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Developmental Brain Research, 63 (1991) 201-207

Elsevier Science Publishers B.V. All rights reserved. 0165-3806/91/$03.50


ADONI S 016538069151356S
BRESD 51356
201
Neonatal nicotine exposure induces permanent changes in brain
nicotinic receptors and behaviour in adult mice
Agnet a Nor dber g 1, Xi ao Zhang 1, Ander s Fr edr i ksson 2 and Per Er i ksson 3
Departments o f Pharmacology 1, Zoophysiology 3 and Toxicology 2, Uppsala University, Uppsala (Sweden)
(Accepted 16 July 1991)
Key words: Nicotine; Neonatal treatment; Brain; Nicotinic receptor subtype; Development; Behaviour; Mouse; [ 3H]Nicotine
The effects of neonatal nicotine exposure on spontaneous and nicotine-induced behaviour in 4-month-old mice and on the development of
brain nicotinic receptors were studied. The behaviour study showed that mice treated with nicotine 66/~g (-)nicotine base/kg body weight
(bw) s.c. twice daily between 10 and 16 days postnatally displayed a hypoactive condition, whereas mice treated with saline displayed a
hyperactive condition. When the nicotinic receptors in the brain cortex were analyzed, the displacement curves for [ 3H]nicotine/(-)nicotine
revealed an almost equal proportion of high- and low-affinity binding sites in 17-day-old mice, while the high-affinity sites predominated in
4-month-old mice, with affinity constants for both high- and low-affinity binding sites 10 times higher in 4-month-old mice than in 17-day-
old-mice. A decrease in the number of nicotinic receptors was observed from day 17 to 4 months, mainly of the low-affinity nicotinic type.
Interestingly, the displacement curves in neonatally nicotine-treated mice showed only one population of high-affinity binding sites in 17-day-
and 4-month-old mice though the total binding sites in 4-month-old mice were the same for the neonatally nicotine-treated and saline-treated
mice. These results indicate that neonatal nicotine treatment prevents the development of low-affinity nicotinic sites in the brain and this
earlier exposure to nicotine induces a different behaviour response in adult animals to a test dose of nicotine. Days 10-16 postnatally appear
to be a critical period for the effects of nicotine on the brain.
INTRODUCTION
Ni cot i ne is one of t he mos t c o mmo n l y used depen-
dence- pr oduci ng subst ances 15. I t i nduces mul t i pl e effect s
in t he br ai n whi ch ar e t hought t o be mai nl y medi at ed
vi a ni cot i ni c r ecept or s 28. Re pe a t e d ni cot i ne expos ur e in-
duces t ol er ance and changes t o t he ni cot i ni c r ecept or s in
t he br ai n. Mat ur e r odent s t r eat ed subchr oni cal l y wi t h
ni cot i ne show a si gni fi cant i ncr ease in t he numbe r of
hi gh-affi ni t y ni cot i ni c r ecept or s i n ar eas whi ch ar e ri ch
in ni cot i ni c r ecept or s 33. I n addi t i on, a shift in t he pr o-
por t i on of l ow-t o-hi gh-affi ni t y ni cot i ni c r ecept or s has
be e n obs er ved in t he r at br ai n af t er ni cot i ne t r e a t me nt 31.
An at t empt t o vi sual i ze ni cot i ni c r ecept or s i n an i n vi vo
st udy in man usi ng t he pos i t r on emi ssi on t omogr a phy
t echni que has r eveal ed a hi gher upt a ke of l ~C-ni cot i ne
in t he br ai ns of s moker s f ol l owi ng i nt r avenous i nj ect i on
t han in t hose of nons moke r s 3. Si mi l arl y an i ncr eased
numbe r of ni cot i ni c r ecept or s have be e n me a s ur e d in
aut ops y br ai n tissue f r om s moker s c ompa r e d t o non-
s moker s 5. St udi es in exper i ment al ani mal s as well as
man i ndi cat e an i ncr ease in t he n u mb e r of hi gh-affi ni t y
ni cot i ni c r ecept or s in t he br ai n f ol l owi ng ni cot i ne expo-
sure. The i ncr ease of ni cot i ni c r ecept or s i n ani mal s af t er
ni cot i ne t r eat ment is accompani ed by i ncreases in cort i -
cal i n vi vo r el ease of acet yl chol i ne 29 and behavi our al
st i mul ant effect s o f nicotineXS.
Dur i ng t he de ve l opme nt of ma mma l s t her e are peri -
ods whi ch ma y be critical f or nor mal mat ur at i on. On e
of t hese per i ods appear s t o be t he per i nat al per i od when
t he br ai n r api dl y gr ows 7. Thi s st age of de ve l opme nt is
char act er i zed by t he mat ur at i on of axonal and dendr i t i c
out gr owt h and est abl i shment of neur al connect i ons , syn-
apt ogenesi s and al so mul t i pl e bi ochemi cal changes whi ch
will t r ans f or m t he f et o- neonat al br ai n i nt o t hat of a ma-
t ur e adul t . Dur i ng this per i od t he chol i ner gi c syst em is
r api dl y devel opi ng and gr adual l y i ncreasi ng t he numbe r
of muscar i ni c 14'2, and ni cot i ni c r ecept or s 14'21'34'35"39'4.
I n vi t r o l i gand bi ndi ng st udi es and mol ecul ar bi ol ogy
st udi es have r eveal ed s ubt ypes of ni cot i ni c r ecept or s in
t he mat ur e br ai n ( f or r evi ew see refs. 28 and 37). I n
cont r ast t o t he muscar i ni c chol i ner gi c r ecept or s, hi gh-af-
finity ni cot i ni c r ecept or s ar e pr es ent i n c ompa r a bl e
amount s pr enat al l y 4. De ve l opme nt a l st udi es usi ng var-
i ous ni cot i ni c l abel l ed l i gands i ndi cat e di f f er ent t i me
cour ses f or t he ni cot i ni c s ubt ypes in t he br ai n 14'21'4. A
dr op in t he n u mb e r of hi gh-affi ni t y ni cot i ni c r ecept or s
has be e n obs er ved i n t he br ai n at bi r t h 4 f ol l owed by a
Correspondence: A. Nordberg, Department of Pharmacology, Box 591, S-751 24 Uppsala, Sweden, Fax: (46) (18) 559718.
202
g r a d u a l i n c r e a s e i n t h e n u mb e r o f b i n d i n g si t es t o a dul t
age. Du r i n g t h e fi rst p o s t n a t a l da ys onl y hi gh- a f f i ni t y ni c-
ot i ni c r e c e p t o r s a r e me a s u r a b l e whi l e b o t h hi gh- a n d
l ow- a f f i ni t y s i t es a r e me a s u r e d i n a dul t r o d e n t br a i ns 4.
Th e p h y s i o l o g i c a l r ol e o f t h e s e d i f f e r e n t s u b t y p e s o f r e-
c e p t o r s ha s n o t ye t b e e n c l a r i f i e d a l t h o u g h t h e hi gh- a f -
f i ni t y s i t es a r e c o n s i d e r e d t o b e ma i n l y p r e s y n a p t i c , me -
di a t i ng r e l e a s e o f n e u r o t r a n s mi t t e r s s uch as a c e t y l c h o l i n e
( AC h ) 3,38.
A d e t e c t a b l e a mo u n t o f n i c o t i n e ha s b e e n me a s u r e d
i n t h e b r e a s t mi l k o f s mo k i n g n u r s i n g wo me n 6. Co t i n i n e ,
t h e ma j o r me t a b o l i t e o f n i c o t i n e , ha s al s o b e e n t r a c e d i n
t h e u r i n e o f i nf a nt s wh o s e p a r e n t s a r e s mo k e r s 32. Th e
p h a r ma c o l o g i c a l s i gni f i c a nc e i n t h e b r a i n o f t hi s e n v i r o n -
me n t a l e x p o s u r e o f i nf a nt s t o n i c o t i n e a nd t h e pos s i bl e
c o n s e q u e n c e s l a t e r o n i n l i f e a r e st i l l u n k n o wn . I n t e r e s t -
i ngl y, n e u r o d e g e n e r a t i v e di s e a s e s s uc h as Al z h e i me r ' s
di s e a s e i n v o l v e def i ci t s i n n i c o t i n i c r e c e p t o r s 23"25'27'28'36.
We h a v e a l r e a d y f o u n d t h e d e v e l o p i n g c h o l i n e r g i c sys-
t e m t o b e s e ns i t i ve t o e n v i r o n me n t a l t o x i c a n t s 12a3 wi t h
c o n s e q u e n c e s i n t h e a d u l t a n i ma l 9 q l . Thi s mo t i v a t e d us
t o s t u d y t h e i nf l ue nc e o f n e o n a t a l n i c o t i n e e x p o s u r e o n
t h e d e v e l o p me n t o f s u b t y p e s o f n i c o t i n i c r e c e p t o r s i n t h e
mo u s e b r a i n a n d o n t h e b e h a v i o u r a l r e s p o n s e s o f a dul t
mi c e .
MATERI ALS AND METHODS
Materials
Materials were obtained from the following sources: pregnant-
NMRI mice from ALAB, Sweden; (-)-[N-Methyl-3H]nicotine (62
Ci/mmol) and 1-Quinuclidinyl[phenyl-4-aH]benzilate ([3H]-QNB)
from Amersham, U. K. ; (-)ni cot i ne di-(+)-tartrate salt and car-
bamylcholine chloride (carbachol) from Sigma, U. S. A.
Animal model
The experimental animal model has been described by us ear-
lier TM. 10-day-old male mice (NMRI) were given ( - ) nicotine 66
/*g nicotine base (nicotine-bi-(+)-tartrate) per kg body weight (bw)
s.c. twice daily (08.00 and 17.00 h) between the 10th and the 16th
postnatal day. Controls received 10 ml/kg bw s.c. of 0.9% NaCI ve-
hicle in the same manner. Each treatment group consisted of mice
from 3- 4 different litters 8. The litters, adjusted within 24 h to 8-10
mice of both sexes, were kept together with their respective moth-
ers until the age of 4 weeks, when male mice were removed and
raised in groups of 4 in a room for only male mice with a 12 h
lighffl2 h dark cycle and at a temperature of 22 C.
Behavioural tests
At adult age of 4 months, the mice were subject to both spon-
taneous and nicotine-induced behavioural tests. The tests were per-
formed between 08.00 and 12.00 h under the same light and tem-
perature conditions as the housing. Mot or activity, observed as
locomotion, rearing and activity, was measured over 3x20 rain in
an automated device consisting of cages (40x25x15 cm) placed
within two series of infrared beams (Rat-O-Matic, ADEA Elek-
tronik AB, Uppsala, Sweden) as described earlier 2. Nicotine-in-
duced behaviour was studied by using two different doses of nico-
tine (40/~g and 80/*g nicotine base per kg bw s.c.). These doses
were chosen since they both induce hyperactivity when given to
adult NMRI mice z~.
Nicotinic and muscarinic receptor binding u~.~~z~ ,,
About one week afler the behavioural tcs~ thv mimals ~erc
killed by decapitation. The brains were taken ~ut, from which the
cortices were dissected. For the [3H]nicotine binding assa,,, the cot--
tices were homogenized in 50 mM Tris-HCl buffer / pl t 8.1)) and
washed twice with the same buffer at 35,1)01} g for I(1 min at a. C
The studies involving increasing conccntralions o[ unlabclled
(-)ni cot i ne were performed by incubating aliquots ( 10tl/A) of mem-
brane suspension containing 0. 2-0. 3 mg protein with 5 nM [ 3H]ni-
cotine and unlabelled (-)ni cot i ne in 50 mM Tris-HCl buffer (pl l
8.0) at 4 C for 40 min. The samples were then liltered through
Whatman GF/ C glass filters presoaked with 0.05% polyethylen-
imine solution. The filters were washed 3 times with assay buffer
and the radioactivity entrapped on the filters was counted in ;a
scintillation counter. The specific binding was calculated by sub-
traction of the values for nonspecific binding in the presence of 10 ~
M unlabelled ( - ) nicotine from the total binding. All samples were
assayed in triplicate. Three to four mice cortices from each group
were used in the binding assay,
For muscarinic receptor binding assays, the brain cortices were
homogenized in Na-K phosphate buffer (pH 7.4) and washed twice
with the same buffer at 35.0(}0 g for 10 min at 4 C. The membrane
preparations (0.15-0.2 mg protein) were incubated with 0.22 nM
[3H]QNB in Na-K phoshatc buffer for 90 min at 25 "C. The sam-
ples were then treated in the same way as for the [3H]nicotine
binding assay. The nonspecific binding was defined in the prescnce
of 1 0 - 6 M atropine. All samples were assayed in triplicate.
Data analysis
ANOVA with a split-plot design was used for statistical evalua-
tion of the data obtained from the behavioural test 16 Pairwise test-
ing between nicotine and control groups was performed with Tukey
HSD tests (a = 0 . 0 1 ) 17.
The data obtained from receptor competition assays were ana-
lyzed using the EBDA/ LI GAND program with a Macintosh com-
puter. Data were fitted to single and multiple site models and com-
pared for statistically significant differences. The model chosen was
that which best described the data at the 0.05 level of significance.
The high and low affinity binding sites were designated R u and RI.
and their corresponding affinity constants K H and KI. respectively.
RESULTS
Effect of neonatal nicotine exposure on spontaneous and
nicotine-induced behaviour in adult mice
Th e r e s ul t s f r o m l o c o mo t i o n , r e a r i n g a n d t ot a l act i v-
i t y v a r i a b l e s i n 4 - mo n t h - o l d ma l e mi c e a f t e r e x p o s u r e t o
6 6 / ~ g n i c o t i n e b a s e / k g b w s. c. t wi c e dai l y o r t o 10 ml
0. 9% Na Cl / k g b w s. c. as c o n t r o l b e t we e n t h e 10th a nd
16t h p o s t n a t a l da ys a r e s h o wn i n Fi gs . 1 a nd 2. Du r i n g
t h e fi rst 60 mi n , t he mi c e we r e o b s e r v e d f or s p o n t a n e -
o u s mo t o r a c t i vi t y ( Fi g. 1). I n s a l i n e - t r e a t e d mi c e a de -
c r e a s e i n a c t i vi t y o v e r t i me wa s o b s e r v e d i n r e s p o n s e t o
t h e d i mi n i s h e d n o v e l t y o f t h e t est c h a mb e r s . Th i s was
al s o t r ue f or t h e n e o n a t a l l y n i c o t i n e - t r e a t e d mi c e a nd n o
s i gni f i cant d i f f e r e n c e f r o m s a l i n e - t r e a t e d a n i ma l s wa s ob-
s e r v e d . Th e r e s p o n s e t o n i c o t i n e in n e o n a t a i l y s a l i ne -
a n d n i c o t i n e - t r e a t e d mi c e is g i v e n in Fi g. 2. Th e mi c e
we r e g i v e n a s i ngl e i n j e c t i o n o f 40 o r 80 /~g n i c o t i n e
b a s e / k g b w s. c. o r 10 ml 0. 9% Na Cl / k g b w s. c. a n d o b -
s e r v e d f or a n o t h e r 60 mi n p e r i o d ( 6 0 - 1 2 0 mi n f r o m
b a s e l i n e ) . T h e r e we r e s i gni f i cant g r o u p x p e r i o d mt e r -
203
actions F25,210 = 28.97, F25,21o = 30.50, F25,210 = 42.36
for t he l ocomot i on, rearing and activity variables, re-
spectively. Pairwise testing bet ween nicotine (40 and 80
/~g) and saline-injected mice showed a significant dose-
rel at ed increase in response t o nicotine in t he saline-
t reat ed mice. In t he neonat al l y ni cot i ne-t reat ed mice t he
opposite was observed, i.e. bot h doses of nicotine caused
a hypoact i ve condition in the animals.
Effect of neonatal nicotine exposure on receptor binding
properties
When nicotinic recept ors were analyzed in t he cere-
bral cortex of saline-treated animals on day 17, almost
equal proport i ons of high- and low-affinity nicotinic re-
ceptors were measured, while t he high-affinity sites pre-
domi nat ed at 4 mont hs of age (Fig. 3 and Table I). A
loss in the number of nicotinic recept ors, mainly of the
low-affinity t ype, was observed bet ween 17 days and 4
mont hs (Table I). The affinity constants observed for
4O0
= 3 o o 1
z
0
=E
8 leo

0
C
800
~ 200-
N C N C N
O-
C N C N C N
5000
t
~ 2 5 0 0 .
~ 1 2 5 0 -
0
I--
O.
C N C N C N
0 - 2 0 2 0 - 4 0 4 0 - 6 0 TIME
Fig. 1. Spont aneous behavi our in 4-mont h-ol d NMRI mal e mice
aft er receiving 66/~g ( - ) ni cot i ne base/ kg bw s.c. twice daily (08.00
and 17.00 h) bet ween t he 10th and 16th post nat al days. Cont rol s
received 10 ml 0.9% NaC1/kg bw s.c. The t r eat ed groups cont ai ned
mice from 3- 4 di fferent litters. For a descri pt i on of t he measure-
ment of mot or activity, see Mat eri al s and Met hods. N, ni cot i ne
t r eat ment ; C, controls.
bot h high- and low-affinity binding sites were 10 times
higher at 4 mont hs t han at day 17 (Table I).
Only one popul at i on of high-affinity binding sites was
seen in the cortices of neonatally ni cot i ne-t reat ed mice
bot h on day 17 and at 4 mont hs (Fig. 2 t op and Table
I). The number of nicotine binding sites was significantly
lower in neonatally ni cot i ne-t reat ed mice compared to
controls on postnatal day 17 (Table I). The total num-
ber of nicotinic binding sites was comparabl e in saline
and neonatally nicotine-treated mice at 4 mont hs but the
low affinity component was lacking in the neonatally
t reat ed group (Fig. 2 middle and Table I).
To investigate whet her neonat al nicotine t reat ment se-
100-
~
6 ( ~ -
400-
~ 2 0 0 -
0
' 54500.
~ 3 0 ( X ) .
~ 1500.
0
.
i ~ i _
CCCNNN CCCNNN
S L HS L H S L HS L H
, ) i
. e
CCCNNN CCCNNN
S L HS L H S L HS L H
n
CCCNNN CCCNNN
S L HS L H S L HS L H
6 0 - 8 0 8 0 - 1 0 0
J ~ _ l i
CCCNNN
S L HS L H
_ l ~ _ i
CCCNNN
S L HS L H
nl nl
CCCNNN
S L HS L H
1 0 0 - 1 2 0 TIME
Fig. 2. Ni cot i ne-i nduced behavi our in 4-mont h-ol d NMRI mal e
mice aft er receiving 66/ zg ( - ) ni cot i ne base/kg bw s.c. twice daily
(08.00 and 17.00 h) bet ween t he 10th and 16th post nat al days.
Cont rol s received 10 ml 0.9% NaCl/kg bw s.c. The t r eat ed groups
cont ai ned mice from 3- 4 di fferent litters. For a descri pt i on of t he
measur ement of mot or activity, see Mat eri al s and Met hods. The
ni cot i ne-i nduced behavi our was st udi ed by using two di fferent doses
of ni cot i ne, 40 and 80/~g/kg bw s.c., and 10 ml 0.9% NaCl/kg bw
s.c. For statistical eval uat i on ANOVA with split-plot design was
used 16. Ther e were significant group x peri od i nt eract i ons for t he
l ocomot i on, reari ng and activity vari abl es [F2s.21o = 28.97, F2s.210
= 30.50, F2s.210 = 42.36, respectively]. Pairwise testing bet ween
nicotine- (40 and 80/ ~g) and saline-injected mice was per f or med
with t he Tukey HSD tests (a = 0.01) 16. The di fferent t r eat ment s
are i ndi cat ed by: N, ni cot i ne ( 66/ t g) ; C, cont rol ; S, Saline; L, nic-
ot i ne 40/~g; H, ni cot i ne 80/~g; and t he statistical difference from
saline is i ndi cat ed by t he asterisks, **P <~ 0.01.
204
. = .
. . =
O
gl,
100
80
60
40
20
0
-10
o , a
-9 -8 -7 -6 -5 -4
100
b
o NaC!
40
20
e o
0 i i . . . . . l i i i i , f I l l , , ~ ~ - -
-10 -9 -8 -7 -6 -5 -4
Log ( ( - ) ni cot i ne) [M]
Fig. 3. The displacement by (-)nicotine of [3H]nicotine binding to
cortical membranes of (a) 17-day-old and (b) 4-month-old mice
which had received either 66/~g (-)nicotine/kg bw s.c. or saline
twice daily (08.00 and 17.00 h) between postnatal days 10 and 16.
Different concentrations of (-)nicotine were incubated with 5 nM
[3H]nicotine and cortex preparations at 4 C for 40 min. Each point
represents the mean values of 3-4 independent experiments, and
the standard arrows were less than 10% of each value. The pro-
portion of high (Rn) and low (RL) affinity binding sites and their
corresponding affinity constants are summarized in Table I,
lectively affects the nicotinic receptors i n the brai n, the
muscari ni c receptors were anal ysed using [3H]QNB/car-
bachol compet i t i on bi ndi ng assays. Dat a obt ai ned from
compet i t i on curves i n cortical tissue from bot h ni cot i ne-
t reat ed ani mal s and cont rol s were best fitted to a two-
bi ndi ng site model . No significant differences in the pro-
port i on of bi ndi ng sites or affinity const ant s were found
bet ween the two groups (Fig. 4).
DISCUSSION
The results of these experi ment s show t hat neonat al
exposure to low doses of ni cot i ne bet ween days 10 and
16 i nduces a per manent di sorder of the brai n funct i on of
adult mice, reveal ed as changes i n behavi our and i n nic-
otinic receptors bi ndi ng properties.
The recept or bi ndi ng dat a obt ai ned in this investiga-
~0
..=
"0
.=_.
r~
5
8
100
80
60
40
0 NaCI %
20 Ni coti ne
0
0 J n , m l ~ , , ~ m ~ l J , ~ i m . l , , , , , , . I , j , , , a . l , , l , i . J ,
-9 -8 -7 -6 -5 -4 -3 -2 -I
Log (carbaehoi) [M]
Fig. 4. Displacement by carbachol of [3H]QNB binding to :the cor-
tical membranes of 4-month-old mice treated with either 66 #g
(-)nicotine/kg bw s.c. or saline between postnatal days i0 and 16~
Different concentrations of carbachol were incubated with 0.22 nM
[3H]QNB and cortex preparations at 25 C for 90 rain. Each point
represents the mean value of 4 independent experiments. The pro-
portion of high- and low-affinity (Rr6 Re) binding sites and corre-
sponding affinity constants (KH; KL) were calculated: for nicotine-
treated mice, KH = 42.8 +- 20 k~M, K L = 2.0 0:5 mM, RH =
54.5 7.0%, and RL = 45.5 +- 7.0%; for controls, K H = 17.3 _+8.1
/zM, K L = 1.4 -+ 0.7 mM, R H = 44.5 5.4%, and R t = 55.5 +- 5.4%.
t i on furt her support earlier studies on dynami c changes
in nicotinic receptors duri ng devel opment and mat ura-
t i on of the brai n. In cont rast to ot her recept or systems,
nicotinic receptors are measurabl e i n r odent s in fairly
high amount s bot h pre- and postnatally. Nicotinic ligands
such as [3H]bungarotoxin and [3H]tubocurarine have
shown maxi mal bi ndi ng in the mouse brai n around day
TABLE I
Effect of neonatal nicotine exposure on the proportion of high
(Rn) and low (Rr) affinity binding sites and their affinity con-
stants (KH; KL) in the cerebral cortex of mice
Values are means -+- S.E.M. of 3-4 independent experiments car-
ried out in triplicate.
KH KL RH RL RTotat
(riM) (pmol/g protein)
Saline-treated
17-day 0.38_+0.15 14.4_+3.8 25.93.0 21.2+--3.1 47.t3A
(55%) (45%)
20-day 0.700.23 14,06.3 2%3--3:6 12.24.4 39.54.0
(69%) (31%)
4-month 3.60+-1.10 12342 22.2_+6.1 5.5_+1.4 27:7-+5:4
(80%) (20%)
Nicotine-treated
17-day 2.06---0.22 35.8---1.5"
4-month 2.97---0.36 28.0+1.0
*P < 0,02 compared t o R t o t a ! for saline-treated 17-day-old mice,
Student's t-test.
12 aft er bi rt h 14. This is at a time poi nt when transmis-
sion processes (including EEG) are measurabl e 17 sug-
gesting that t he nicotinic recept ors possibly have t rophi c
effects. Interestingly, t he binding sites of ot her nicotinic
ligands such as [3H]nicotine and [3H]ACh are present in
t he brain prenat al l y, decrease at bi rt h and increase post-
natally 4. The different time courses observed for t he
nicotine ligands indicated di fferent time courses in t he
devel opment of subtypes of nicotinic recept ors. The
[3H]ACh binding showed no not abl e change with age
(bet ween days 3 and 20 aft er birth) in mouse cort ex 21
while t he [3H]nicotine binding increased gradually up to
adult level within t he first 3 weeks of birth40. The dif-
ference bet ween [3H]ACh and [3H]nicotine binding is
also reflected in the relative proport i on of high- and low-
affinity [3H]nicotine binding sites observed in t he brain
on days 3 and 17 aft er birth. In the present study t he
ratio of high- t o low-affinity sites was 55:45 on day 17
while only high affinity sites were present on day 3 40 .
Fr om these studies it can be speculated t hat the maj or
increase in nicotinic recept ors from day 3 to day 17 is
due to an increase in the number of low-affinity sites.
The fact t hat [3H]ACh only labels high-affinity nicotinic
r ecept or sites I also explains the earlier observat i on of
t he unchanged number of [3H]ACh binding sites be-
t ween day 3 and day 2021. Losses in t he number of nic-
otinic recept ors have been observed in t he human brain
with normal aging 26'27. It is evi dent from t he present
study t hat aging processes coul d start fairly earl y in mice
with a loss of low-affinity nicotinic binding sites from day
17 to 4 mont hs with a concomi t ant decrease in affinity.
Accordi ng to this devel opment and change of nico-
tinic recept ors during t he mat urat i on of t he brain, the
significance of t he envi ronment al exposure of nicotine in
infants and possible consequences l at er on in life is of
special interest. An animal model t hat has been used
previously is to t reat mice during t hei r peri od of rapid
brain growth, occurring during t he first 2 to 3 weeks af-
t er birth 7. In previous studies we have r epor t ed that en-
vi ronment al pollutants such as DDT and pyret hroi ds,
which are pot ent neurot oxi cant s in bot h ver t ebr at e and
i nvert ebrat e species, cause mar ked changes in bot h be-
havi our and bi ochemi st ry of adult mice when adminis-
t er ed orally in low doses during t he ' brain growth
spurt '9-11. In the present study t he mice were t reat ed
with low doses of nicotine twice daily during t he post-
natal peri od days 10-16, in or der to see if this exposure
would induce any per manent effect on brain function or
brain cholinergic receptors. Interestingly, this t r eat ment
induced a significant decrease in t he t ot al number of nic-
otinic binding sites 17 days aft er birth in mice t reat ed
neonat al l y compar ed t o cont rol mice. This decrease is
probabl y due to a loss of low-affinity binding sites (Ta-
205
ble I). This early nicotine exposure did not change t he
number of nicotinic recept ors but changed t he propor-
tion of subtypes of nicotinic recept ors in 4-month-old
mice: two binding sites were seen in cont rol animals and
onl y high-affinity binding sites in neonatally nicotine-
t reat ed animals. It is known t hat r epeat ed exposure to
nicotine in adult animals increases the number of high-
affinity sites and t hei r proport i on compar ed to low-affin-
ity sites 31. Thus it is possible t hat exposure to nicotine
from postnatal days 10-16 promot es an increase in the
number of high-affinity nicotinic recept ors and t hereby
prevent s the devel opment of low-affinity sites since this
is the critical time for the devel opment of low-affinity
sites. Since the proport i on of low-affinity sites decreases
with age in naive animals, t he consequence of neonat al
exposure to nicotine might be an accel erat ed aging pro-
cess. The absence of effect of neonat al nicotine exposure
on muscarinic recept ors supports t he t heory that t he ef-
fect of neonat al nicotine exposure is selective for t he
nicotinic recept ors in t he brain. An i mport ant question
is whet her t he high- and low-affinity nicotinic sites rep-
resent two separate molecules or are convertible? The
finding in this study t hat neonat al nicotine t reat ment al-
ters t he devel opment of t he sites makes the assumption
of separate r ecept or molecules mor e likely which is also
support ed by t he molecule biology studies which have
reveal ed a whole family of genes coding for the nicotinic
r ecept or subtypes 28'37. Furt her studies will reveal
whet her t he effect of neonat al t reat ment is selective for
certain brain areas.
It is well known t hat different nicotine doses induce
different effects when given to adult animals. Since we
have studied the effect of nicotine on mot or activity in
mice earlier and found t hat hyperactivity is observed
with low doses of nicotine and hypoactivity with higher
doses of nicotine 23, we chose similar doses of nicotine in
t he behavi oural experi ment in this study. We found t hat
in contrast to t he results in cont rol animals, where hy-
peractivity was seen, 40 and 80 #g nicotine base in neo-
natally nicotine-exposed animals induced hypoactivity.
This might indicate supersensitivity to nicotine in adult
animals neonat al l y exposed to nicotine. One possible ex-
planation for t he changes in behavi our due to neonat al
nicotine t reat ment might be t hat nicotine acts on more
t han one popul at i on or subtype of nicotinic recept ors in
the CNS and these different subtypes show different sen-
sitivities to varying doses of nicotine 19. Since the low-af-
finity sites were absent in neonat al l y ni cot i ne-t reat ed an-
imals it is tempting to correl at e t he increased sensitivity
to nicotine in these animals with t he high-affinity sites.
These recept ors, although t hey are easily desensitized,
might be functionally active inducing various pharmaco-
logical effects which might be opposite to or di fferent
206
from the effects of the low-affinity sites. Very little is still
known about the physiological funct i ons of the high- and
low-affinity sites. The high-affinity sites have, however,
been suggested to be mai nl y presynaptic, medi at i ng re-
lease of several neurot ransmi t t ers such as ACh and
dopami ne 3'4'29'38. To ext rapol at e these findings to the
human situation, it is qui t e plausible that ni cot i ne expo-
sure duri ng the growth and devel opment of the brai n can
influence the devel opment of subtypes of nicotinic re-
ceptors in the brai n. The predi sposi t i on for devel opment
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