Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice

Ž .Brain Research 853 2000 41–48www.elsevier.comrlocaterbres

Research report

Exposure to nicotine during a defined period in neonatal life inducespermanent changes in brain nicotinic receptors and in behaviour of

adult mice

Per Eriksson ), Emma Ankarberg, Anders FredrikssonDepartment of EnÕironmental Toxicology, Uppsala UniÕersity, NorbyÕagen 18A, S-752 36 Uppsala, Sweden¨

Accepted 12 October 1999

Abstract

Neonatal exposure to low doses of nicotine has been shown to prevent the development of low-affinity nicotine-binding sites, and toelicit a different behaviour response to nicotine in the mice as adults. This study has identified a defined period during the development ofneonatal mouse brain for the induction of these permanent changes. Neonatal mice, aged either 3, 10, or 19 days were exposed to

Žnicotine, 66 mg nicotine-baserkg b.wt., s.c. twice daily, on 5 consecutive days. In the cerebral cortex, high- and low-affinity HA and. Ž3 .LA nicotine-binding sites were assayed H-nicotinernicotine in neonatal male mice aged 8, 15, and 24 days and in adult mice aged 4

months. Spontaneous behaviour and nicotine-induced behaviour were observed in 4-month-old male mice. The spontaneous behaviourtest did not indicate any difference between saline- and nicotine-treated mice, whereas the nicotine-induced behaviour test revealed ahypoactive response to nicotine, though only in mice given nicotine on days 10–14. The response of controls and the other age categoriesto nicotine was an increased activity. At no time during the neonatal period could LA nicotine-binding sites be found following nicotinetreatment, but the persistence of this effect was evident only in adult mice exposed on days 10–14. q 2000 Elsevier Science B.V. Allrights reserved.

Keywords: Developmental neurotoxicity; Nicotine; Nicotinic receptor; Behavior; Neonatal; Adult

1. Introduction

The development of an organism includes periods thatcan be critical for its normal maturation. In many mam-malian species, rapid growth of the brain occurs duringperinatal development, the so-called ‘brain growth spurt’Ž . w xBGS 13 . In the human, this period begins during thethird trimester of pregnancy and continues throughout thefirst 2 years of life. In mouse and rat, this period isneonatal, spanning the first 3–4 weeks of life, duringwhich the brain undergoes several fundamental develop-mental phases, viz. maturation of axonal and dendriticoutgrowth, establishment of neural connections, synapto-genesis, multiplication of glia cells with accompanying

w xmyelinization, and cell, axon and dendrite death 13,31 .The BGS is associated with numerous biochemical changesthat transform the feto-neonatal brain into that of themature adult. This is also the stage of development when

) Corresponding author. Fax: q 46-18-518843; e-mail:[email protected]

w xanimals acquire many new motor and sensory faculties 8 ,w xincluding advances in spontaneous motor behaviour 9 .

During this period too, the cholinergic transmitter systemundergoes rapid development in the neonatal rodentw x4,11,24 when gradually increasing numbers of muscarinicand nicotinic receptors are found in the cerebral cortex and

w xhippocampus 23,24,32,33,41,49 . Perinatal brain develop-ment has been found vulnerable to various toxic agentsw w xxsee 16 . In earlier studies, we found the developingcholinergic system to be sensitive to low doses of nicotine,and also to various environmental toxicants such as DDT

Ž .and pyrethroids agents known to affect neuronal activity ,Ždiisopropylfluorophosphate DFP, an acetylcholinesterase

. w xinhibitor and polychlorinated biphenyls 2,17–19,21,43 ,all leading to persistent behavioural changes as well aseffects on the cholinergic nicotinic receptors or cholinergicmuscarinic receptors. The induction of behavioural andcholinergic muscarinic receptor disturbances in the adultanimal appears to be limited to a short period duringneonatal development, around postnatal day 10, and fol-lowing doses that apparently have no permanent effects

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02231-3

( )P. Eriksson et al.rBrain Research 853 2000 41–4842

w xwhen administered to the adult animal 1,2,21 . Further-more, neonatal exposure to a low dose of a neurotoxicagent can lead to increased susceptibility in the adults to

w xan agent having a similar neurotoxic action 26,27,51 .Nicotine makes its impact on human health as a compo-

nent of tobacco products. Underweight newborns, highrates of perinatal morbidity, mortality and Sudden InfantDeath Syndrome, and persistent defects in learning andbehaviour may all be associated with to maternal smokingw w xxsee 47 . Nicotine is one of the most commonly used

w xaddictive substances 26 . At least two major classes ofbrain nicotinic receptors in the vertebrate brain have beencharacterized, using radiolabelled ligands: those having

w3 x w3 xhigh affinity for H nicotine and H acetylcholine, andw125 x w xthose with high affinity for I bungarotoxin 41,54,56 .

Low-affinity nicotine-binding sites, also present in thehuman and rodent brain, resemble a-bungarotoxin-binding

w xsites 41,42,56 . In an earlier study we found that neonatalexposure to nicotine, between postnatal days 10 and 16prevented the development of low-affinity nicotine-bindingsites in the cerebral cortex and that this neonatal exposureto nicotine induced a different behaviour response to nico-

w xtine in the adult animals 43 . Test doses of nicotine knownto elicit increased activity normally caused decreased ac-tivity in mice treated neonatally with nicotine.

With regard to these previously observed effects onpermanent changes in cholinergic muscarinic receptors andbehaviour induced during defined periods of neonatal braindevelopment, the present study was undertaken to assessthe effects of nicotine exposure at different neonatal ageson nicotine-binding sites during the neonatal period andthe possible persistent effects extended into adult age onnicotinic receptors and behaviour.

2. Materials and methods

Pregnant NMRI mice were obtained from B and K,Sollentuna, Sweden. Each litter, adjusted within 24 h tocomprise 8–12 mice and to contain offspring of either sexin about equal numbers by euthanizing excess pups, waskept together with its respective dam in a plastic cage in aroom with an ambient temperature of 228C and a 12r12-hlightrdark cycle. At the age of 4 weeks, pups wereweaned and the males were placed and raised in groups of4–7 in a room for male mice only. The animals were

Ž .supplied with standardized pellet food Ewos, Sweden andtap water ad libitum.

Neonatal male mice, aged 3, 10, or 19 days were givenŽ . Ž Ž .y nicotine, 66 mg nicotine-base nicotine-bi- q -tartrate,

. ŽSigma, USA per kg b.wt. s.c. twice daily 08.00 and.17.00 on 5 consecutive days. Mice serving as controls

received, in the same manner, 10 mlrkg b.wt. of the 0.9%NaCl vehicle. Each treatment group consisted of micefrom 3–4 different litters.

The study was divided into two parts, one for neonatalanimals and one for adults. In the neonatal study, thenicotinic receptors in the cerebral cortex were analysed inthe different age categories 24 h after the last injection ofnicotine. In the adult study, the different neonatally treatedgroups of mice were observed for both spontaneous andnicotine-induced behaviour. Nicotinic receptors were alsoanalysed in the adult animals.

2.1. Spontaneous behaÕiour

Spontaneous behaviour was tested in the male mice atw xthe age of 4 months, as previously described 21 . A total

of 8 mice, randomly selected from 3 or 4 different litters,were tested once only, the tests being performed between 8and 12 a.m. under the same ambient light and temperatureconditions. Motor activity was measured over 3=20 min

Žin an automated device consisting of cages 40=25=15. Žcm placed within two series of infrared beams low level

. Žand high level Rat-O-Matic, ADEA Elektronik AB, Upp-. w xsala, Sweden 3 . Locomotion: registered when the mouse

moved horizontally through the low-level grid of infraredbeams. Rearing: vertical movement was registered at a rateof 4 c.p.s., whenever and as long as a single high-levelbeam was interrupted, i.e., the number of counts obtainedwas proportional to time spent rearing up. Activity: a

Ž .pick-up mounted on a lever with a counter-weight withwhich the test cage was in contact registered all types ofvibration within the test cage, i.e., those caused by mouse

Ž .movements, shaking tremors and grooming.

2.2. Nicotinic receptor binding assay

In the neonatal study, male mice were killed by decapi-tation on postnatal day 8, day 15 or day 24. In the adultstudy the neonatally treated mice were killed at the age of4 months. Mice to be used in the receptor studies were notused in the behavioural tests. The brain was dissected onan ice-cold glass plate into cerebral cortex and a crudesynaptosomal P2 fraction was prepared, having protein

w w xxcontents of about 2–3 mgrml measured according to 35w xas described earlier 20 . The P2 fractions were kept frozen

Ž . Ž .y258C until assayed within 4 months .Nicotinic receptor binding sites in the cerebral cortex

ŽŽ . wwere measured using tritium-labelled nicotine y - N-3 xmethyl- H -nicotine, specific activity 3.03 TBqrmmol,

.Amersham International, Bucks, UK following the methodw xof Nordberg et al. 43 . An aliquot of the P2 fraction was

w3 x Ž .incubated with H nicotine 5 nM for 40 min at 48C in 50Ž .mM Tris–HCl buffer pH 8.0 in a total volume of 1.0 ml.

Ž .Eighteen different concentrations of unlabelled y nico-Ž y3 y10 .tine 10 –10 M were used to measure the propor-

tions of high- and low-affinity binding sites and theircorresponding affinity constants. Each binding was deter-mined in triplicate. The incubation was terminated by rapidfiltration over a Whatman GFrC filter treated with 0.05%

( )P. Eriksson et al.rBrain Research 853 2000 41–48 43

polyethyleneimine to eliminate displaceable filter binding.The filters were washed three times with 2-ml portions ofice-cold buffer. After washing, the filters were dried andplaced in mini-scintillation vials. A 5-ml amount of

R Ž .Quickzint 2000 Zinsser Analytic, UK liquid scintilla-tion fluid was added to each vial, and the radioactivity

Žcounted in a scintillation analyser Packard Tri-Carb 1900. Ž .CA . To determine the proportions of high HA and low

Ž .LA affinity binding sites, the data from the competitivew3 x Ž .displacement of H nicotine by unlabelled y nicotine

were fitted by a nonlinear least-squares method as de-w xscribed by Birdsall et al. 7 . The computerized two-site

model in Graph PadR was used to calculate the percent-ages of HA- and LA-binding sites with correspondingaffinity constants. The two-site model was used in our

w xearlier studies 20 .

2.3. Statistical analysis

Spontaneous behaviour: the data were subjected to aŽ .split-plot ANOVA analysis of variance and pairwise

Fig. 1. Spontaneous behaviour in 4-month-old NMRI male mice exposedto nicotine, 66 mg nicotine baserkg b.wt., s.c. twice daily, on 5 consecu-tive days, at an age of either 3, 10, or 19 days. Controls received 10 ml0.9% NaClrkg b.wt., s.c. Each treatment group contained 24 mice from6–8 different litters. For measurements of spontaneous behaviour, seeSection 2. For statistical evaluation, ANOVA with split-plot design was

w xused 30 . There were no significant group=period interactionsw Ž . Ž . Ž . xF 10,276 s 0.69, F 10,276 s 1.73, F 10,276 s 1.53 for‘locomotion’, ‘rearing’, and ‘total activity’, respectively.

Fig. 2. Nicotine-induced behaviour in 4-month-old NMRI male miceŽexposed to nicotine, 66 mg nicotine baserkg b.wt., s.c. twice daily 08.00

.and 17.00 between the 3rd and 7th postnatal days. Controls received 10ml 0.9% NaClrkg b.wt., s.c. Each treatment group contained 8 micefrom 3–4 different litters. For measurement of spontaneous behaviour,see Section 2. The nicotine-induced behaviour was studied by using twodifferent doses of nicotine, 40 and 80 mgrkg b.wt. s.c., and 10 ml 0.9%NaClrkg b.wt. s.c. For statistical evaluation, ANOVA with split-plot

w xdesign was used 30 . There were significant group=period interactionsw Ž . Ž . Ž . xF 10,60 s16.56, F 10,60 s19.88, F 10,60 s16.16 , for the variables‘locomotion’, ‘rearing’ and ‘total activity’, respectively. Pairwise testing

Ž .between nicotine-injected 40 and 80 mg and saline injected mice wasŽ .performed with the Tukey HSD tests a s0.01 . The different injections

Ž . Ž . Ž .are indicated by: S saline; L nicotine 40 mg; H nicotine 80 mg; andthe statistical difference vs. saline is indicated by asterisks, UU P F0.01.The height of the bars represents the mean valueqS.D.

testing between nicotine- and vehicle-treated groups wasŽperformed with Tukey’s HSD honestly significant differ-

. w xence test 30 .Nicotinic receptor binding: in evaluating the 1- or 2-site

binding model, the data were subjected to a goodness-of-fitwtest, based on the ‘extra sum of squares’ principle Draper

w xxand Smith, 1966 in Ref. 40 . Comparisons of the percent-age values and the affinity constants were made withStudent’s t-test and the Mann–Whitney U-test, respec-tively.

Body weight data were subjected to One-way ANOVA.

3. Results

There were no clinical signs of toxic symptoms in thetreated mice throughout the experimental period. Nor were


there any significant differences in body weight gain,between the nicotine- and vehicle-treated mice, withineach age category, during the treatment period or at adultages.

The results from the behavioural variables ‘locomotion’,‘rearing’ and ‘total activity’ in 4-month-old NMRI malemice exposed to 66 mg nicotine-baserkg b.wt. s.c. twicedaily for 5 consecutive days at an age of either 3, 10, or 19days, and from corresponding controls that received onlythe vehicle, 0.9% NaCl, are shown in Figs. 1–4. Duringthe first 60 min, the mice were observed regarding sponta-

Ž .neous motor activity Fig. 1 . In mice given saline, in thethree different age categories, there was a significant de-crease in activity over time in response to the diminishednovelty of the test chambers. This also applied to micegiven nicotine, regardless of age category, and there were

w Ž .no significant group=period interactions F 10,276 s


.and 17.00 between the 10th and 14th postnatal days. Controls received10 ml 0.9% NaClrkg bw s.c. Each treatment group contained 8 micefrom 3–4 different litters. For measurement of spontaneous behaviour,see Section 2. The nicotine-induced behaviour was studied by using twodifferent doses of nicotine, 40 and 80 mgrkg b.wt. s.c., and 10 ml 0.9%NaClrkg b.wt. s.c. For statistical evaluation, ANOVA with split-plot

w xdesign was used 30 . There were significant group=period interactionsw Ž . Ž . Ž . xF 10,60 s42.52, F 10,60 s36.28, F 10,60 s111.61 , for the vari-ables ‘locomotion’, ‘rearing’ and ‘total activity’, respectively. Pairwise

Ž .testing between nicotine-injected 40 and 80 mg and saline injected miceŽ .was performed with Tukey HSD tests a s0.01 . The different injections



.and 17.00 between the 19th and 23rd postnatal days,. Controls received10 ml 0.9% NaClrkg bw s.c. Each treatment group contained 8 micefrom 3–4 different litters. For measurement of spontaneous behaviour,see Section 2. The nicotine-induced behaviour was studied by using twodifferent doses of nicotine, 40 and 80 mgrkg b.wt. s.c., and 10 ml 0.9%NaClrkg b.wt. s.c. For statistical evaluation ANOVA with split-plot

w xdesign was used 30 . There were significant group=period interactionsw Ž . Ž . Ž . xF 10,60 s19.25, F 10,60 s19.18, F 10,69 s27.42 , for the vari-ables ‘locomotion’, ‘rearing’ and ‘total activity’, respectively. Pairwise

Ž .testing between nicotine-injected 40 and 80 mg and saline injected miceŽ .was performed with Tukey HSD tests a s0.01 . The different injections


Ž . Ž . x0.69, F 10,276 s1.73, F 10,276 s1.53 for ‘locomo-tion’, ‘rearing’, and ‘total activity’, respectively. The re-sponse to nicotine in the three different age categoriestreated neonatally with nicotine or saline is illustrated inFigs. 2–4. The mice were given a single injection of 40 or80 mg nicotine-baserkg b.wt. s.c. or 10 ml 0.9% NaClrkg

Žb.wt. s.c. and observed for another 60 min period 60–120.min from base line . In mice treated with nicotine between

days 10 and 14 there were significant group=periodw Ž . Ž .interactions F 10,60 s 42.52, F 10,60 s 36.28,

Ž . xF 10,60 s111.61 for ‘locomotion’, ‘rearing’, and ‘totalactivity’, respectively. Pairwise testing between saline-in-

Ž .jected and nicotine-injected 40 and 80 mg mice showed asignificant dose-related increase in response to nicotine insaline-treated mice, but in the neonatal nicotine-treatedmice the response was the converse, i.e., both doses ofnicotine caused a decreased activity, compared with saline


Ž .injection Fig. 3 . In mice exposed neonatally to nicotinebetween days 3 and 7, or between days 19 and 23, therewere significant group=period interactions for ‘locomo-tion’, ‘rearing’, and ‘total activity’ in the 3-day-olds:w Ž . Ž . Ž . xF 10,60 s16.56, F 10,60 s19.88, F 10,60 s16.16 ,

w Ž . Ž .and in the 19-day-olds: F 10,60 s19.25, F 10,60 sŽ . x19.18, F 10,69 s27.42 . However, pairwise testing be-

Ž .tween saline- and nicotine-injected 40 and 80 mg miceshowed a significant dose-related increase in response tonicotine in both saline- and nicotine-treated mice and therewere no significant difference between saline- and nico-

Ž .tine-treated mice Figs. 2 and 4 .The effects of exposure to nicotine at different neonatal

ages on receptor-binding properties in the mice neonatallyand as adults were analysed by evaluating goodness-of-fit

Table 1Effects of exposure to nicotine at different neonatal ages on the propor-tion of high and low affinity binding sites and their affinity constants inthe cerebral cortex of neonatal and adult micea

Treatment n High-affinity site Low-affinity site Goodness-of-fitage Ž . Ž .% k nM % k mM 1-site vs. 2-site

NeonatalDays 3–7NaCl 4 77.8"6.8 5.2 22.2"6.8 18.0 pF0.05Nicotine 3 95.0"0.9 19.8 – – pG0.1

Days 10–14NaCl 3 71.3"14.6 5.02 28.7"14.6 5.02 pF0.01Nicotine 3 96.8"3.6 16.8 – – pG0.1

Days 19–23NaCl 3 86.7"13.0 3.98 13.3"13.0 7.26 pF0.01Nicotine 3 95.6"1.3 8.16 – – pG0.1

AdultDays 3–7NaCl 3 76.2"3.9 3.44 23.8"3.9 1.66 pF0.01Nicotine 3 86.9"1.0 6.58 13.1"1.0 1.99 pF0.01

Days 10–14NaCl 3 82.6"7.2 3.36 17.4"7.2 1.62 pF0.025Nicotine 3 100"3.0 7.96 – – not fitted

Days 19–23NaCl 3 90.0"3.3 7.73 10.0"3.3 5.18 pF0.01Nicotine 3 85.8"3.7 6.99 14.2"3.7 5.37 pF0.01

a Male NMRI mice received nicotine, 66 mg nicotine baserkg b.wt., s.c.twice daily, on five consecutive days, at an age of either 3, 10, or 19days. Controls received 10 ml 0.9% NaClrkg b.wt., s.c. Neonatal micewere killed 24 h after the last injection, and adult mice at the age of 4

w3 x Ž .months. The binding parameters were estimated from H nicotiner y -nicotine competition curves performed on P2 fractions pooled from threeand two mice in the neonatal and adult mice, respectively, as described

w xearlier 43 . The percentage values are means"S.D. and the affinityconstants are geometric means. The statistical evaluations between nico-tine- and saline-treated mice, in each age-category, of the percentagevalues and the affinity constants were made with Student’s t-test and theMann–Whitney U-test, respectively. The goodness-of-fit is based on the

w‘‘extra sum of squares’’ principle Draper and Smith, 1966, as referred tow xby Munson and Rodbard 40 . The p-values are obtained from the

tabulated F-distribution.

of the total material for each respective treatment to one-and two-site models, based on the ‘extra sum of squares’

wprinciple Draper and Smith, 1966, as cited by Munson andw xxRodbard 40 . The evaluation in neonatal mice, exposed to

nicotine between days 3–7, 10–14 or 19–23 and killed 24h after the last injection, revealed a significant increaseŽ .PF0.05 or PF0.01 in binding data from control micein all three age categories, but not in binding data from

Ž . Ž .mice given nicotine PG0.1 Table 1 . Therefore, in thecompetition binding assay, only one population of bindingsites, high affinity, was observed in the cerebral cortex ofneonatal mice exposed to nicotine. The adult mice at the

Žage of 4 months revealed a significant increase PF0.025.or PF0.01 in binding data from control mice in all three

age categories and also in mice given nicotine betweendays 3–7 or 19–23. Strikingly, in adult mice exposed tonicotine between days 10–14, the binding data revealed no

Ž .significant not fitted, PG0.1 increase, and only onepopulation of binding sites was seen, namely high affinity.In the age categories where both HA and LA binding siteswere detected, no significant change was observed in theaffinity constants between nicotine- and saline-treated mice.

4. Discussion

We have seen earlier that neonatal exposure to lowdoses of nicotine affects the NAChR in the neonatal mousebrain, leading to permanent disorder of brain function ofadult mice, revealed as changes in behaviour and in bind-

w xing properties of nicotinic receptors 43 . The presentinvestigation has shown that induction of these distur-bances in the mouse seems to be limited to a short periodof time during neonatal development. Even though theresults of nicotine treatment was that LA binding sitescould not be found during the neonatal period, the persis-tence of this effect up to an adult age of 4 months wasseen only in mice that were exposed on days 10 to 14.Furthermore, at the adult age of 4 months the alteredspontaneous behavioural response to nicotine was ob-served only in mice given nicotine between days 10 and14.

The persistent effects caused by exposure on days 10–14appear not to be related to differences in uptake androrretention of nicotine in this age group, compared with theother two age groups, as the nicotine exposure in alltreatment groups caused a lack of LA binding sites duringthe neonatal period. Earlier studies have demonstrateddynamic changes in nicotinic receptors during develop-ment and maturation of the brain. Specific binding ofw3 xH nicotine has been detected in mouse brain during the

w3 xlate prenatal period. At birth there is a drop in H -nicotine binding sites for then increase during a period of 4

w x Žweeks when the adult level is reached 57 . With increas-ing age, from 1 month to 14 months in the rat cerebral

w3 x .cortex, the H nicotine binding is known to decrease. Atbirth, only HA binding sites can be detected in mouse


cerebral cortex, but between postnatal days 5 and 17, LAw xnicotine-binding sites became detectable 41 . Recent stud-

ies in rat have also revealed persistent effects on LAnicotine-binding sites in adults following neonatal expo-

Ž . w xsure to nicotine 0.1 mgrkg b.wt., s.c. twice daily 38 .Several recent studies have shown the complexity of the

development of nAChR subunit gene expression at mRNAlevel. Expression of mRNA for a2, a3, a4, a7, b2 andb4 is evident in rodent brain during neonatal developmentw x14,34,38,46,55 . It has been suggested that the predomi-nant nAChR subtype in rodent brain consists of a4 andb2 subunits and binds to nicotinic agonists with high

w xaffinity 25,54 , while a7 subunit may be the main compo-w xnent of a-BgT binding 10,44,45 . Whether the changes in

nAChR, seen after nicotine exposure, are linked to changesin mRNA levels of different subtypes is not known. Miao

w x 3et al. 38 found a persistent increase in H-nicotine-bind-ing sites and a lack of LA binding sites in rat cerebralcortex after neonatal exposure to nicotine, but these changescould not be correlated to different mRNA levels of vari-ous nAChR subunits. This lack of correlation has also been

w xreported in mouse after chronic exposure to nicotine 36 .Ž .However, prenatal nicotine exposure 2 mgrkgrday in

rat has been shown to transiently increase nAChR subunitsa7, a4 and b2 mRNA in brain, the effect being most

w xpronounced on postnatal day 14 46 . A null mutation ofthe a7 subunit has disclosed the absence of high-affinity125I-a-BgT sites, but the high-affinity nicotine sites in the

w xbrain were not detectably affected 44 . It has been sug-gested that the expression of the a7 subunit may becorrelated with differences in nicotine binding, nicotine-in-

w xduced seizures, and nicotine preference 39,50 , and low-affinity nicotine-binding sites resembled to a-bungarotoxin

w xbinding sites 41,42,56 . That is why the observed effectson low-affinity nicotine-binding sites observed in the pre-sent study are of particular interest in defining the func-tional role of low-affinity nicotine sites, as adult micelacking these sites showed quite the opposite behaviouralresponse to nicotine, compared with controls.

The mechanisms of the observed developmental neuro-toxic effects of nicotine during a critical phase of braindevelopment are of special interest and remain furtherstudies. In vitro experiments have shown that nicotine caninduce apototic cell death in undifferentiated hippocampalprogenitor cells but spares the same cells when they are

w xdifferentiated 6 . This apoptotic effect appeared also de-w xpendent on calbindin in regulation of Ca and activationi

of a-BgT-AChRs. When administered during the neonatalperiod, nicotine has also been shown to inhibit DNA

w xsynthesis in different regions of the rat brain 37 . In theirstudy, the period of rapid cell replication was suggested tobe the most sensitive to nicotine. Developmental exposureduring gestational and early postnatal life has been shown

Ž .to affect ornithine decarboxylase ODC activity. ODCcatalyses the initial step in the formation of polyamines,which are major intracellular regulators of cell develop-

ment. The most sensitive period in these experiments wasa developmental stage corresponding to the proliferation ofnicotine receptors and the timing of receptor control of cell

w xreplication and differentiation 48 . In those studies, theŽnicotine doses given are known to affect body weight 6

.mgrkgrday for several days and to cause vasoconstric-Ž .tion acute dose of 9 mgrkg b.wt. . Prenatal exposure to

Ž .nicotine 6 mgrkgrday can lead to such behaviouralw xchanges as hyperactivity in the offspring 53 . Long-term

changes in cognitive function, together with effects on thenoradrenergic system, have also been reported in adult ratsexposed to nicotine during gestation. In the present study,the very low doses of nicotine given, namely 0.2 mgrkg

Ž .b.wt. for 5 days equivalent to 66 mg of nicotine-free basedid not affect weight gain during the different neonatalexposure periods or cause any change in adult weight.Although this exposure did not affect spontaneous be-haviour in 4-month-old animals, nicotine doses known tocause hyperactive behaviour in adult animals did causehypoactive behaviour in adult mice treated neonatally withnicotine. Adult mice exposed on postnatal days 10–14displayed altered behaviour when provoked with renewedexposure to nicotine; this was not seen when the neonatalexposure occurred on days 3–7 or 19–23. The increasedsusceptibility to toxic agents at adult age in animals ex-posed during neonatal life indicates that neonatal exposureto toxic agents can potentiate andror modify the reactionto adult exposure to xenobiotics. In recent studies, weobserved that neonatal exposure to neurotoxic agents, DDTand pyrethroids, leads to increased susceptibility in adultsto develop additional behavioural disturbances, as well aschanges in cholinergic receptors and mRNA of muscarinic

w xcholinergic receptor subtypes 22,27,28,51,52 .The time window for induction of the persistent effects

in neonatal mouse coincides in humans with a periodstarting with the third trimester and continuing for severalmonths after birth. This low-dose exposure is relevant as itis also known that nicotine is transferred via mothers milk,and at concentrations higher than the maternal plasma

w xnicotine level 12 . A similar critical stage during neonataldevelopment, with consequences for the cholinergic sys-tem and behaviour, has also been seen regarding thedevelopment of muscarinic cholinergic receptors, togetherwith persistent derangement of behaviour in adults. Agentsknown to cause such effects are DDT and bioallethrinw x w x1,21 and DFP 2 . In these studies the persistent effectsdid not appear to be related to different amounts of the

Ž .compound present DDT or to the different degree ofŽ .acetylcholinesterase inhibition DFP during the different

stages of neonatal brain development. These data and thepresent paper show that the developing cholinergic systemis susceptible, during a short period of time, to be perma-nently affected.

The cholinergic transmitter system is involved in nu-w xmerous behavioural phenomena 29 and correlates closely

w xwith cognitive functions 5,15 . Whether exposure of hu-


mans to nicotine during a critical stage of perinatal devel-opment may play any part in the development of addictionto nicotine andror in the development of neurodegenera-tive disorders such as Alzheimer’s disease or an acceler-ated aging process is of special concern.

In summary, the present study indicates a defined criti-cal phase during the neonatal development of nicotine-bi-nding sites in the mouse brain and a possible role oflow-affinity nicotine-binding sites in the response to nico-tine in adults, with consequences for behavioural reactionsin adults.

Acknowledgements

This study has been supported by grants from the Bankof Sweden Tercentenary Foundation, the Swedish Councilfor Work Life Research, the Foundation for StrategicEnvironment Research and the Swedish EnvironmentalProtection Board. Miss Anna Pettersson is akcnowledgedfor excellent technical assistance.

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Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice