram of the metabolism of dopamine. Abbreviations: DA =dopamine; DOPAC = 3,4-dihydroxyphenylacetic acid; HVA = homovanillic acid; 3-MT = 3-methoxytyramine; COMT = catechol-O-methyltransferase, MAO = monoamine oxidase.Wood and Altar (1988), with permission.

In dopaminergic nerve terminals dopamine is metabolised intraneuronally to the

corresponding aldehyde by MAO. The aldehyde is subsequently oxidised to 3,4-

dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase. After diffusing out

of the neurons DOPAC can be further metabolised to homovanillic acid (HVA) by

COMT. Dopamine, released into the synaptic cleft, can be inactivated both by

reuptake into the dopaminergic nerve terminals, and by inactivation involving COMT.

The extraneuronal dopamine metabolite, 3-methoxytyramine (3-MT) is formed from

dopamine by COMT and can be further metabolised to HVA by MAO and aldehyde

dehydrogenase (Fig. 2.3).

27

DOPAC can be used as an indicator of intraneuronal synthesis and metabolism of

dopamine (Roffler-Tarlov et al. 1971) since a substantial amount of DOPAC is derived

from the newly formed pool of dopamine (Zetterström et al. 1988). HVA is formed by

both MAO and COMT, indicating the sum of dopamine synthesis, metabolism and

release (Roffler-Tarlov et al. 1971; Westerink and Spaan 1982). As HVA is

predominantly derived from O-methylation of DOPAC, the changes in the tissue and

extracellular content of HVA follow those of DOPAC in rats (Westerink 1985; Wood

and Altar 1988). In mice, however, DOPAC and HVA are not formed as consequtive

products in the same metabolic pathway, in that little DOPAC is converted to HVA in

mouse brain (Sharman 1973). Tissue contents as well as extracellular concentration of

3-MT can be used as an index of dopamine release (Kehr 1976; Wood and Altar 1988)

provided that the effects of post mortem changes have been eliminated.

2.1.6 Nicotine and brain dopaminergic systems

Nicotine appears to differentially affect dopamine release, metabolism and the

electrophysiological properties of dopaminergic neurons in nigrostriatal and

mesocorticolimbic dopaminergic systems. Nicotine enhances striatal and limbic

dopamine turnover and metabolism. Nicotine increases in vitro 3H-dopamine release

from striatal slices and synaptosomes (Arqueros et al. 1978; Grady et al. 1992; Rapier

et al. 1988; Westfall 1974) and from minced preparations of nucleus accumbens

(Rowell et al. 1987). Nicotine increases in vivo striatal DOPAC and HVA

concentrations (Freeman et al. 1987; Haikala et al. 1986; Lichtensteiger et al. 1976;

Nose and Takemoto 1974; Roth et al. 1982) and limbic, mainly accumbal, DOPAC

concentrations (Grenhoff and Svensson 1988). Nicotine has been reported to increase

the rate of disappearance of striatal and accumbal dopamine after blockade of

synthesis by α-methyl-p-tyrosine (Andersson et al. 1981b; Lichtensteiger et al. 1982).

Further, nicotine elevates the extracellular dopamine levels in both striatal (Damsma et

al. 1988; Imperato et al. 1986; Toth et al. 1992) and limbic brain areas (Benwell and

Balfour 1992; Imperato et al. 1986; Mifsud et al. 1989; Nisell et al. 1994) as measured

28

by in vivo microdialysis. Systemic or local infusion of nicotine also stimulates the

firing frequency of midrain dopaminergic neurons (Calabresi et al. 1989; Clarke et al.

1985a; Grenhoff et al. 1986; Lichtensteiger et al. 1982). A dose-response comparison

between substantia nigra and VTA reveals that systemic nicotine produces a larger

increase in firing frequency of dopaminergic neurons in VTA (Mereu et al. 1987;

Westfall et al. 1989). This correlates well with the observations that acutely

administered nicotine preferentially activates limbic dopamine metabolism, turnover

and release (Andersson et al. 1981a; Brazell et al. 1990; Grenhoff and Svensson 1988;

Imperato et al. 1986).

Nicotine-induced release of DA in vitro from rat striatal slices and synaptosomes has

been reported to attenuate (Grady et al. 1994; Rowell and Hillebrand 1994; Schulz and

Zigmond 1989) during or after prolonged nicotine exposure suggesting that the

desensitization of the nAChRs is involved. It has been hypothesized that at least two

different nAChR subunit complexes are involved in nicotine-induced dopamine

release from rat striatal synaptosomes, one of which has a α3β2 subunit composition

(Kaiser et al. 1998; Kulak et al. 1997) and the other has a α4β2 subunit composition

(Sharples et al. 2000). In contrast, others (Grady et al. 1997) have suggested that both

the transient and persistent phases of nicotine-induced dopamine release from striatal

synaptosomes are mediated by only one type of receptor, which contains both α3 and

α4 as well as β2 subunits.

The effect of chronic nicotine pretreatment in vivo on nicotine-induced changes in

dopaminergic activity seems to depend on the response measured, dosing schedule and

species. During prolonged constant nicotine infusion the acute nicotine-evoked

increase of extracellular dopamine in the NAcc and in the dorsal striatum was

inhibited and the elevation of accumbal dopamine metabolites was attenuated

suggesting desensitization of the receptors mediating nicotine-induced mesolimbic and

nigrostriatal DA responses (Benwell and Balfour 1997; Benwell et al. 1995). On the

other hand, Damsma et al. (1989) and Nisell et al. (1996) reported no change in the

nicotine-induced increase of DA-release in the NAcc of rats when nicotine was given

by repeated injections. After intermittent nicotine treatment even sensitization of the

29

nicotine response in the striatum, the NAcc and the prefrontal cortex has been reported

(Balfour et al. 1998; Benwell and Balfour 1992; Marshall et al. 1997; Reid et al. 1996;

Shoaib et al. 1994). In mice, nicotine-induced changes in striatal dopamine metabolism

were abolished at 24 hours after withdrawal from 7-week oral nicotine administration

suggesting the development of tolerance (Pietilä et al. 1996).

Nicotine has variable effects on locomotor activity depending on animal species, drug

dosage, route of administration and duration of treatment. Acute systemic

administration of nicotine in rats produces a biphasic effect on locomotor activity, so

that an initial depressant activity is replaced by a stimulant action (Clarke and Kumar

1983; Stolerman et al. 1973). In mice, acute nicotine usually decreases locomotor

activity (Collins et al. 1988a; Hatchell and Collins 1980) and only few authors have

reported a stimulating effect of acutely administered nicotine (Nordberg and Bergh

1985; Sparks and Pauly 1999). In rats, the locomotor stimulant effects of nicotine have

been suggested to depend upon its ability to stimulate mesolimbic dopamine release

(Benwell and Balfour 1992; Clarke 1990). However, recent studies have shown that

the relationship between the stimulatory effects of nicotine on locomotor activity and

mesolimbic dopamine is more complex than orginally thought (Balfour et al. 1998).

Tolerance towards nicotine’s depressant effect on locomotor activity has been

demonstrated in rats and in mice (Benwell et al. 1995; Clarke and Kumar 1983; Marks

et al. 1983; Pietilä et al. 1998). However, a more pronounced stimulatory effect

(reverse tolerance or behavioural sensitization) has been demonstrated in chronically

treated rats (Clarke and Kumar 1983; Ksir et al. 1985) and more recently, in mice

(Gäddnäs et al. 2000). In this mouse study, the increased locomotor activity in the

nicotine-treated mice correlated to increased striatal concentrations of DOPAC and

HVA, indicating enhanced striatal metabolism. This finding suggests that the cerebral

dopaminergic systems might be important in the mediation of the nicotine-induced

locomotor hyperactivity in mice as they have been shown to be of importance in rats

(Clarke 1990; Pietilä and Ahtee 2000).

30

2.3 Nicotine and c-fos

2.3.1 Immediate-early gene c-fos

Extrinsic signals can modulate cell function in different ways. Excitation of neurons

has two general consequences. The first is the short-lived response of the cell that is

elicited immediately after application of the stimulus and which has no requirement for

ongoing protein synthesis. An example of such a response is an increase or a decrease

in the firing rate of a particular neuron. Secondly, relatively brief periods of excitation

can bring about longer-term responses that are dependent upon protein synthesis and

that involve alterations in the expression of specific genes (Curran and Morgan 1987;

Morgan and Curran 1991a).

Genes that are activated rapidly and transiently after cell stimulation and whose

expression cannot be prevented by protein synthesis inhibitors are known as the

immediate-early genes (IEGs) (Sheng and Greenberg 1990). IEGs are believed to

encode transcription factors, which will, in turn, modify the expression of other genes

known as target genes (Sheng and Greenberg 1990; Fig. 2.4). Target gene expression

can then modify the phenotype of the cell in question (Herrera and Robertson 1996).

Target genes are also involved in the processes such as learning and memory, drug

tolerance/sensitisation (Hughes and Dragunow 1995). Neurotransmitters can have

differential effects on the same target gene in different cell types or on different genes

in the same cell by inducing distinct patterns of IEG production or by causing IEGs to

undergo different post-translational modifications (Esterle and Sanders-Bush 1991).

31

Figure 2.4 Proposed model for the role of cellular IEGs in stimulus-response coupling.Extracellular ligands (L) interact with receptors (R) and activate second messenger systemsvia membrane-transducing components (T). Second messengers elicit rapid alterations thatlead to short term response. The same second messengers, directly or indirectly, act to inducetranscription of the cellular IEGs (A-C). IEGs may be components or regulators of the signaltransduction cascade (e.g. receptors, G-proteins) or they may promote the transcriptionalactivation of further target genes (1-7); e.g. those encoding ion channels, neuropeptideprecursors etc., that cause changes in long-term responses. Potential regulation of IEGs bynegative feedback control is indicated by dotted lines. Curran and Morgan (1987), withpermission.

The IEG that was discovered first is the well-characterized proto-oncogene c-fos

(Curran et al. 1983), which encodes a nuclear phosphoprotein (Fig. 2.5). Fos protein

undergoes extensive post-translational modification and forms a dimeric complex with

Jun, the protein encoded by c-jun. (Curran and Morgan 1987). Dimerization occurs

through an α-helical domain that is termed a leucine zipper. Fos-Jun heterodimers

32

exhibit sequence-specific DNA binding to the canonical motif TGACTTCA. Several

studies have established (Curran and Franza 1988) that this DNA sequence is the

binding site for the transcription factor activator protein 1 (AP-1).

Figure 2.5 Induction of the Fos protein is elicited by extracellular stimuli such asneurotransmitters. These agents produce signals that lead to a transcriptional activation of thec-fos gene. mRNA accumulates transiently, the Fos protein is synthetized and transported tothe nucleus and extensively modified. In the nucleus, Fos forms a protein complex with oneof the several cellular proteins and becomes associated with chromatin Curran and Morgan(1987), with permission.

Indeed, Fos and Jun, in addition to a number of Fos- and Jun-related proteins, are

constituents of AP-1 (Curran and Franza 1988). Thus, Fos acts in concert with other

immediate-early gene products to regulate transcription of other target genes (Morgan

and Curran 1991a).

33

The intracellular mechanisms that lead to activation of c-fos gene are somewhat

unclear (Sheng and Greenberg 1990). The activation of many second messenger

pathways leads to early response gene expression and these may include stimulation of

adenylate cyclase, protein kinase C and tyrosine kinases; Ca2+ entry into cells; and

mobilisation of intracellular Ca2+. The biochemical events that couple these second

messenger systems to early response gene expression remain unknown (Sharp et al.

1993b). However, intracellular Ca2+ appears to play a central role in neuronal cells for

coupling neuronal depolarisation to c-fos expression (Sheng and Greenberg 1990).

Ca2+ entry through voltage-activated Ca2+ channels and the resultant activation of a

calmodulin-dependent protein kinase, or a similar enzyme, and phosphorylation of

CRE-binding protein (CREB), which initiates the transcription of c-fos, have been

strongly suggested as the means by which depolarization induces the c-fos gene (Sharp

et al. 1993b).

Some of the early studies suggested the use of Fos immunostaining (Fos IS) as an

indicator of neuronal activity (Dragunow and Faull 1989; Hunt et al. 1987; Sagar et al.

1988). Several more recent studies have suggested (Herrera and Robertson 1996;

Hughes and Dragunow 1995) that Fos IS provides a method to map the pattern of

postsynaptic stimulation within the intact nervous system with single cell resolution

(Morgan and Curran 1991b) since Fos-staining is localized to the cell nucleus (Sagar

et al. 1988). A potential use of Fos staining is to define the cellular targets of

neuroactive substances, like nicotine. The basal expression of Fos is relatively low in

most brain regions and Fos is expressed rapidly and transiently after a variety of

physiological and pharmacological stimuli (Sagar et al. 1988). These stimuli include,

for example, seizure activity (chemically and electrically induced), kindling, brain

injury (e.g. mechanical), sensory stimulation (noxious, visual, olfactory,

somatosensory), stress, learning, the induction of LTP and pharmacological activation

of specific receptor agonists and antagonists (Herrera and Robertson 1996; Hughes

and Dragunow 1995). Fos-IS is most informative when novel stimuli are applied or

when the animal is stimulated after a period of sensory deprivation, whereas after

prolonged stimulation it will yield negligible results (Chaudhuri 1997). Fos expression

is an anatomical technique for metabolic mapping similar in some respects to 2-

34

deoxyglucose autoradiography (2-DG), which is a measure of total glucose

consumption in brain and therefore, useful for mapping regional changes of glucose

metabolism (Sharp et al. 1993b). Although lacking the quantitative potential of the 2-

DG method, Fos expression offers cellular resolution (Sharp et al. 1993b) which is not

achieved by the 2-DG method.

Mapping the functional activity in the brain with Fos or other IEG products has two

major drawbacks: limited cell-type expression and stimulus-coupling uncertainty

(Chaudhuri 1997). Only a subset of neurons may express a particular transcription

factor (e.g. Fos) and neurons in some brain structures may not express it at all (Labiner

et al. 1993). Thus, Fos cannot be considered to be a general marker of neural activity

that can be applied throughout the central nervous system (Chaudhuri 1997). A causal

link between IEG expression and a particular triggering event is often difficult to

establish, since IEG expression can be influenced by multiple receptor systems and by

different signal transduction pathways. Furthermore, with in vivo preparations it is

difficult to establish whether the pattern of Fos expression was produced by the

specific stimulus that was applied or by some non-specific feature of the experimental

condition or even by an unrelated mental or endocrine event. To verify the stimulus-

response association, control animals that are handled similarly to the experimental

animals and carefully controlled experimental conditions should be used (Chaudhuri

1997; Dragunow and Faull 1989). However, these controls can only resolve the

uncertainty at a regional level; they do not permit a positive association to be made at

the cellular level for any of the individual Fos-stained neurons (Chaudhuri 1997).

Though caution should be exerted in overinterpreting the presence or absence of Fos-

IS in a particular situation, the maps of Fos expression may still prove useful (Morgan

and Curran 1991b). A dual-activity mapping technique may provide an internal control

and thus resolve the above-mentioned problems at the cellular level. The technique

relies on the different spatial locations of transcription factor mRNA and protein

(cytosolic and nucleic, respectively) and the different temporal patterns of their

expression (Chaudhuri 1997). To date, this has only been tested succesfully with one

IEG, namely zif268 mRNA and protein in monkeys (Chaudhuri 1997) but not with c-

fos.

35

2.3.2 Acute nicotine and c-fos

Acute nicotine administered using various protocols (i.p., s.c., i.v.-infusion, local

microinfusions) activates different groups of rat brain areas (see Table 2.3).

Table 2.3 Rat brain areas activated after various acute nicotine administrationprotocols.

Fos/

c-fos

Nicotine dose Time Brain areasactivated

Reference

Fos 2 mg/kg/h i.v. 60 min SC, MT, IPN Ren and Sagar 1992

c-fos 1.5 mg/kg i.p. 30 min PC, CG, MH, DG, PVN,LC

Sharp et al. 1993a

Fos 0.1 mg/kg i.v. 60 min Cg, PVN, SON, NTS-A2, NTS-C2, LC, ACe

Matta et al. 1993

Fos 0.4 mg/kg s.c. 120 min SMN, MT, IPN, CLI, SC Pang et al. 1993

Fos 1 mg/kg s.c. 120 min Cg, PC, CPU, NAcc Kiba and Jayaraman1994

Fos 30 µM, 300 µMinto SON

80 min SON Shen and Sun 1995

Fos 0.09-0.18 mg/kgi.v.

60 min NTS-A2, NTS-C2, LC,PVN

Valentine et al. 1996

Fos 8.0 µg/side intoVTA

120 min NAcc Panagis et al. 1996

Fos 0.35 mg/kg s.c. 90 min Cg, FC, PaC, OC, PC,NAcc, CPU, MH, ACe,ThN, SC

Mathieu-Kia et al.1998

ACe = central nucleus of amygdala, Cg = cingulate cortex, CLI = caudal linear nucleus, CPU= caudate putamen, DG = dentate gyrus, FC = frontal cortex, IPN = interpeduncular nucleus,LC = locus coeruleus, MH = medial habenula, MT = medial terminal nucleus of theaccessory optic tract, NAcc = nucleus accumbens, NTS-A2, NTS-C2 = nucleus tractussolitarius, OC = orbital cortex, PC = piriform cortex, PaC = parietal cortex, PVN =hypothalamic paraventricular nucleus, SC = superior colliculus, SMN = supramamillarynucleus, SON = supraoptic nucleus, ThN = anteroventral and lateroposterior thalamic nuclei,VTA = ventral tegmental area.c-fos = Northern gel analysis for c-fos mRNA, Fos = Fos protein immunohistochemistry.

It has been reported that the pattern of Fos-labeling is dependent upon both the route

and mode of drug administration (Bullitt 1990; Hunt et al. 1987). Only in one study

has the nature of the Fos-positive neurons activated by acute administration of nicotine

36

been determined. In this report the neurons of the ventral midbrain area, in which acute

nicotine increased the number of Fos-positive nuclei, were not found to be

dopaminergic (Ren and Sagar 1992). In many brain areas nicotine-induced c-fos

expression is blocked by mecamylamine which suggests its mediation by central

nAChRs (Kiba and Jayaraman 1994; Pang et al. 1993; Ren and Sagar 1992).

2.3.3 Intermittent nicotine administration, Fos and Fos-related antigens

There are very few studies concerning the effect of chronic nicotine treatment on Fos-

immunostaining. Two studies report the effects of intermittent chronic nicotine

treatment with injections; 0.35 mg/kg s.c 3 times a day for 14 days (Mathieu-Kia et al.

1998) and 0.5 mg/kg s.c. once daily for 12 days (Nisell et al. 1997). In both studies a

challenge dose of nicotine was given next day after intermittent nicotine treatment.

Both studies reported the sensitization of the effect of nicotine on Fos-IS after the

challenge dose in the medial prefrontal cortex (PFC) and in the shell of NAcc. In

addition, Mathieu-Kia and collaborators (1998) reported the sensitization in the Cg,

the PaC and the core of the NAcc. Nisell et al. (1997) also found that the chronic

nicotine treatment itself increased the Fos-IS in the medial PFC.

Merlo Pich and collaborators have studied the effect of nicotine self-administration on

the expression of Fos protein in various brain areas (Pagliusi et al. 1996; Pich et al.

1997; 1998). These authors have compared the effects of self-administered nicotine to

nicotine given with injections (= passive nicotine treatment, same amount that was

self-administered). Nicotine self-administration increased Fos expression in most of

the brain regions that were activated by passive nicotine treatment (Pich et al. 1997), in

particular in the CPU, the shell and the core of NAcc, the lateral septum, the Cg and

the PFC. In contrast to passively administered nicotine, nicotine self-administration

did not increase Fos expression in the hypothalamus, the LC, the DG and the amygdala

(Pagliusi et al. 1996). These differences are important, since immediate early gene

expression can represent both the direct pharmacological effects of nicotine on neural

37

networks and the activation of drug-seeking behaviour that controls nicotine-

consumption, which includes the reinforcing properties of nicotine (Pich et al. 1999).

The gene expression maps of the Fos-related antigens (FRAs) expressed in the

terminal fields of the mesocorticolimbic dopaminergic pathway of rats trained for

nicotine self-administration have also been studied (Pich et al. 1997). FRAs consist of

a heterogenous group of proteins of the c-fos family that heterodimerize with members

of the c-jun family to produce AP-1 complexes. FRAs and in particular the 35 kDa

component recently identified as ∆-Fos, are medium-late onset genes and their

products persist for several days in the nucleus of target neurons (Hope et al. 1994).

The gradual development and long-lasting temporal properties of the chronic FRAs

make them candidates for the transcriptional mediators underlying some of the long-

lasting adaptations such as dependence (Hope et al. 1994). Compared with control rats,

increased expression of FRA immunoreactivity was found in the anterior CG, the PFC,

the NAcc, the medial CPU, but not in the amygdala of rats self-administering nicotine

(Pich et al. 1997). Only in rats self-administering nicotine were selective increases of

activity-dependent FRA signals found in the SC, a region enriched with nAChRs but

with less dopamine receptors, supporting the selectivity of nicotine effects.

2.4 Brain areas in which nicotine activates c-fos

2.4.1 Dopaminergic target areas

Nicotine increases the number of Fos-positive nuclei in most striatal and limbic

dopaminergic target areas, namely in the striatum, NAcc, Cg, ACe and PFC as

discussed in Sections 2.3.2 and 2.3.3. The striatum is usually divided into dorsal

striatum and ventral striatum (including the NAcc). Dorsal striatum receives afferents

from sensorimotor cortex and dopaminergic inputs from the pars compacta of the

substantia nigra (Heimer et al. 1995). The striosomes and matrix are the major

neurotransmitter-specific compartments of the caudate-putamen and have different

input-output connections (Robertson et al. 1991). The NAcc is divided to the core and

38

shell regions which are differentially innervated by mesencephalic dopaminergic fibres

and are distinguished by differences in basal and stimulated dopamine levels as well as

in dopamine-mediated synaptic responses (Heimer et al. 1991; Meredith et al. 1992).

The NAcc receives its afferents from cerebral cortex, intralaminar thalamic nuclei and

mesencephalic dopamine cells (Heimer et al. 1995). The core of the NAcc projects to

the dorsolateral part of the ventral pallidum, which in turn projects to the subthalamic

nucleus and substantia nigra. The shell of the NAcc projects to the ventromedial part

of ventral pallidum, which further projects to the VTA (Deutch and Cameron 1992).

Cg and PFC are among the terminal regions of the mesocorticolimbic DA pathway

(Zilles and Wree 1995). The nicotine-induced increase of Fos expression has been

reported to be mediated via dopamine D1 receptors in striatum (Kiba and Jayaraman

1994), NAcc and PFC (Nisell et al. 1997). Both direct and indirect stimulation of

dopamine receptors increases Fos protein expression, mainly in striatum, after

administration eg. of cocaine or amphetamine (Hughes and Dragunow 1995). Further,

non-competitive NMDA antagonists, CPP and MK-801, have been reported to block

the nicotine-induced increase of Fos expression in striatum and NAcc (Kiba and

Jayaraman 1994). Both NAcc and CPU receive glutamatergic pathways from several

regions of cerebral cortex (Walaas 1981).

ACe forms part of the mesolimbic DA system; it is a major target of the DA pathway

originating largely in the A10 and A9 (Kilts et al. 1988; Loughlin and Fallon 1983)

and provides input to substantia nigra (Gonzalez and Chesselet 1990; Price and

Amaral 1981) whilst also receiving catecholaminergic input from the brainstem (Moore

and Bloom 1979). ACe is involved in several stress-induced behavioural and autonomic

responses (Brown and Gray 1988) that may depend on central catecholamine release.

ACe also influences the secretion of adrenocorticotropin (ACTH) by modifying the

negative feedback of glucocorticoids (Dunn and Whitener 1986). Nicotine may activate

ACe either directly or indirectly by stimulating the dopaminergic or noradrenergic

afferent pathways (Matta et al. 1993). Nicotine might also stimulate the release of

corticotrophin-releasing hormone (CRH) from ACe neurons (Matta et al. 1997).

39

2.4.2 Other brain areas

Superior colliculus (SC) and medial terminal nucleus of the accessory optic tract

(MT). The nicotine-induced Fos expression in visual structures can be mediated via

nAChRs present in high density in MT and on afferent retinal fibers (Clarke et al.

1985b; Prusky and Cynader 1988; Wada et al. 1989). The nicotine receptors in the MT

are predominantly presynaptic (London et al. 1985). Nicotine in all probability acts

presynaptically or in the retina itself to stimulate the release of a neurotransmitter,

possibly glutamate, from retinal ganglia cell axon terminals in the MT (Canzek et al.

1981) and in the SC (Binn 1999). The interaction of nicotine with the components of the

visual system can support the evidence for improved performances in visual information

processing observed in smokers and non-smokers receiving nicotine via cigarette smoke

or subcutaneous injection (Foulds et al. 1996; Parrot and Graig 1992).

Hypothalamic paraventricular nucleus (PVN). Although nicotine is a potent activator

of the HPA axis, as manifested by an increase in serum adrenocorticotropic hormone

(ACTH) and corticosterone levels (Balfour 1989; Balfour et al. 1975; Benwell and

Balfour 1979; Conte-Devolx et al. 1981; Weidenfeldt et al. 1989) nicotine does not

directly act in the PVN. Nicotine indirectly stimulates the release of corticotropin-

releasing hormone (CRH) from the parvocellular region of the PVN (pcPVN) by

acting on brainstem noradrenergic structures in a dose-dependent and regionally

selective manner (Matta et al. 1990b; 1995). The CRH neurons located in pcPVN send

their axons to the portal capillaries in the median eminence and subsequently stimulate

ACTH release from the anterior pituitary gland (Swanson et al. 1983). Systemic or

i.c.v. administration of nicotine induces NA release in the PVN in conscious rats

(Matta et al. 1995; Sharp and Matta 1993) and subsequent ACTH secretion

(Andersson et al. 1983; Matta et al. 1990a; 1990b). Mecamylamine given directly into

the PVN of rats did not antagonize the nicotine-induced elevation of extracellular

noradrenaline (NA) concentrations as measured by in vivo microdialysis (Fu et al.

1997). In contrast, nicotine-induced NA release was blocked when mecamylamine was

administered i.p. or directly into the fourth ventricle. Thus, systemic nicotine was

concluded to act on nAChRs in brainstem catecholaminergic regions rather than on

40

presynaptic nAChRs in order to elicit NA release from axon terminals (Matta et al.

1998). Nicotine induces both Fos protein expression (Matta et al. 1993; 1998) and c-

fos mRNA response (Sharp et al. 1993a) in the PVN.

Supraoptic nucleus (SON). Nicotine is well known to elevate systemic vasopressin

levels (Andersson et al. 1983; Zbuzek and Zbuzek 1991). Vasopressin in addition to

regulating fluid and electrolyte balance (Sladek and Sladek 1985) also potentiates the

effects of CRH on ACTH release (Prouxl et al. 1984). Magnocellular neurons, whose

perikarya are located principally within the SON, PVN and accessory nuclei of the

hypothalamus synthesize vasopressin and oxytocin (Silverman and Zimmerman 1983)

whilst cholinergic innervation mediates the vasopressin release within SON (Amstrong

1985). In the SON, microinfusion of nicotine induces Fos protein expression mainly in

the magnocellular vasopressin-releasing neurons (Shen and Sun 1995). Shioda et al.

(1997a) have localized both nAChR α4 subunit mRNA expression and

immunoreactivity in the magnocellular neurons in the SON. Nicotine seems to activate

vasopressin-releasing neurons via nAChRs composed of α4 subunits and has also been

linked to stimulation of the cyclic AMP- protein kinase A-regulated Ca2+-signaling

pathway (Shioda et al. 1997b). On the other hand, nAChR α7 subunit mRNA has been

detected in the SON (Séguéla et al. 1993), and acetylcholine-evoked voltage clamp

currents in the SON were abolished by nAChR α7 subunit selective antagonist

methyllycaconitine but not by dihydro-β-erythroidine, an α4β2 nAChR subunit

selective antagonist (Zaninetti et al. 2000).

Interpeduncular nucleus (IPN). IPN appears to occupy a central position in the

midbrain limbic system (Morley 1986). Most of the connections of the IPN are related

to the neural circuits of the limbic system and its midbrain connections. For example, a

direct pathway has been demonstrated from IPN to the hippocampus, the main

integrative centre of the limbic system (Contestabile and Flumerfelt 1981). Thus, IPN

is one of centres in the network of connections that convey and modulate the input

from the limbic forebrain regions to the limbic midbrain area (Contestabile and

Flumerfelt 1981), and is not only involved in avoidance and emotional behaviour

(Morley 1986) but also in arousal (Hentall and Gollapudi 1995). It has been reported

41

that IPN partly mediates the nicotinic depression of locomotor activity and is involved

in dampening the nicotinic arousal mechanisms (Hentall and Gollapudi 1995). In

humans, nicotine’s effects have been found to depend on the baseline level of arousal

at the time an individual uses tobacco (Rosecrans and Karin 1997).

The cholinergic habenulo-interpeduncular pathway, i.e. the neurons sending their

axons from medial habenula to the IPN, possesses high density of 3H-nicotine binding

sites (Clarke et al. 1985b; London et al. 1985), that are either pre- or postsynaptic

nAChRs (Brown et al. 1984; Mulle et al. 1991). The IPN strongly expresses mRNAs

for α2, α3, α4, α5, α7, β2 and β4 nAChR subunits (Deneris et al. 1989a; Séguéla et

al. 1993; Wada et al. 1989; 1990a). Moreover, in a quantitative in vivo 3H-nicotine

binding study the density of nicotinic binding sites in IPN was one of the highest of

any brain region assayed (London et al. 1985).

42

3 AIMS OF THE STUDY

The main purpose of this study was to investigate in vivo the functional changes

(desensitization and long-lasting inactivation) induced by chronic nicotine infusion

and withdrawal in the nAChRs involved in the mediation of nicotine’s central effects.

The detailed aims were:

To map the brain areas activated by systemic administration of acute nicotine using an

immunohistochemistry procedure to estimate the expression of Fos protein encoded by

immediate-early gene c-fos in rats. To clarify further the nature of nicotine-induced

Fos protein expression in brain areas that are likely to be involved in coping with

stress. The neural pathways activated by nicotine were studied by examining possible

interaction of diazepam and nicotine on their ability to induce Fos-expression (I).

To investigate the presynaptic effects of chronic nicotine infusion and withdrawal on

nicotine-induced changes in dopaminergic transmission by determining the

concentrations of dopamine and its metabolites in various brain areas of rats and mice

(II, IV). To further determine the nature of reduced response to nicotine –

desensitization or tolerance which is overcome by increasing the dose – the

hypothermic response of mice to nicotine was determined during chronic infusion

(IV).

To evaluate how chronic nicotine treatment and its withdrawal influence the acute

nicotine-induced Fos protein expression in various rat brain areas (II, III). In

particular, the immunostaining of Fos protein in dopaminergic target areas was studied

to determine whether the nicotine-induced changes in dopamine metabolism could be

detected in postsynaptic neurons at the level of immediate-early gene producs (II).

43

4 MATERIALS AND METHODS

The methods used have been described in detail in the respective communications.

4.1 Animals

Male Wistar rats (I, II, III) and male HsdHan:NMRI mice developed at the National

Marine Research Institute (Bethesda, MD, USA) (IV) were used. Animals were bred

locally in the Laboratory Animal Centre of the University of Helsinki. Mice were

housed in groups of six to eight and rats in groups of four to six in a cage at an ambient

temperature of 20-22 °C. The animals had ad libitum access to standard diet and tap

water. The experimental protocols were approved by the Committee for Animal

Experiments of the Faculty of Science of the University of Helsinki.

4.2 Experiments

4.2.1 Chronic administration of nicotine

Nicotine was administered chronically via osmotic minipumps (rats) and via nicotine-

releasing reservoirs (mice). Osmotic minipumps (Alzet 2001) containing saline

(= sham-operated rats) or nicotine hydrogen tartrate solutions (= nicotine-infused rats)

were implanted subcutaneously to rats under halothane anesthesia (II, III). Nicotine

was infused at a dose of 4 mg/kg/day for 7 days. The dose refers to the base. Rats were

housed singly after surgery. To mice (-) - nicotine was administered chronically for 7

days using subcutaneously implanted nicotine releasing reservoirs (IV) as described

for rats by Erickson et al. (1982) which were modified for treatment of mice according

to Leikola-Pelho et al. (1990). Control mice were sham-operated under the same

experimental conditions i.e. s.c. implanted reservoirs contained saline instead of

nicotine. Mice were housed in original groups after surgery.

44

4.2.2 Withdrawal from nicotine treatment

In rats withdrawal was induced by removing the minipumps surgically on the seventh

day of chronic nicotine treatment. In mice the removal of implanted reservoirs in order

to induce withdrawal proved to be impossible due to tissue growth at the lesioned site.

4.2.3 Nicotine challenge experiments

For injections, (-)-nicotine base was diluted with saline (0.9% NaCl solution), and the

pH of the final solution was adjusted to 7.0 – 7.4 (I − IV). 10 − 12 week-old rats were

given single acute doses of 0.5 and 1 mg/kg nicotine or saline subcutaneously (I). To

prevent the peripheral effects of nicotine, which are likely to occur with the dose of 1

mg/kg, rats were pretreated with hexamethonium (10 mg/kg, i.p.) dissolved in saline.

Rats were perfused intracardially 60 min after the acute nicotine administration and

brains were prepared for immunohistochemistry.

Rats received either saline or acute nicotine (0.5 mg/kg s.c.) 60 min before

decapitation (II) or intracardial perfusion (II, III) on the seventh day of nicotine

infusion with the minipumps still in place and after 24-h or 72-h withdrawal. Mice

received either saline or acute nicotine (1 or 2 mg/kg s.c.) 60 min before decapitation

on the seventh day of the infusion with the reservoirs still in place (IV). The acute

challenge experiments were performed at an ambient temperature of 20-24 °C.

4.2.4 Measurement of rectal temperatures

The rectal temperatures of mice (IV) were measured on the seventh day of chronic

treatment (either saline- or nicotine-infused mice) before and at 30 and 60 min after

acute challenge injection (saline or nicotine). The rectal temperatures were measured

with an electric thermocouple (Ellab Instruments, Copenhagen, Denmark) inserted

2.5 cm into the rectum. ∆T is the mean difference of rectal temperatures measured

45

before an acute injection and 30 or 60 min after acute treatment, the last measurement

being immediately before decapitation.

4.2.5 Dissection of brain

Mice and rats were killed by decapitation and their brains rapidly removed. After

decapitation of rats the striatum and limbic forebrain (containing inter alia the

olfactory tubercles, medial part of the nucleus accumbens, the central nucleus of

amygdala and part of the paleocortex) were dissected on an ice-cold plate and

collected on dry ice within 5 min (II). The striata of mice were dissected and frozen

within 3 min (IV). The striatal brain area dissected from the mice consisted of dorsal

striatum as well as ventral striatum, which included the nucleus accumbens. Tissues

were weighed and stored at - 80 °C until assayed.

4.2.6 Preparation of plasma samples

Before collecting and preparing the blood samples (II, IV), all laboratory equipment

was carefully rinsed with ethanol (99.9% w/w) and kept in an oven at 40-100 ºC for at

least 12 h before use in order to remove nicotine contamination. After decapitation, the

trunk blood of the mice and rats was collected and the plasma separated from blood by

centrifugation. The plasma samples were stored at - 80 ºC until assayed. The samples

were prepared as described by Curvall et al. (1982) with slight modifications, as

described earlier by Leikola-Pelho et al. (1990) and Pekonen et al. (1993).

4.2.7 Preparation of brain sections for immunohistochemistry

The nicotine- or saline-infused rats received saline or 0.5 mg/kg of nicotine s.c. one

hour before the pentobarbital (100 mg/kg, i.p.; Orion Pharma, Espoo, Finland, 5-10

min before perfusion). The experiments were performed under the same conditions i.e.

46

control and nicotine-treated rats were handled similarly, at the same time of the day

and room temperature was always the same. Rats were always injected in another

room than the one where the perfusions were carried out to minimise stress. Rats were

then perfused intracardially with 0.9% phosphate-buffered saline (PBS) followed by

4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB) pH 7.4. The brains were

dissected out and postfixed with the same fixative for 4 h at room temperature. The

brains were immersed in a 20% sucrose solution at 4 °C until used. The 40 µm sections

were cut on a cryostat.

4.2.8 Drugs

The drugs were (-)-nicotine base (Fluka AG, Buchs, Switzerland), nicotine

hydrogentartrate (Sigma Chemical Company, St. Louis, Missouri, USA),

hexamethonium hydrobromide (Fluka AG, Buchs, Switzerland) and diazepam

(Diapam® 5 mg/ml inject, Orion Pharma, Espoo, Finland).

4.3 Analytical methods

4.3.1 Estimation of plasma concentrations of nicotine and cotinine using GC-MS

(II, IV)

The concentrations of nicotine and its main metabolite cotinine were determined by a

gas chromatographic-mass spectrometric (GC-MS) method using selected ion

monitoring (SIM) as earlier described by Leikola-Pelho et al. (1990) and Pekonen et

al. (1993). GC-MS analyses were performed on an Hewlett Packard 5970 quadrupole

MS coupled to an Hewlett Packard 5890 GC using an SE-54 fused silica column (15

m). In the GC-MS analyses fragment ions of m/z 84 (nicotine), 98 (cotinine) and 129

(quinoline) were used.

47

4.3.2 Measurement of tissue contents of do pamine and its metabolites using

HPLC-EC (II, IV)

The striatal (II, IV) and limbic (II) concentrations of DA and its metabolites DOPAC

and HVA were measured using high-performance liquid chromatography (HPLC) with

electrochemical detection after Sephadex G-10 gel chromatographic clean-up of

samples. Tissue samples were homogenised in 1 ml 0.2 M HClO4, and pH of the

homogenates was adjusted to 2.4 by KOH/HCOOH buffer. After centrifugation

(15 min, 28 000 x g, 4°C) 1 ml of supernatant was put on an acidic (0.01 M HCl)

Sephadex G-10 column (bed height 8 mm) prepared in a long Pasteur pipette. After the

supernatants had passed through the columns, the compounds were eluted, and a

200 µl portion of each collected fraction was injected through a high-pressure injection

valve in a HPLC column. The C-18 reverse phase (Spherisorb ODS 2 µm) HPLC

column (25 cm, 4.6 mm i.d.) was connected to the electrochemical detector

(Coulochem II, ESA). The assay is described in detail by Haikala (1987).

4.3.3 Fos-immunohistochemistry (I, II, III)

In Study I the sections were first incubated in 1.5% normal goat serum (in 0.1 M PB)

for 60 min to block non-specific staining. Sections were then incubated in primary fos-

antibody diluted 1:2000 in PBS (pH 7.2; 0.1% sodiumazide) for 14 − 16 h at room

temperature. Sections were processed with the avidin-biotin method (Vectastain Kit,

Vector Laboratories, Burlingame, CA) with diaminobenzidine (Sigma Chemicals,

St.Louis, MO, USA) as the chromagen, mounted on gelatine/chrome alume-coated

slides, air dried, dehydrated through graded ethanols to xylene and coverslipped with

DePex (BDH Laboratory Supplies, Poole, U.K.). For the Studies II and III and study

described in section 5.4.1 we had to change the staining protocol due to the problems

with excessive background staining with new lots of primary antibody. Also, different

counting method for the Fos-positive nuclei was used. The sections were first

incubated in 2% normal rabbit serum (NRS, Vector Laboratories, Burlingame, CA,

USA; in PBS + 0.5% Tween 20) for 60 min to block non-specific staining. The

48

sections were then incubated in primary fos-antibody diluted in 1:1000 (experiments

during chronic nicotine) or 1:2000 (withdrawal experiments) in PBS (in 0.5% Tween

20 + 4% NRS) for 72 h at 4°C. The sections were processed with the avidin-biotin

method with diaminobenzidine as above. The sections were processed and mounted as

described above.

The primary antibody used was a sheep polyclonal antibody to fos oncoproteins to a

synthetic peptide with amino-acid sequence: Met - Phe - Ser - Gly - Phe - Asn - Ala -

Asp - Tyr - Glu - Ala - Ser - Ser - Ser - Arg - Cys, selected from a conserved region of

mouse and human c-fos N-terminal (van Straaten et al. 1983). This sequence has no

homology with other members of nuclear transcription factors recently cloned

(Mathieu-Kia et al. 1998). The Fos-antibody was obtained first from Cambridge

Research Biochemicals, UK (OA-11-823, OA-11-824), then later, after the company

had changed its name, Genosys Biotechnologies Inc., UK (OA-11-824). Controls for

the immunostaining, which included omission of either primary or secondary antibody,

demonstrated no Fos-immunostaining. OA-11-823/824 has been used widely in the

literature and the primary preabsorbtion with purified antigen, which demonstrated no

Fos-IS, has been done several times (Bernard et al. 1993; Berretta et al. 1992; Graybiel

et al. 1990; Johnson et al. 1992).

In Study I the number of Fos-positive nuclei was counted manually using a light

microscope at 10 x magnification. A group mean (± S.E.M) was determined from the

counts of 3 − 4 rats in each treatment group. In the subsequent studies the Fos-positive

nuclei were counted with a 10x objective with the assistance of a LEICA QWin image

analysis system on selected brain areas within a rectangular area of 480 x 360 µm2. A

group mean (± S.E.M) was determined from the counts of 4 − 7 rats in each treatment

group. The mean number of Fos-positive nuclei of both hemispheres was calculated

from one of three adjacent sections, but all three were visually inspected under

microscope and were required to show similar pattern of Fos IS. The atlas of Paxinos

and Watson (1986) was used to identify the brain areas in all three studies (Table 4.1).

49

Table 4.1 Rat brain areas where the Fos-immunostaining was determined.

Brain area Plate Co-ordinates

AP DV

Cingulate cortex 11 -1.0 -3.0Nucleus accumbens 13-14 -1.2 -6.6Caudate-putamen 17 -1.8 -3.6Central amygdala 29 -4.0 -8.0Paraventricular nucleus 25 0 − -1.0 -8.0Supraoptic nucleus 23 -1.8 -9.6Medial terminal nucleus 39 -1.2 -8.0 − -9.0Interpeduncular nucleus 41 0 -9.0AP = anterior-posterior; DV = dorsal-ventral. Paxinos and Watson (1986).

4.3.4 Statistical analysis

As the the concentrations of dopamine and its metabolites (II, IV) were estimated in

several separate experiments, the statistical analyses were carried out by three-way

analysis of variance (ANOVA) (experiment x chronic treatment x acute challenge). As

no significant interactions between the experiment and other factors were observed,

the randomised block two-way ANOVA was performed using experiments as blocks.

If there were significant chronic x acute nicotine interactions (P<0.1), the analysis was

continued by comparing appropriate cell means with linear contrasts. The Fos-

immunostaining data were analysed by Kruskall-Wallis nonparametric ANOVA

followed by the Mann-Whitney U-test (I, II) and by two-way ANOVA followed by

linear contrast analysis (II, III). The effect of nicotine challenge doses on rectal

temperatures was analysed by two-way ANOVA for repeated measurements. As

significant chronic x acute x time interactions (P<0.1) were found, the Tukey’s post

hoc comparisons at timepoints were made (IV). The data concerning plasma nicotine

and cotinine concentrations were analysed by Student’s t-test (II, IV).

50

5 RESULTS

5.1 The plasma concentrations of nicotine and cotinine in rats and mice (II,IV)

The plasma concentrations of nicotine and cotinine in rats and mice treated acutely and

chronically with nicotine as well as withdrawal of nicotine in rats are summarised in

Table 5.1. The nicotine and cotinine concentrations both in rats and mice were clearly

elevated on the seventh day of chronic nicotine infusion.

Table 5.1 The plasma concentrations of nicotine and cotinine on the seventh day ofchronic treatment (saline or nicotine) and at 1 h after nicotine administration in ratsand mice . Also shown are the concentrations at 1 h after nicotine administration inrats withdrawn of nicotine (24 h).

RATS Saline-infused Nicotine-infused

Acutetreatment

Nicotine (ng/ml) Cotinine (ng/ml) Nicotine (ng/ml) Cotinine (ng/ml)

On the 7thdaySaline 0 0 76 ± 7 858 ± 142

Nicotine(0.5 mg/kg)

80 ± 22 103 ± 25 114 ± 24 639 ± 106

After 24 hwithdrawalSaline 0 0 0 22 ± 12

Nicotine(0.5 mg/kg)

78 ± 6 119 ± 15 79 ± 6 149 ± 15

MICE Saline-infused Nicotine-infused

Acutetreatment

Nicotine (ng/ml) Cotinine (ng/ml) Nicotine (ng/ml) Cotinine (ng/ml)

On the 7thdaySaline 0 0 207 ± 32 1366 ± 258

Nicotine(1 mg/kg)

96 ± 14 222 ± 31 301 ± 56 1861 ± 253

Nicotine(2 mg/kg)

421 ± 35 1529 ± 284

The results are from Study II and IV, and unpublished (nicotine 2 mg/kg in mice).

51

In rats, acute nicotine did not clearly increase the plasma nicotine or the cotinine

concentrations with nicotine-releasing minipumps still in place. Further, at 24 hours

after removal of the minipumps no nicotine and only traces of cotinine were found in

the plasma. Acutely administered 0.5 mg/kg nicotine elevated the plasma nicotine and

cotinine concentrations in the withdrawn rats to the same degree as in the control rats.

In mice, the plasma nicotine concentrations measured on the seventh day of the

chronic nicotine treatment with nicotine-releasing reservoirs still in place agree well

with the previous results of Leikola-Pelho et al. (1990).

5.2 Nicotine-induced hypothermia in mice (IV)

During the 7-day chronic nicotine administration the rectal temperatures of nicotine-

infused mice did not differ significantly from those of the saline-infused control mice.

The effects of acute nicotine administration (1 and 2 mg/kg s.c.) on rectal temperatures

of both saline-infused control and nicotine-infused mice are shown in Fig. 5.1.

Figure 5.1 The effects of 1 mg/kg and 2 mg/kg of acute nicotine on the body temperature ofthe control mice and of the nicotine-infused mice at an ambient temperature of 20-22°C. Themeans ± S.E.M of 12-15 mice are shown. The asterisks show the significant differences(* P<0.05, ** P<0.01) between acute nicotine administration and control mice, the circleshows significant difference (o P<0.05) between acute nicotine administration and chronicnicotine infusion.

52

Attenuation of the acute nicotine-induced hypothermia during chronic nicotine

infusion was observed at 30, but not at 60 min after injection. Thus, the nicotine-

infused mice recovered more slowly from hypothermia than the saline-infused mice.

The acute nicotine dose of 1 mg/kg decreased the rectal temperature of nicotine-

infused mice significantly less than that of control mice. The dose of 2 mg/kg

decreased the rectal temperature of nicotine-infused mice to a similar level as found in

control mice.

5.3 The tissue concentrations of dopamine and it metabolites, DOPAC andHVA

5.3.1 The effect of nicotine challenge during chronic nicotine administration in

mice and rats (II, IV)

The effect of acute nicotine challenge given on the seventh day of chronic nicotine

infusion on dopamine metabolites DOPAC and HVA in mice and rats is summarised

in Table 5.2. Both in saline-infused control mice and rats the acute challenge doses of

nicotine increased the striatal concentrations of DOPAC and HVA. In mice dose-

dependency was studied and indeed, it was found that 1 mg/kg of nicotine only

increased striatal DOPAC, whereas the 2 mg/kg dose increased both DOPAC and

HVA concentrations. In rats, it was possible to assay the concentrations of DOPAC

and HVA in limbic brain areas separately and in this case a 0.5 mg/kg dose of nicotine

increased both DOPAC and HVA more clearly (limbic DOPAC by 49 − 70% and

HVA by 66 − 86%) than in striatal brain areas (striatal DOPAC by 24 − 40% and

HVA by 20 − 46%).

Neither in mice nor in rats did nicotine increase striatal DOPAC and HVA

concentrations when acute nicotine challenge was given on the seventh day of chronic

nicotine infusion (Table 5.2, II; Fig. 2, IV; Fig. 1). In contrast, in the limbic areas of

rats acute nicotine somewhat increased, but significantly less than in the control rats,

HVA concentration although it did not elevate DOPAC concentration on the seventh

day of chronic infusion (Table 5.2, II; Fig.3).

53

Chronic nicotine treatment itself did not alter striatal DOPAC and HVA concentrations

either in rats or in mice (II; Fig. 2, IV; Fig. 1). Instead, limbic HVA concentration was

significantly elevated in rats with minipumps still in place (II; Fig. 3). Striatal and

limbic DA concentrations were not altered by any of the treatments in either rats or in

mice (II; Figs. 2 and 3, IV; Fig.1).

Table 5.2 The effect of acute nicotine given on the seventh day of chronic nicotine orsaline infusion on striatal and limbic dopamine metabolites, DOPAC and HVA, inmice and rats.

MICE RATS

STRIATAL STRIATAL LIMBIC

DOPAC HVA DOPAC HVA DOPAC HVA

Acute nicotine to saline-infused control animals

0.5 mg/kg - - ↑↑↑ ↑↑ ↑↑ ↑↑↑

1 mg/kg ↑↑ 0 - - - -

2 mg/kg ↑↑↑ ↑↑ - - - -

Acute nicotine on the seventh day of chronic nicotine infusion

0.5 mg/kg - - 0*** 0** 0 ↑↑*

1 mg/kg 0** 0 - - - -

2 mg/kg 0*** 0** - - - -

Table summarises the results from Study II (Figs. 2 and 3) and from Study IV (Fig. 1).The arrows show significant increases by acute nicotine: ↑↑ P<0.01 and ↑↑↑P<0.001. The asterisks show the significant differences (** P<0.01, *** P<0.001)between nicotine-treated and control animals at 60 min after an acute nicotinechallenge dose given s.c. (linear contrasts after 2-way randomised block ANOVA). Adash (-) indicates not measured, 0 indicates no change induced by acute nicotine.

54

5.3.2 The effect of nicotine challenge on rats withdrawn from chronic nicotine

administration (II)

The effect of an acute nicotine challenge on the striatal and limbic dopamine

metabolites DOPAC and HVA in nicotine-withdrawn rats (24 h and 72 h) is

summarised in Table 5.3.

Nicotine-withdrawal did not alter striatal or limbic DA, DOPAC and HVA

concentrations (II; Figs. 2 and 3). At 24 h and at 72 h after removal of the minipumps,

acute nicotine significantly elevated striatal DOPAC and HVA in the nicotine-infused

rats to almost the same extent as in the saline-infused rats. Acute nicotine

administration did not elevate the limbic DOPAC concentration after 24-h withdrawal,

but after 72-h withdrawal DOPAC concentrations were found to have increased. After

24-h and 72-h withdrawal the nicotine-induced elevations of HVA in the nicotine-

infused rats were similar to those of the controls.

Table 5.3 The effect of an acute nicotine challenge (0.5 mg/kg s.c. 60 min) on thestriatal and limbic dopamine metabolites, DOPAC and HVA at 24 and 72 h afterremoval of the osmotic nicotine-releasing minipumps (= nicotine-withdrawn rats).

STRIATAL LIMBIC

DOPAC HVA DOPAC HVA

Acute nicotine to saline-infused control rats

↑ or ↑↑ ↑ or ↑↑ ↑↑↑ ↑↑↑

Acute nicotine in nicotine-withdrawn rats (24 h)

↑ ↑↑ 0* ↑↑

Acute nicotine in nicotine-withdrawn rats (72 h)

↑ ↑ ↑↑ ↑↑

Table summarises the results from Study II (Figs. 2 and 3). The arrows showsignificant increases of acute nicotine: ↑ P<0.05; ↑↑ P<0.01 and ↑↑↑ P<0.001. Theasterisk shows the significant difference (* P<0.05) between nicotine-treated andcontrol animals at 60 min after an acute nicotine challenge dose given s.c. (linearcontrasts after 2-way randomised block ANOVA). 0 indicates no change induced byacute nicotine.

55

5.4 The expression of Fos protein in rat brain areas

5.4.1 Naïve and saline-injected rats (unpublished results)

To test whether injection and handling before perfusion affected the number of Fos-

positive nuclei in different rat brain areas, two rats were given saline s.c. (0.2 ml/100g)

and perfused thereafter along with two rats that underwent perfusion only (Table 5.4.).

All rats were given pentobarbital (100 mg/kg i.p.) 10 min before perfusion.

Table 5.4 The number of Fos-positive nuclei in different brain areas in naïve andsaline-treated male Wistar rats.

Saline-injected rats Naïve rats

rat I rat II rat I rat II

SON 0/0 0/0 0/0 0/0

MT 0/0 5/16 0/0 8/3

IPN 1 21 14 8

VLG 0/0 0/0 0/0 0/0

ACe 0/0 5/6 0/0 0/0

PVN 18/13 14/16 19/18 8/11

The figures give numbers of nuclei counted on left/right side of the brain.ACe = central nucleus of amygdala, SON = supraoptic nucleus, PVN = hypothalamicparaventricular nucleus, MT = medial terminal nucleus of the accessory optic tract,VLG = ventral lateral geniculate and IPN= interpeduncular nucleus.

5.4.2 Acute nicotine (I and unpublished results)

In study I, an acute dose of 1 mg/kg of nicotine (s.c. 60 min; pretreated with

hexamethonium to prevent the nicotine-induced hypoxia) tended to increase Fos

immunostaining (Fos IS) in the interpeduncular nucleus (IPN; number of Fos-positive

56

nuclei 115 ± 34, mean ± SEM, n=3) as compared with control rats treated with saline

(+ hexamethonium) under the same conditions (45 ± 2, n=3). In the medial terminal

nucleus of the accessory optic tract (MT) where Fos-positive nuclei were found in only

one control animal, the increase from 1 ± 1 (n=4) to 96 ± 23 (n=4) was statistically

significant (P<0.01) (I, Fig. 1). Fos IS was also studied in two other areas in the central

visual system: no Fos IS was detected in the superior colliculus (SC) of either control or

nicotine-treated rats. Nicotine did not increase the number of Fos-positive nuclei in the

ventral lateral geniculate nucleus (VLG; saline: 39 ± 15, NIC: 32 ± 8, n=4). Nicotine

tended to increase Fos IS in the stress-related hypothalamic paraventricular nucleus

(PVN; NIC: 336 ± 54 vs. saline: 281 ± 33, n=4) (I, Fig. 2 and 3) and supraoptic nucleus

(SON; NIC: 89 ± 7 vs. saline: 44 ± 19, n=4) (I, Fig. 2 and 4). Nicotine did not induce

Fos IS in the central nucleus of amygdala (ACe; 100 ± 13, n=4) as compared with

control rats (95 ± 21, n=4) (I, Fig. 2 and 5).

Table 5.5 The effect of an acute nicotine challenge (0.5 mg/kg, s.c., 60 min) on thenumber of Fos-positive nuclei (mean ± S.E.M.) in various brain areas of male Wistarrats as compared with saline-treated control rats.

PFC Cg NAcc CPU ACe

Control 38.5± 4.0 54.0±13.2 28.2±2.7 15.2±4.7 15.2±5.2Acutenicotine

66.8± 7.8 88.0±9.6 81.7±10.2** 44.0±11.0* 74.0±22.1*

SON PVN MT SC IPNControl 11.5±2.0 35.5±5.7 0.5±0.2 2.8±0.9 1.2±0.6Acutenicotine

60.3±11.5** 373.5±40.4** 86.0±8.3** 166.2±13.9** 162.5±9.5**

*P<0.05, **P<0.01 (Kruskall-Wallis followed by Mann-Whitney U-test), n= 5-6.PFC = prefrontal cortex, Cg = cingulate cortex, NAcc = core of nucleus accumbens ,CPU = dorsomedial caudate-putamen, ACe = central nucleus of amygdala, SON =supraoptic nucleus, PVN = hypothalamic paraventricular nucleus, MT = medialterminal nucleus of the accessory optic tract, SC = superficial gray layer of superiorcolliculus and IPN= interpeduncular nucleus.

Table 5.5 summarizes the results obtained from rats treated with a challenge dose of

0.5 mg/kg, s.c., 60 min (unpublished). This challenge dose was later used in the

chronic nicotine and withdrawal experiments. With this smaller nicotine dose the

critical signs of hypoxia were not seen and therefore, the administration of

57

hexamethonium, which might interfere with the nicotine’s effects on Fos expression,

was not necessary. When hexamethonium was not used, nicotine elevated Fos IS

significantly in all brain areas studied, except in the PFC and in the Cg. On

examination of the dopaminergic target areas, the increase of Fos-expression by acute

nicotine occurred mainly in the core part of the NAcc, whereas no change in the

number of Fos-positive nuclei was observed in the shell (Data not shown). Therefore,

Fos-positive nuclei were only counted in the core of NAcc.

5.4.3 Acute nicotine and diazepam (I)

Table 5.6 summarizes the results from an acute study in which the effects of 1 mg/kg

of nicotine (s.c.) were studied in rats pretreated with 10 mg/kg diazepam (i.p.).

Table 5.6 The changes in the number of Fos-positive nuclei (calculated as percentagesof corresponding controls) in different rat brain areas after acute nicotine and/or acutediazepam.

TreatmentBrain area

nicotine (nic) diazepam (diaz) nic + diaz

SON + + + + + + + + + + + +

PVN + + + + + +

MT + + + + + + + + + + + + + +

IPN 0 - - 0

VLG 0 + + + +

ACe + + + + + +

For abbreviations see Table 5.40 0 − +20% + + + + +100 − +500%+ +20 − +50% + + + + + +500 −+ + +50 − +75% - - -50 − -75%+ + + +75 − +100%

58

5.4.4 Chronic nicotine-infusion and withdrawal (II, III)

During 7-day chronic nicotine infusion

The effects of an acute nicotine challenge dose given on the seventh day of chronic

saline- or nicotine infusion on Fos expression were studied in the brain areas shown in

Table 5.7. Constant nicotine infusion itself did not alter the Fos-IS in any of the brain

areas studied.

On the seventh day of chronic nicotine infusion the effect of acute nicotine on the

number of Fos-positive nuclei varied, as shown in Table 5.7. In the PVN, SON and

CPU of rats infused chronically with nicotine, acute nicotine had no effect. In the

NAcc and IPN there was an increase of Fos-immunostaining (IS) which, however, was

significantly less than in saline-infused control rats. In the ACe and Cg acute nicotine

increased Fos-IS in nicotine-infused rats to the same extent as it did in saline-infused

rats.

Table 5.7 The effect of an acute nicotine (0.5 mg/kg s.c. 60 min) on Fos-immuno-staining in various brain areas of control and nicotine-treated rats on the seventh day ofchronic nicotine infusion (4.0 mg/kg/day) with the minipumps still in place.

PVN SON CPU NAcc IPN Cg ACe

Acute nicotine on the seventh day of saline-infusion

↑↑↑ ↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑ ↑↑↑

Acute nicotine on the seventh day of chronic nicotine infusion

0*** 0*** 0*** ↑*** ↑↑↑*** ↑↑ ↑↑↑

Table summarises the results from Study II (Fig. 4) and from Study III (Fig. 1). The arrowsshow significant increases of acute nicotine: ↑ P<0.05, ↑↑ P<0.01 and ↑↑↑ P<0.001.0 indicates no change induced by acute nicotine as compared with corresponding acute salinecontrols. The asterisks show the significant differences (*** P<0.001) between nicotine-treated and control animals at 60 min after an acute nicotine challenge dose given s.c.. Thestatistical values were calculated by 2-way ANOVA followed by linear contrasts and thus thesignicances somewhat differ from Study II, Fig. 4.CPU = dorsomedial caudate-putamen, NAcc = core of nucleus accumbens, Cg = cingulatecortex, ACe = the central nucleus of amygdala, IPN = interpeduncular nucleus, PVN =hypothalamic paraventricular nucleus, SON = supraoptic nucleus.

59

Nicotine withdrawal

Neither 24-h nor 72-h withdrawal from nicotine altered the number of Fos-positive

nuclei in the CPU, NAcc, Cg and ACe (II, Fig. 4) and 72-h withdrawal did not alter

Fos expression in the PVN, SON or IPN (III, Fig.1). No increases of Fos-IS were seen

in the CPU, in the NAcc or in the Cg when acute nicotine was given to rats withdrawn

for 24 h from 7-day nicotine infusion. However, in the ACe of these nicotine-

withdrawn rats acute nicotine increased Fos IS in the same way as in control rats (II,

Fig. 4). At 72 h after removal of the nicotine-releasing minipumps acute nicotine

elevated Fos IS in the same way as in control rats in all seven brain areas studied (II,

Fig. 4 and III, Fig. 1).

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6 DISCUSSION

6.1 The plasma concentrations of nicotine and cotinine

Osmotic minipumps release nicotine steadily throughout the period they are intended

to be used when implanted in rats. In this study we used minipumps designed for 7-day

administration so that on the seventh day the plasma nicotine concentration was 76 ±

7 ng/ml (n=7) at an infusion rate of 4 mg/kg/day. This correlates well with the study of

Benwell et al. (1995) where they used minipumps designed for 14-day administration.

These authors obtained the plasma concentrations of nicotine 87 ± 12 ng/ml on the

14th day of the infusion at the rate of 4 mg/kg/day. The dose of chronic nicotine

(4 mg/kg/day) resulted in plasma nicotine levels similar to peak levels found in arterial

blood following the inhalation of smoke from cigarettes (Henningfield et al. 1993).

The plasma nicotine concentrations on the seventh day of infusion were almost

equivalent to plasma concentrations found at 60 min after one single injection of

nicotine (0.5 mg/kg, s.c., Study II, Table 1). It has been reported that brain levels of

nicotine were about three times higher than those in the blood (Benowitz 1990; Rowell

and Li 1997; Sastry et al. 1995). Thus, the plasma nicotine concentrations determined

in this study implicate high cerebral nicotine concentrations in rats.

Earlier, in our laboratory Leikola-Pelho and collaborators (1990) determined the

plasma concentrations of nicotine and cotinine in mice chronically infused with

nicotine using the same administration protocol as in Study IV. Chronic nicotine was

administered via subcutaneously implanted nicotine-releasing reservoirs for 1 – 29

days. During the first seven days of chronic infusion, nicotine concentrations in the

plasma decreased steadily from a peak value of 480 ng/ml to 200 ng/ml (Leikola-Pelho

et al. 1990). In Study IV the plasma concentrations of nicotine in mice obtained on the

seventh day with the reservoirs still in place (207 ± 32 ng/ml, n=12) correlate well

with the results of Leikola-Pelho et al. (1990). The determination of plasma nicotine

and cotinine concentrations is essential when comparing the nicotine-induced changes

in these two studies, because the reservoirs used were home-made. When compared to

61

rats or humans, a higher dose of nicotine is required in mice because of the rapid

metabolism of nicotine in mice (Rowell and Li 1997).

6.2 Nicotine-induced elevation of striatal and limbic DOPAC and HVAconcentrations

In this study the striatal and limbic concentrations of DA and its metabolites DOPAC

and HVA were estimated in rats and the striatal ones in mice. The advantage of

estimating the post mortem tissue concentrations of dopamine metabolites is that

enables us to study the dopaminergic transmission simultaneously from several brain

areas and from animals that have not experienced extra surgery and anaesthesia. In this

respect, the animals are experimentally naïve. There are disadvantages of this method,

in that it is not possible to study the time-course of drug-induced alterations in

neurotransmitter release in one animal and to correlate the changes of neurotransmitter

release in real time with the behaviour of the animal. Furthermore, in mice very few

studies have utilized the in vivo microdialysis technique to determine DA metabolism,

since accurate implantation of probes in mouse brain coupled with measurement of

DA and its metabolism in the extracellular fluid sensitively enough is difficult to

accomplish.

In agreement with the study of Grenhoff and Svensson (1988) acute nicotine

(0.5 mg/kg, s.c.) in rats (II) elevated the tissue concentrations of limbic dopamine

metabolites somewhat more than those measured in striatum.

In sham-operated mice both acute nicotine doses (1 and 2 mg/kg) increased DOPAC

concentration, and the larger acute dose of 2 mg/kg also increased HVA. These

findings agree with those of Haikala et al. (1986) in that striatal DOPAC concentration

is more susceptible to the effects of nicotine than striatal HVA. Thus, it could be that

nAChRs involved in the regulation of the intraneuronal DA metabolism differ in their

sensitivity from those involved in impulse-mediated DA release as suggested by

Leikola-Pelho et al. (1990). The differences we found in the effect of acute nicotine on

62

limbic DOPAC and HVA concentrations both in nicotine-treated and nicotine-

withdrawn rats, (see Tables 5.2 and 5.3) further support this hypothesis.

6.3 The effect of chronic nicotine and its withdrawal on the nicotine-inducedchanges in cerebral dopamine metabolism

6.3.1 The desensitization of nAChRs regulating cerebral dopamine metabolism

The nicotine-induced elevations of the limbic DOPAC and HVA were reduced when

acute nicotine was given to rats infused with nicotine. These findings agree with

microdialysis studies done in rats (Benwell and Balfour 1997; Benwell et al. 1994;

1995), where the effect of acute nicotine challenge on extracellular concentration of

DA in the dorsal striatum and those of DA, DOPAC and HVA in NAcc was attenuated

during constant nicotine infusion. Furthermore, in contrast to saline-infused animals

acute nicotine treatment induced no changes in striatal DA metabolism in rats and

mice on the seventh day of continuous nicotine infusion. These phenomena could be

due to the continuous presence of an agonist, in this case nicotine, desensitizing the

nicotinic acetylcholine receptors (nAChRs) regulating the dopaminergic neurons. In

our studies, the attenuation of nicotine's effect on DA metabolism occurred with

prolonged exposure of rats and mice to nicotine concentrations similar to those found

in the plasma of heavy smokers (Table 5.1). Furthermore, such plasma concentrations

indicate high cerebral nicotine concentrations in rats and mice as noted in section 6.1.

Lippiello et al. (1995) observed that nicotine tends to stabilize nAChRs in the high

affinity conformation which is related to the process of functional desensitization

(Marks et al. 1983; see section 2.1.4). Thus, our experiments indicate that nAChRs

involved in the control of striatal and limbic dopamine metabolism and turnover were

desensitized during constant nicotine infusion.

According to the studies performed in mice, the desensitization of nAChRs regulating

DA metabolism seems to be dose-dependent. The results of Leikola-Pelho et al.

(1990), demonstrating that a 3 mg/kg acute dose of nicotine did not any more increase

63

DOPAC and HVA as compared with smaller doses and even decreased them during

chronic nicotine, supports this suggestion.

In nicotine-infused rats we found differences in the levels of striatal and limbic DA

metabolites during chronic infusion as well as after acute nicotine administration

These results suggest that at least some of the limbic nAChRs mediating nicotine’s

effects on dopamine turnover are less easily desensitized than the striatal ones. This

finding agrees with the suggestion of Henningfield et al . (1996) based on in vitro

experiments (Brodie 1991; Grady et al. 1994; Rapier et al. 1988) that somatodendritic

nAChRs located on mesolimbic DA neurons appear to desensitize much less readily

than nAChRs located on the terminals of the nigrostriatal pathway. In vitro the subunit

composition of nAChRs determines the degree to which receptors are desensitized by

various concentrations of nicotine (Fenster et al. 1997), for example, the α4 subunit

has been found to be more sensitive to desensitization than α3. Dalack et al. (1998)

suggested that there may be more α3-subunit containing receptors in the NAcc than in

the dorsal striatum, based on functional studies of Brioni et al. (1995) and Mirza et al.

(1996) where nicotine agonists ABT-418 and isoarecolone were used. ABT-418 and

isoarecolone have much higher affinity for receptors containing α4-subunit than those

containing α3 (Papke et al. 1997). Treatment with ABT-418 is three times less potent

at activating ventral tegmental area neurons than nicotine (Brioni et al. 1995), while

isoarecolone is much less potent than nicotine at stimulating dopamine release in the

nucleus accumbens (Mirza et al. 1996).

6.3.2 The long-lasting inactivation of nAChRs regulating cerebral dopamine

metabolism in rats

After 24-h and 72-h withdrawal from nicotine infusion, acute nicotine elevated striatal

DOPAC and HVA to the same extent in nicotine-infused and saline-infused rats. The

loss of function of the nAChRs regulating striatal DA metabolism during chronic

nicotine infusion is thus a reversible phenomenon recovering, within 24-h of

withdrawal. Acute nicotine also elevated limbic HVA concentrations similarly in the

64

control and in the nicotine-withdrawn rats. However, the nicotine-induced elevation of

limbic DOPAC was still abolished after 24-h withdrawal, but not after 72-h

withdrawal. Thus, nAChRs mediating nicotine-induced elevation of limbic DOPAC

were still inactivated after 24-h withdrawal when no nicotine and only traces of

cotinine were detected in the plasma. The lack of response of limbic DOPAC to acute

nicotine could be due to long-lasting inactivation of nAChR function which in in vitro

experiments has been distinguished from reversible receptor desensitization and which

is caused by the prolonged pretreatment or high concentrations of nicotine (Rowell and

Duggan 1998). These long-lived inactive receptor states are likely to underlie the

development of tolerance to nicotine (Fenster et al. 1999b).

We could not demonstrate any nicotine-induced sensitization in either striatal or limbic

DA metabolism either when nicotine was infused constantly or withdrawn for 24 or 72

hours. This agrees with previous findings when nicotine was given by constant

infusion (Benwell et al. 1995; Marshall et al. 1997). The effect of chronic nicotine

treatment seems to depend on the method of administration. Intermittent

administration protocols resulted in sensitization of the response (Balfour et al. 1998;

Cadoni and Di Chiara 2000; Nisell et al. 1996), whereas constant infusion produces

desensitization as found in this study (Benwell et al. 1995; Marshall et al. 1997).

6.4 Fos protein expression: brain areas activated by acute nicotineadministration

The primary Fos antibody used in this study was raised against amino acids 2-16 of the

N-terminal region of the cFos-protein, thus it indicates the activation of expression of

the c-fos gene only (Herrera and Robertson 1996; van Straaten et al. 1983).

In this study the basal levels of Fos expression in control rats, handled in a way similar

to the rats in acute and chronic nicotine experiments but given saline instead of

nicotine, were rather low. The prefrontal and cingulate cortices showed somewhat

more Fos IS than was found in control rats. Additionally, the excessive background

staining in these two brain areas somewhat interfered with the counting of the Fos-

65

positive nuclei. Since the basal Fos expression was low (Tables 5.4 and 5.5) in the

stress-related areas PVN, SON and ACe (Senba and Ueyama 1997) we can assume

that the animals did not experience stress caused by experimental conditions.

Acute nicotine administration increased the number of Fos-positive nuclei in striatal

and limbic dopaminergic target areas, namely in the dorsomedial CPU, the core of

NAcc, Cg and in the ACe. Also, acute nicotine tended to increase the Fos expression

in the PFC (Table 5.5). In agreement with earlier experiments (Nisell et al. 1997) acute

nicotine at the dose of 0.5 mg/kg did not significantly increase Fos-expression in the

shell of the NAcc. The nicotine-induced increase in Fos protein expression in

dopaminergic target areas could indicate the postsynaptic effects of nicotine-induced

dopamine release in nigrostriatal and mesocorticolimbic dopaminergic systems. The

observation that the D1 receptor antagonist SCH23390 blocks nicotine-induced Fos

expression in striatum, NAcc and PFC, supports this suggestion (Kiba and Jayaraman

1994; Nisell et al. 1997).

Acute nicotine increased the number of Fos-positive nuclei in the SC and in the MT

which nuclei are known to be included in the visual system. These results agree with

earlier studies that have consistently described nicotine’s effect on the visual system

measuring either local cerebral glucose utilization (Grünwald et al. 1987; London et al.

1988) or Fos immunostaining (Mathieu-Kia et al. 1998; Pang et al. 1993; Ren and Sagar

1992). Furthermore, as shown earlier, acute nicotine increased Fos expression in the

hypothalamic PVN, SON and IPN, a nucleus connected to the midbrain limbic system

(see Sections 2.3.2 and 2.3.3).

6.5 The interaction of nicotine and diazepam in the induction of Fos expression

The findings that nicotine increases Fos expression in stress-related brain areas PVN,

SON and ACe led us to study the effects of diazepam on nicotine-induced Fos

expression. Diazepam in this case significantly activated Fos IS in the PVN, SON and

ACe. However, it did not increase Fos IS in the MT, IP and VLG areas. Diazepam,

when administered prior to nicotine, did not prevent nicotine-induced Fos expression in

66

any brain area studied. Furthermore, we found no significant interactions between the

acute effects of diazepam and nicotine on Fos IS, although nicotine tended to antagonise

the diazepam-induced elevation of Fos IS in ACe.

In this study, the rather large doses of diazepam had significant effect on Fos expression

in stress-related brain areas. Niles and co-workers noticed that 5 mg/kg diazepam

increased Fos expression, as measured by Western blotting, in the striatum but not in

hippocampus (Niles et al. 1997). Fos protein induction is thought to reflect neuronal

activation and typically occurs in response to various convulsant agents (Niles et al.

1997). Thus, it is somewhat paradoxical that the anticonvulsant diazepam increased the

number of Fos-positive nuclei in various brain areas. Lack of interactions between

diazepam and nicotine in inducing the expression of Fos suggests that these drugs

activate different sets of neurons in these brain areas. In the pcPVN nicotine activates

neurons which are known to mediate ACTH release. In the SON nicotine activates both

vasopressinergic and oxytocinergic neurons (Matta et al. 1993). At present it is not clear

which subclasses of neurons diazepam activates in these brain areas. Further, previous

studies indicate no relationship between the density of benzodiazepine binding sites and

the extent to which diazepam decreased stress-induced c-fos expression (Beck and

Fibiger 1995). Balfour (1991) has earlier concluded that nicotine’s anxiolytic properties

are unlikely to be associated with GABAA receptors that are thought to mediate the

anxiolytic properties of the benzodiazepines. Certainly further experiments are needed to

clarify the effects of diazepam in the induction of Fos protein. As the diazepam dose

used was quite large, it would be interesting to study the dose-response especially at

smaller doses of diazepam.

67

6.6 The effect of chronic nicotine treatment and its withdrawal on nicotine-induced Fos protein expression

6.6.1 Dopaminergic target areas

Chronic nicotine treatment

The attenuation of nicotine-induced Fos IS in the CPU might be due to desensitization

as discussed in section 6.3.1 regarding striatal dopamine during the chronic nicotine

infusion. In contrast to the findings in the CPU, acute nicotine administration caused a

slight increase in the number of Fos-positive nuclei in the NAcc of rats during chronic

nicotine infusion and no decrease of nicotine-induced Fos IS was found in the limbic

areas, Cg and ACe. Furthermore, as discussed above, limbic HVA was somewhat

elevated by acute nicotine during chronic nicotine treatment. Thus, in the same way to

nicotine’s effects on limbic dopamine metabolism, its Fos expression increasing effect

in the limbic brain areas seems to be less easily attenuated during chronic nicotine

treatment than its effects in the CPU area. Thus, this could indicate that nAChRs

mediating nicotine's postsynaptic effects in the striatal areas might desensitize more

easily than the ones in the limbic areas. The observed differences in the striatal and

limbic areas between nicotine’s effects on both DA metabolites and Fos expression

suggest that nAChRs and their receptor states in these brain areas might differ. As

nAChR states vary according to subunit composition (Cachelin and Jaggi 1991; Hsu et

al. 1996) subtypes of nAChRs may be differentially involved in the acute and chronic

effects of nicotine and their associated cellular compensatory events (Balfour 1994).

Nicotine-withdrawal

Withdrawal itself (24 or 72 h) did not change Fos expression. After 24-h withdrawal

acute nicotine did not increase the Fos IS in the CPU, the NAcc or in the Cg. The

inability of nicotine to induce Fos-expression in the CPU and NAcc could be due to

the long-lasting inactivation of nAChRs as discussed in section 6.3.2. (Rowell and

Duggan, 1998). It should be noted that on the seventh day of treatment when there was

a marked amount of nicotine present, acute nicotine did not increase Fos IS in the CPU

and only increased Fos IS to a small degree in the NAcc. Further, the inability of

68

nicotine to induce Fos expression in the NAcc seems to correlate with the finding that

acute nicotine did not elevate limbic DOPAC of rats withdrawn for 24 h. The inability

of acute nicotine to induce Fos expression in the CPU does not correlate with our

findings on DA metabolites. Dopaminergic pathways are not necessarily the only ones

involved in mediating nicotine’s effects on Fos-protein expression during withdrawal.

Cortical and thalamic glutamatergic inputs to the striatum may modulate the

dopaminergic activation of postsynaptic intracellular mechanisms leading to changes

in Fos expression (Harlan and Garcia, 1998). Acute nicotine’s effects on DA

metabolites were found to be similar to its effects on Fos expression in that both were

fully restored within 72 h after withdrawal of nicotine infusion.

6.6.2 The relationship of nicotine-induced changes between dopamine

metabolism and Fos protein expression in dopaminergic target areas

Interestingly, nicotine’s effects on Fos protein expression in dopaminergic target areas

seem to correlate with the changes in dopamine release and metabolism. In the present

study, the attenuation of both nicotine-induced dopamine metabolism and Fos protein

expression in the CPU area during constant nicotine infusion was demonstrated in rats,

when there were marked nicotine and cotinine concentrations present in the plasma.

Furthermore, evidence for a long-lasting inactivation of nAChRs mediating nicotine’s

effects on limbic dopamine metabolism and on Fos-expression in striatal and limbic

areas was found during the 24-h withdrawal, when there was no nicotine and only

traces of cotinine in plasma of these rats. Nisell and coauthors observed the

sensitization of both dopamine release in vivo and Fos protein expression in the PFC

after intermittent nicotine administration for 12 days in rats (Nisell et al. 1996; 1997).

In the NAcc, no sensitization of either response was observed (Nisell et al. 1996;

1997). It seems that at least, in the case of brain dopaminergic systems, the functional

status of nAChRs involved in nicotine’s effects correlates well with the level of c-fos.

Thus, Fos expression could not only map the location of activity but could also

indicate the pattern of postsynaptic stimulation in these neuronal pathways.

69

6.6.3 Other brain areas

The effects of 7-day chronic nicotine infusion and 72-h withdrawal on the nicotine-

induced increase in Fos expression were also studied in the PVN, SON and IPN. Fos

protein expression in response to acute nicotine was abolished in the PVN and SON on

the 7th day of nicotine infusion. This phenomenon could be caused by desensitization

of nAChRs located in these hypothalamic areas or in the regions regulating the

functions of these areas. After 72-h withdrawal, nicotine’s effects on Fos protein

expression were restored showing that the desensitization of nAChRs is a transient and

reversible phenomenon.

The increase of c-fos mRNA levels has been found to be desensitized in several brain

areas, including the PVN, when two intermittent nicotine injections (i.p.) were given at

2-h intervals (Sharp et al. 1993a). Sharp and Beyer (1986) demonstrated that by

repeating nicotine administration, nicotine-induced release of ACTH was almost

totally abolished. Also, nicotine-induced NA release in the PVN was found to be

reduced by 50 − 60% when two nicotine injections were given at 100 min intervals

either i.p. or directly into the fourth ventricle (Matta et al. 1995; Sharp and Matta

1993). All these effects were concluded to result from desensitization of nAChRs. Our

results show that the nAChRs mediating the effects of nicotine in the PVN or SON

seem to be desensitized or tolerant during chronic nicotine infusion as estimated by the

Fos protein expression. This method does not reveal the actual location of the nAChRs

that were desensitized; whether it occurs in NA-releasing neurons originating from the

brainstem or within the PVN is unknown. Both α4 and even more likely α7 subunit

containing nAChRs, which are now suggested to mediate nicotine’s effects in the SON

(Zaninetti et al. 2000), are reported to desensitize during nicotine exposure (Fenster et

al. 1997).

The attenuation of Fos IS in the IPN was clear but not as pronounced as in the PVN

and SON, because acute nicotine still significantly increased the Fos IS during chronic

nicotine infusion. This correlates quite well with our observations that in rats infused

70

chronically with nicotine, acute nicotine still, to some degree, increased the Fos IS in

the NAcc and furthermore, no decrease of nicotine-induced Fos IS was found in the

Cg and ACe of these rats indicating that limbic nAChRs do not readily desensitize

during chronic nicotine treatment.

These findings suggest that the levels of desensitization of nAChRs in various brain

areas differ. The nAChRs in the hypothalamic areas, PVN and SON, or in their input

areas regulating nicotine’s effects on hormone secretion seem to be desensitized more

easily than the nAChRs in the limbic areas controlling behaviour such as in the IPN.

Thus, there seems to be variations in the functional states and/or in the subunit

combinations of nAChRs mediating nicotine’s effects in brain.

6.7 The development of tolerance in nicotine-infused mice

The hypothermic effect of nicotine is mediated by central nAChRs (Mansner et al.

1974) and can be prevented by elevating the ambient temperature (Haikala et al.

1986). In several studies chronic nicotine treatment was found to induce tolerance to

the nicotine-induced decrease of body temperature (Mansner et al. 1974; Marks and

Collins 1985). Further, in mice withdrawn for 24 h from a seven-week oral nicotine

administration schedule (Pietilä et al. 1996), acute nicotine challenge induced

significantly less hypothermia than in controls indicating the development of tolerance

to the hypothermic effect of nicotine. Tolerance also developed to the locomotor-

depressant effects of acute nicotine in these mice (Pietilä et al. 1998). In Study IV,

acute nicotine doses of 1 and 2 mg/kg induced hypothermia in a dose-dependent

manner which was at its maximum at 30 min after administration of the drug. In mice

infused chronically with nicotine from subcutaneously implanted nicotine-releasing

reservoirs for 7 days, tolerance was found to the hypothermic effect of the acute

nicotine dose of 1 mg/kg but not to the dose of 2 mg/kg. Previously no tolerance was

observed to the hypothermic effect of large or repeated (3 mg/kg or 1 x 4 mg/kg s.c.)

acute nicotine doses after 7-day chronic nicotine infusion in mice (Leikola-Pelho et al.

1990). Thus, we found that increasing the nicotine dose overcomes the attenuation of

71

the hypothermic effect of nicotine. This suggests that tolerance, in the classical sense,

i.e. an effect that is characterised by the fact that increasing the dose of the drug

overcomes the diminution of its effect following repeated administration, develops to

nicotine-hypothermia during chronic treatment. In contrast, during chronic nicotine

treatment, acute nicotine did not increase the concentrations of striatal DA metabolites

suggesting that desensitization is the main mechanism by which the nAChRs

regulating striatal DA metabolism, are inactivated under the present experimental

conditions. Thus, the nAChRs involved in these two effects differ.

72

7 SUMMARY AND CONCLUSIONS

In the present study both desensitization and long-lasting inactivation of the nAChRs

involved in the mediation of nicotine’s central effects on cerebral dopamine

metabolism and on Fos expression were demonstrated in vivo in rodent brain during

chronic nicotine administration and its withdrawal.

1) In rat acute administration of nicotine induced Fos IS both in the CPU and limbic

dopaminergic areas. Acutely administered nicotine also induced Fos IS in both the PVN

and SON, which are stress-related brain areas, and in MT and IPN. Diazepam induced

Fos IS in all stress-related areas studied (PVN, SON, ACe), but not in any of the other

brain areas studied. No significant interactions were found between the acute effects of

diazepam and nicotine on Fos expression and this finding suggests that these drugs

activate different sets of neurons within the stress-related brain areas.

2) The results from studies performed in the rat suggest that the degree of

desensitization of nAChRs in various brain areas differs. The nAChRs in the striatal

areas seem to be desensitized more easily than those in the limbic areas during

prolonged nicotine infusion. Thus, in the CPU area neither the elevation of DA

metabolites nor the expression of Fos protein was affected by acute nicotine during

chronic nicotine infusion. In contrast, acute nicotine administration caused some

elevation of both limbic HVA and Fos expression in the NAcc of rats during chronic

nicotine infusion. No reduction of nicotine-induced Fos expression was found in the

Cg and ACe, and only a slight reduction was found in the IPN which are known to be

brain areas involved in regulation of various behavioural reactions. Further, the

nAChRs in the hypothalamic areas, PVN and SON, or in their input areas regulating

nicotine’s effects on hormone secretion also seem to be more easily desensitized than

the nAChRs in the limbic areas.

We also found evidence for a long-lasting inactivation of nAChRs mediating

nicotine’s effects in vivo. Thus, at 24 h after withdrawal acute nicotine did not elevate

limbic DOPAC, but striatal DOPAC and HVA as well as limbic HVA were elevated to

the same degree as in control rats. Furthermore, acute nicotine did not increase the Fos

73

expression in the CPU or NAcc, although in ACe its effects were similar to that in

controls.

After longer withdrawal (72 h) nicotine’s effects on cerebral dopamine metabolism

and Fos protein expression were restored showing that these functional changes of

nAChRs are reversible phenomena.

3) In mice, the responses of striatal DA metabolism and body temperature to acute

nicotine were both reduced during constant nicotine infusion. However, the tolerance

to nicotine’s hypothermic effect could be overcome to some degree by increasing the

dose of nicotine, suggesting that tolerance, in the classical sense, develops in nAChRs

involved in thermoregulation during chronic nicotine treatment. In contrast, during

chronic nicotine treatment acute nicotine did not increase the concentrations of striatal

DA metabolites suggesting that desensitization is the main mechanism by which

nAChRs regulating striatal DA metabolism are inactivated under the present

experimental conditions. Thus, the nAChRs involved in these two effects differ.

In conclusion, there seems to be variations in the functional states and/or in the subunit

combinations of nAChRs mediating nicotine’s effects in brain. It has been

hypothesized that smokers, seeking the most rewarding effect, adjust their way of

smoking so that they reach the appropriate combination of stimulated and desensitized

nAChRs. The long-lasting inactivation of nAChRs is one of the important factors in

the development of tolerance to nicotine. The variations in responses to nicotine may

prove to be useful when studying the protective effect of nicotine in Parkinson’s

disease as well as in its reinforcing and dependence-producing properties.

74

ACKNOWLEDGEMENTS

This work was carried out at the Division of Pharmacology and Toxicology,

Department of Pharmacy, University of Helsinki.

I express my deepest gratitude to my tutor and supervisor, Professor Liisa Ahtee, for

her inspiring enthusiasm and encouragement. Her profound knowledge of

neuropharmacology combined with great teaching skills have made the completion of

this work possible. I will always value the hospitality shown to me by Prof. Ahtee

whilst preparing papers to be published.

Docent Jouni Sirviö and docent Sampsa Vanhatalo offered constructive criticism on

the manuscript and valuable suggestions which led to its improvement, and to them I

extend my sincere thanks. I am furthermore grateful to Mr. Adrian Mogg (University

of Bath, United Kingdom) for revising the language of the manuscript.

I am deeply grateful to the co-authors of these studies, M. Sc. Tiina Seppä, M. Sc.

Helena Gäddnäs and M. Sc. Sirpa Lahtinen, for their excellent collaboration and

fruitful discussions during the study. To Ph. D. Kirsi Pietilä I want to extend my

sincere thanks for first being my tutor and then for excellent collaboration in the field

of nicotine research. To M. Sc. Mikaela Moed I express my appreciation for her

capable assistance during the course of her pro gradu study. Further, I wish to thank

Ms. Marjo Vaha for her skilful assistance in all stages of this work.

I was introduced to the world of Fos immunohistochemistry during a stay at the

University of Cambridge, where I visited the Department of Anatomy, the laboratory

of Dr. Joe Herbert in spring 1992. I am deeply grateful to him and his skilful staff for

guidance in my first steps in the field of immunohistochemistry. Further thanks go to

Prof. Pertti Panula and his staff at the Department of Anatomy, University of Helsinki

at that time to help me with setting up the method. I wish to thank Ph. Lic. Juhani

Tuominen for his advice concerning statistical problems.

75

Special thanks go to M. Sc. Ulla-Mari Parkkisenniemi for great friendship both in the

work and leisure. I wish also to thank the entire staff of the Department of

Pharmacology and Toxicology for creating a friendly and inspiring atmosphere in

which it has been pleasant to work.

Finally, I owe my warmest gratitude to my husband Arto and to our children Laura and

Riikka for their encouragement, support and never-ending love during the many years of

the research. They certainly have kept my feet firmly on the ground. Also, the support,

encouragement and love of my dear parents Raili and Jussi and my dear brother Timo

has been essential. Further, I want to express sincere thanks to Minna Nieminen for

being invaluable friend and support during all these years.

Financial support from University of Helsinki, the Finnish Cultural Foundation and the

Research Council for Health of the Academy of Finland is gratefully acknowledged.

Helsinki, July 2000

Outi Salminen

76

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