Developmental Brain Research, 58 (1991) 223-229 223 Elsevier

BRESD 51219

Inhibition of D N A synthesis in neonatal rat brain regions caused by acute nicotine administration

B.J. McFarland, F.J. Seidler and T.A. Slotkin

Department of Pharmacology, Duke University Medical Center, Durham, NC 27710 (U.S.A.)

(Accepted 23 October 1990)

Key words: Deoxyribonucleic acid; Nicotine

Perinatal exposure to nicotine has been shown to cause morphological and neurobehavioral abnormalities in developing brain. In the current study, neonatal rats were given an acute injection of nicotine (3 mg/kg) at 1,3, 8, 10 or 15 days of age, and [3H]thymidine incorporation into DNA examined over the 30-min period after drug administration. Three brain regions were used that differ in their timetables of cell maturation and in their concentrations of nicotinic receptors. Nicotine inhibited DNA synthesis in all brain regions but with a rank order of effect corresponding to the concentration of nicotinic receptors, namely midbrain + brainstem /> cerebral cortex > cerebellum. Superimposed on this hierarchy, periods of rapid cell replication were more sensitive to nicotine, so that drug effects in the cerebellum, which develops last, became significant past the point at which nicotine no longer affected DNA synthesis in the other regions. The inhibitory effect of nicotine was also found in fetal brain on gestational day 20 after injection of nicotine to pregnant rats. Studies with adrenergic and ganglionic blocking agents and with 100% 02 indicated that autonomic and respiratory actions of nicotine, including ischemia, cardiac arrhythmias and hypoxia, could not solely account for the inhibition of DNA synthesis in neonatal brain. In contrast, injection of a small amount (2/~g) of nicotine directly into the central nervous system readily caused inhibition; the same small dose given systemically had no effect. These data suggest that nicotine damages the developing brain, in part, through direct actions on cell replication.

INTRODUCTION

It is widely recognized that maternal tobacco use, in

addition to retarding fetal growth, elicits a variety of

neurochemical and behavioral deficits in the offspring 5' 12,15,17-21,27-29. A number of studies suggest that nicotine

acts directly on immature nerve cells through nicotinic

receptors present early in fetal and neonatal develop-

ment 12'2°'29. Thus, although smoking or acute injection of

nicotine can cause fetal damage through ischemia and

hypoxia, abnormalit ies of brain development can be

detected at nicotine exposure levels that do not evoke

these secondary actions and do not cause growth retar- dation19 21.23,29,3o.

Recent work on the development of cholinergic neu-

rotransmission indicates that acetylcholine serves as a

trophic signal regulating the timing of cellular differen-

tiation in targeted brain regions 2'9"19. Consequently, it

has been proposed that the sensitivity of cell maturat ion

to nicotine is a reflection of premature promotion of

differentiation at the expense of replication 19, a mecha-

nism that has been readily demonstrated for other

neurotransmit ter systems 7'32'33. The current study exam-

ines this hypothesis through measurements of incorpora-

tion of [3H]thymidine into DNA after acute administra-

Correspondence: T.A. Slotkin, Box 3813, Duke Univ. Med. Ctr.,

tion of nicotine to neonatal rats. We have selected 3 brain

regions with disparate t imetables for cellular maturat ion

and for acquisition of nicotinic receptors4'8"2°'22"29: mid-

brain + brainstem, which develops earliest and exhibits

the highest receptor concentrat ion, cerebral cortex, with

an intermediate maturat ional t imetable and slightly lower

receptor complement , and cerebellum which develops

last and has the lowest concentrat ion of receptors.

Studies were conducted over the first two weeks of

postnatal life, a period corresponding roughly to neural

development in the last trimester of gestation in man 6. In

addition, measurements were made in fetal brain after

nicotine administrat ion to dams on gestational day 20.

Finally, we have examined the mechanism by which

nicotine affects D N A synthesis, including potential con-

tributions of hypoxia, ischemia or other autonomic

actions of nicotine, as compared to direct actions of the

drug within the central nervous system.

MATERIALS AND METHODS

Timed pregnant Sprague-Dawley rats (Zivic-Miller Laboratories, Allison Park, PA) were housed individually in plastic breeding cages and allowed free access to food and water. On the day of birth (day 0), pups were randomized and redistributed to nursing dams with the litter size maintained at 9-11 pups to ensure standard nutrition.

Durham, NC 27710, U.S.A.

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224

Randomization was repeated every 2-3 days and pups of both sexes were selected from several different cages for each experiment.

Animals were given a subcutaneous injection of nicotine di- bitartrate (9 mg/kg b. wt., equivalent to 3 mg/kg of nicotine free base) or an equal volume (1 pl/g) of isotonic saline-sodium bitartrate vehicle; this dose does not cause neonatal mortality but does produce clear-cut signs of vasoconstriction 27 29. Repeated gesta- tional or neonatal administration of 3 mg/kg of nicotine has been commonly used to evoke both biochemical and behavioral alter- ations in animals s'13"14'18'23'27'28 and has been compared to heavy cigarette smoking in man In'is. Immediately after drug administra- tion, animals were given [3H]thymidine (1 pCi/g, s.c.) and returned to the cages with their dams for the ensuing 30 min. At the end of this period, animals were killed by decapitation and brains dissected into 3 regions: blunt cuts were made through the cerebellar peduncles, whereupon the cerebellum (including flocculi) was removed from the underlying tissue; a cut was made rostral to the thalamus to separate 'cerebral cortex' from 'midbrain + brainstem'.

Tissues were homogenized (Polytron, Brinkmann Instruments, Westbury, NY) in a minimum of 10 vols. of ice-cold water and an aliquot was removed to assess the total uptake of radiolabel. To measure [3H]thymidine incorporation into DNA, a second aliquot was precipitated with 10% trichloroacetic acid, sedimented at 1000 g for 15 min and the resultant pellet washed twice by resuspension and recentrifugation. The final pellet was then digested with hyamine hydroxide and counted.

In some experiments, animals received [3H]leucine (1 pCi/g) to assess protein synthesis, rather than [3H]thymidine; in others, the time of administration of [3H]thymidine was varied to 30 min, 2 h or 4 h after the nicotine, with sacrifice 30 min after administration of radiolabel. For examination of autonomic blocking drugs, animals were pretreated s.c. with one of the following agents 15 min before receiving nicotine (or saline) and [3H]thymidine: phenoxy- benzamine HCI (5 mg/kg), chlorisondamine chloride (5 mg/kg), or a mixture of propranolol HCI (5 mg/kg) + atropine sulfate (2.5 mg/kg). Previous work has detailed the efficacy of these drugs and doses in neonatal rats for producing a-adrenergic, nicotinic gan- glionic, fl-adrenergic and muscarinic antagonism, respectively 7'11"24. The potential role of hypoxia in the effect of nicotine on [3H]- thymidine incorporation was examined by ventilating the cage with pre-warmed, humidified, 100% O z during the 30-min period after the administration of nicotine and [3H]thymidine; details of the gassing procedure have also appeared before, including the dem- onstration of rapid equilibration of neonatal blood gases with the inhaled mixture 7'24"25'34.

Intracisternal drug administration was performed with a 31-gauge x 3 mm needle inserted directly through the overlying skin and musculature, through the foramen magnum into the cisterna magna 16. Animals were given either 6 #g of nicotine di-bitartrate (equivalent to 2 pg of nicotine) or saline-bitartrate vehicle in a total volume of 10 pl.

Data analysis and statistics Because the incorporation of radiolabel into macromolecules is

dependent upon the amount of [3H]thymidine or [3H]leucine taken up by the tissue, synthesis was evaluated as the ratio of trichloro- acetic acid-precipitable label to total label in the tissue ('fractional incorporation'). Effects on incorporation of label into DNA or protein therefore reflect changes in the synthetic rate and not differences in precursor availability 31'33.

Data are shown as means _+ S.E.M. Differences between groups were assessed by analysis of variance (ANOVA, data log-trans- formed wherever variance was heterogeneous) with factors of drug treatment, brain region and age. Where significant drug-related differences were identified in the global test, appropriate lower- order ANOVAs and Duncan's multiple range test were applied post-hoc to identify individual regions or ages at which the nicotine group exhibited differences from controls. Significance was assumed at the level of P < 0.05. For convenience, results are given as the mean _+ S.E.M. of the percent change from control values, but

TABLE l

Incorporation o f [3H]thymidine into DNA in brain regions o f control rats

Data represent means + S.E.M. obtained from 8-22 rats at each age. ANOVA indicates that the incorporation rate is both region- and age-dependent (main effects of region and age and interaction of region × age).

Age Fractional incorporation (days) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Midbrain + Cerebral Cerebellum brainstem cortex

1 0.16+0.02 0.31 +0.02 0.48+0.03 3 0.22 + 0.02 0.33 + 0.02 0.35 + 0.02 8 0.11 + 0.01 0.14+0.01 0.52+0.01

10 0.13+0.01 0.11 + 0.01 0.67_+0.03 15 0.081 + 0.005 0.058 -+ 0.005 0.34 -+ 0.03

EFFECTS OF ACUTE NICOTINE

ON DNA SYNTHESIS

I MIOBRAIN * CEREBRAL 1

0 . . . . .

-1o

-20

i -30

~ -4 o

-50

AGE (doys)

Fig. 1. Effects of nicotine (3 mg/kg), administered acutely at different ages, on [3H]thymidine incorporation into brain region DNA. Data represent means + S.E.M. (as percent change from control values) obtained from 18-30 rats at each age; fractional incorporation rates of control rats appear in Table I. ANOVA indicates a significant reduction in DNA synthesis caused by nicotine (main treatment effect), but in an age-dependent and region-dependent manner (significant interactions of treatment × age and treatment x region x age). Analysis at separate ages was then conducted by two-way ANOVA, which indicates a significant main treatment effect of nicotine at all ages and a significant interaction of treatment x region at 3, 8, 10 and 15 days. Post-hoc analysis by Duncan's Multiple Range Test indicates significantly greater effects in midbrain + brainstem compared to cerebral cortex or cerebellum on day 3, greater effects in midbrain + brainstem and cerebral cortex compared to cerebellum on days 8 and 10, and greater effects in cerebellum compared to the other regions on day 15. The effects of nicotine were also significant in each region considered separately (two-way ANOVA, significant main treat- ment effect in all 3 regions and treatment × age interaction in the midbrain + brainstem). Asterisks denote individual ages at which the nicotine group differs from control.

225

EFFECTS OF ACUTE NICOTINE ON PROTEIN SYNTHESIS - DAY I0

TIME COURSE - DAY i0

(midbroin + broinstem)

-10

o') -2o

~ - 3 0

t .

~-40

-50

Fig. 2. Effects of nicotine administration (3 mg/kg) on postnatal day 10, on incorporation of [3H]leucine into protein in brain regions. Data represent means + S.E.M. (as percent change from control values) obtained from 21 rats. ANOVA indicates no significant effect of nicotine.

statistical comparisons were always conducted on the raw data.

Materials (-)Nicotine di-(+)bitartrate, dl-propranolol HCI and atropine

sulfate were obtained from Sigma Chemical Co. (St. Louis, MO), phenoxybenzamine HCI from Smith Kline & French (Philadelphia, PA) and chlorisondamine chloride from Ciba Pharmaceuticals (Summit, N J). Thymidine [methyl-3H] (specific activity 20 Ci/mmol) and l-leucine[4,5-3H(N)] (specific activity 53 Ci/mmol) were pur- chased from DuPont Medical Products (Wilmington, DE).

RESULTS

In keeping with previous results 3"22"33 the regional

pattern for [3H]thymidine incorporation into control neonatal rat brain had a distinct hierarchy, with highest DNA synthetic rates in the cerebellum, followed by cerebral cortex and midbrain + brainstem (Table I). During the 2-week period postpartum, incorporation fell drastically in midbrain + brainstem and cerebral cortex, but remained high in cerebellum. Administration of nicotine to neonatal rats caused an immediate reduction in the rate of [3H]thymidine incorporation into DNA in all brain regions (Fig. 1). Inhibition by nicotine was regionally-selective (significant interaction of treatment × region × age), with the overall rank-order midbrain + brainstem /> cerebral cortex > cerebellum. Age-depen- dence was also apparent (significant interaction of treat- ment × age) in that the effect in midbrain + brainstem and cerebral cortex reached a peak during the first and second postnatal weeks; in cerebellum, the effect of nicotine was smaller initially, but increased toward the end of the second week. The effect of nicotine appeared to be restricted to DNA synthesis as opposed to that of

-I

-2

cJ i.

-5

TIME AFTER NICOTINE INJECTION

Fig. 3. Time course of effect on [3H]thymidine incorporation into DNA after injection of nicotine (3 mg/kg) on postnatal day 10, evaluated in midbrain + brainstem. Data represent means + S.E.M. (as percent change from control values) obtained from 9-10 rats at each time point. Animals were given nicotine followed by [3H]thymidine immediately, at 30 min, at 2 h or at 4 h after the nicotine, and killed 30 min after the [3H]thymidine injection. ANOVA indicates a significant reduction in incorporation attribut- able to nicotine (main treatment effect) and significant time- dependence of the effect (interaction of treatment x time). Asterisks denote individual time points at which the nicotine group differs from control.

EFFECTS OF ACUTE NICOTINE ON

DNA SYNTHESIS - DAY I0

(midbroin + broinstom)

O~ - I 0

0 JZ o -20

4J

C -30

o L

CL -40

-50

I i n c u b a t o r : no dams

I con - no dom:

ombient

n ic - w i th dom

Fig. 4. Effects of external conditions on inhibition of [3H]thymidine incorporation into DNA caused by nicotine administration (3 mg/kg) in midbrain + brainstem on postnatal day 10. In the left panel, dams were removed from the cages of both the control and nicotine groups after drug administration and the cages placed in a 37 ° incubator. In the right panel, dams were removed only from the control group and the control pups were left at ambient tempera- ture, whereas the nicotine group remained with the dam. Data represent means + S.E.M. (as percent change from control values) obtained from 7-9 rats under each condition and treatment. Asterisks denote that the nicotine groups show significant reduc- tions in DNA synthesis when compared to the controls under the two different conditions.

226

EFFECTS ON ONA SYNTHESIS ON DAY i0 "- PRETREATMENT WITH BLOCKERS OR lOOX OXYGEN

(madbroin + broimst~m)

0

QI

~ - 1 0 0 zz 0

-20

tU -3tl Q tl.

- 4 0

Fig, 5. Effects of pre t rea tmcnt with autonomic blocking agents or 100% 0 2 on inhibition of [3H]thymidine incorporat ion into D N A caused by nicotine (3 mg/kg) in midbrain + brainstem on postnatal day 10. Abbreviations: SAL = saline, NIC = nicotine, PBZ = phenoxybenzamine, PRO = propranolol, ATR = atropine, CH- LOR = chlorisondamine, OXY = 100% O 2. Blocking agents were given 15 rain before nicotine and (3H]thymidine; gassing with 100% 0 2 commenced with drug administration and was continued for the ensuing 30 rain. Data represent means _+ S.E.M. (as percent change from control values) obtained from 6-10 rats in each group. There was significant inhibition (asterisks) in all nicotine-treated groups vs controls (Duncan's Multiple Range Test) and no significant alter- ation of nicotine's effect by autonomic blocking agents or 100% O 2 (two-way ANOVA, no significant interaction of blocker x nicotine treatment). Treatment with blockers alone had no significant effect on DNA synthesis.

other macromolecules: [3H]leucine incorporation into

protein was unaffected on postnatal day 10, an age at

which inhibition of D N A synthesis was significant in all

regions (Fig. 2). The midbrain + brainstem on postnatal day 10 was then selected as a model system for subse-

quent evaluation of the mechanism of action of nicotine

on D N A synthesis because of the magnitude and consis- tency of the nicotine effect, the relatively large amount of tissue available, the high local concentration of nicotinic receptors 2°'29 and the prior identification of nicotine-

induced morphological alterations in this area 1°.

Fig. 3 shows the time course of inhibition of D N A synthesis after acute nicotine administration. The effect was maximal in the 30-min period immediately after injection of nicotine, but remained significant through at least the 4th h. During the course of these experiments, it was noted that nicotine-injected animals frequently felt cold to the touch and were sometimes scattered about the cage by the dam. Accordingly, we undertook studies to test both temperature and caretaking variables. First, animals were removed from their dams, placed in a temperature-controlled incubator at 37 ° , and then given

t3 l./ -20 .¢o c o/ LI -30 L @

Q" -4o

EFFECTS OF ACUTE N]CLJ! ZNE ~,% L.~;' ON DNA SYNTHESIS

:IlI

-50 SUBCUTANEOUS

Fig. 6. Effects of intracisternal or subcutaneous administrat ion of a small dose (2/zg) of nicotine on incorporat ion of [3H]thymidine into DNA in brain regions on postnatal day 10. Abbrevat ions: Mb + bs = midbrain + brainstem, Ctx = cerebral cortex, Cb = cerebellum. Data represent means + S.E.M. (as percent change from control values) obtained from 10-12 rats in each group. A N O V A indicates a significant reduction in D N A synthesis caused by intracisternai nicotine (main t rea tment effect; asterisks for individually significant regions) but not for subcutaneous nicotine.

saline or nicotine along with [3H]thymidine; the inhibi-

tory effect of nicotine was still present despite the fact that neither control nor nicotine groups had any maternal

care during the measurement period and both had their temperatures controlled (Fig. 4, left). In the second

experiment, we subjected the control group to a 'worst

case' situation: dams were removed from the cage and

the control pups were left at ambient temperature

throughout the experiment, whereas the nicotine group remained with their dams (Fig. 4, right). Nevertheless,

the nicotine effect was still present. We followed the

individual nicotine-treated pups that displayed cold skin

or were scattered by the dam and compared D N A synthesis rates to their littermates that were not cold or

scattered; the nicotine effect was equivalent in both

subgroups (data not shown). Finally, effects of a lower dose of nicotine (1 mg/kg) that did not evoke cold skin,

prolonged blanching or altered caretaking, still arrested D N A synthesis: midbrain + brainstem fractional incor- poration = 0.144 + 0.009 in controls (n = 6), 0.104 _+ 0.007 in the nicotine group (n = 7, P < 0.005).

Conceivably, nicotine could influence D N A synthesis in developing brain secondarily to severe ischemia or hypoxia 7a4"25'28'3°. Notably, gasping and blanching of the

skin were observed in some of the nicotine-treated animals. We therefore at tempted to block the peripheral actions of nicotine with phenoxybenzamine, an irrevers- ible a-adrenergic blocker that would interrupt the vaso-

ACUTE MATERNAL NICOTINE -

GESTATIONAL OAY 20

HIDBRhlN ÷ CEREBRAL BRMNSTEN CORTEX

0

-10

~-20

t . .

~.-40 ,

-50

Fig. 7. Effects of acute maternal nicotine administration (3 mg/kg) on gestational day 20, on [3H]thymidine incorporation into DNA in fetal brain regions. Data represent means +_ S.E.M. (as percent change from control values) obtained from fetuses of 10 dams. ANOVA indicates a significant overall reduction in DNA synthesis (main treatment effect); asterisks denote individual regions display- ing significant differences from control.

constrictor effects of the catecholamines released by nicotine (Fig. 5); phenoxybenzamine failed to prevent the fall in DNA synthesis. Similarly, combined fl-adrenergic and muscarinic blockade by pretreatment with propra- nolol + atropine, a treatment that would prevent cardiac arrhythmias associated with nicotinic stimulation of sym- pathetic ganglia, did not block the effects on DNA synthesis, nor did chlorisondamine, a peripherally acting, ganglionic nicotinic antagonist. The blocking agents alone had no impact on DNA synthesis. Hyperoxygena- tion with 100% 0 2 also failed to interfere with nicotine's effects.

These findings suggested that nicotine might be acting on DNA synthesis directly in the central nervous system. To test this hypothesis, we administered a small dose of nicotine (2/~g) directly into the central compartment by intracisternal injection. Based on an average brain weight of approx. 1000 mg in the 10-day-old rat, this dose represents local delivery of 2 mg/kg of brain weight, a lower concentration than with systemic treatment. The intracisternal treatment produced neither blanching or coldness of the skin, nor gasping. Nevertheless, there was a large decrement in DNA synthesis in all 3 brain regions, whereas the same small dose given systemically had no effect (Fig. 6).

Finally, we determined whether nicotine could simi- larly interfere with DNA synthesis in the fetal rat brain, by administering the drug to the pregnant dam on gestationai day 20 (Fig. 7). A robust inhibitory effect was detected in midbrain + brainstem and cerebral cortex; there was insufficient tissue in cerebellum to permit determination of DNA synthesis at this age.

227

DISCUSSION

Results obtained in this study indicate that nicotine

acutely reduces DNA synthesis in the developing brain. Because the effects of a single injection lasted for several hours, inhibitory actions could cumulate with repeated administration, contributing to the morphological, bio- chemical and behavioral abnormalities attributed to developmental exposure to nicotine 5'1°'12-15"17A8-21'27-3°.

Importantly, our results further suggest that the actions are exerted directly within the central nervous system. Thus, on postnatal day 1, before regional disparities in receptor concentrations become marked 29, nicotine's

actions were fairly uniform throughout the 3 brain regions. During the ensuing week, when receptors become selectively more concentrated, viz. midbrain + brainstem /> cerebral cortex > cerebellum, the nicotine

effect reflected these regional differences. The inhibitory actions of nicotine also exhibited a distinct relationship to periods of rapid cell replication: by 15 days of age only

the cerebellum still possessed a high rate of [3H]- thymidine incorporation and the effect of nicotine then became more prominent in that region. Thus, nicotine's effects on DNA synthesis are related to both the concentration of nicotinic receptor sites and the rate of cell replication. This dual relationship can explain, for example, why a region like the cerebellum, despite a much higher rate of DNA synthesis, is affected less than the midbrain + brainstem through the first 10 postnatal days but more on 15 days: the disparity in nicotine receptor concentrations dominates the pattern of drug effects only so long as the latter region maintains a moderate rate of cell replication, but when midbrain + brainstem DNA synthesis falls to very low levels, the effect of nicotine is larger in the cerebellum despite its lower receptor concentration.

Cholinergic receptor mechanisms have been impli- cated in the switch of developing cells from replication to differentiation 2"9A9 and therefore we undertook studies

to determine the mechanism by which nicotine exerts its inhibitory actions on DNA synthesis. Nicotine did not interfere with protein synthesis, suggesting that the effect on DNA represents a specific drug effect on cell replication. Furthermore, administering agents that block nicotine-induced ischemia (phenoxybenzamine), cardiac arrhythmias (propranolol + atropine) or hypoxia (100% 02) all failed to prevent the actions of nicotine on DNA synthesis. Finally, direct administration of a small dose of nicotine directly into the central nervous system reduced DNA synthesis, whereas the same dose administered systemically did not. These results indicate that nicotine directly arrests cell replication in the developing brain. This specific mechanism clearly contributes to the impact

228

of cont inued nicotine exposure on central nervous system

development : r epea ted nicotine adminis t ra t ion, ei ther by

inject ion (which includes hypoxia and ischemia as con-

tr ibuting variables) or infusion (which does not) disrupts

overal l pa t terns of cell repl icat ion and differentiat ion

throughout the brain, leading to abnormali t ies in cell

number and size, in synaptogenesis and synaptic activity, and ul t imately in behavior 12'19-21'27-3°.

It should be noted that our findings do not mean that

ischemia, cardiac arrhythmias or hypoxia cannot affect

D N A synthesis by themselves, but ra ther that nicotine 's

effects are exer ted over and above any per iphera l

actions. Indeed , we have a l ready found that hypoxia or

vasoconstr ict ion alone can affect D N A synthesis 1'7.

These variables may be relat ively more impor tant in the

maternal / fe ta l unit, where we found that fetal brain D N A

synthesis was much more sensitive to nicotine than

neonata l rat brain, despi te lower fetal receptor con-

centra t ions 12"2°'29. Gesta t ional effects of nicotine on cell

repl icat ion may thus represent a compendium of direct

actions on cell repl icat ion and indirect actions on pla-

cental b lood flow and oxygen delivery.

Finally, it is impor tan t to consider whether nicotine

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genesis, gliogenesis or both. Al though the biochemical

techniques used here do not permit definit ive del ineat ion

of these two events, a number of factors suggests that

neurogenesis is included in the effects. Neurogenesis

peaks prenatal ly in the midbrain + brainstem, in the

immedia te per inatal per iod in cerebra l cortex and post-

natally in the cerebel lum 3'22'e6, a pa t te rn that corresponds

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cell populat ions affected by nicotine will clearly be of

value in assessing how effects exer ted on cell biochem-

istry are t ranslated into behavioral deficits.

Acknowledgement. Supported by a grant from the Smokeless Tobacco Research Council.

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