Nicotine injections into the ventral tegmental area ... · Nicotine injections into the ventral tegmental area increase locomotion and Fos-like immunoreactivity in the ... animals

ELSEVIER Brain Research 730 (1996) 133-142

BRAIN RESEARCH

Research report

Nicotine injections into the ventral tegmental area increase locomotion and Fos-like immunoreactivity in the nucleus accumbens of the rat

G. Panagis J, M. Nisell, G.G. Nomikos, K. Chergui, T.H. Svensson D{'l)ovtnl('n[ 0[' Physiolofy and Pharmacology, Dii'ision o/'Pharmacolo,k'Y. Kar¢,linska ln.stitutet. S- 171 77 .S'to~'kh<,hn. Swudun

Accepted 2 April 1996

~tbstract

Systemic administration of nicotine has been shown to increase locomotor activity in rats, an effect which is enhanced by chronic pretreatment with the drug. Furthermore, administration of nicotine either systemically, or locally within the ventral tegmental area (VTA), increases extracellular levels of dopamine (DAY in the nucleus accumbens (NAc). In the present study, we examined the effect of local, bilateral injections into the VTA of nicotine (0.02, 0.2, 2.(1 and 8.0 btg/0.5 txl/side) on locomotor activity of rats in an open field. Nicotine (8.0 #,g/side) significantly increased forward locomotion within 20 min after injection, whereas rearing was not affected. The stimulatory effect of locally applied nicotine was completely blocked by pretreatment with mecamylamine (1.0 mg/kg, s.c.). Repeated intra-tegmental iniections of a subthreshold dose of nicotine (2.0 btg/side every 2 days), gradually increased locomotion, compared to the effect of acute intra-tegmental administration or control injections of saline, after the fifth and sixth injection. The effects of mtra-tegmental injections of nicotine were further investigated on cells in several target areas for the VTA-DA neurons through determination of c-f0s expression by means of Fos immunohistochemistry. Intra-tegmental injections of nicotine (8.0 b~g/side) increased Fos-like immunoreactivity in the NAc, but did not affect the number of Fos-positive nuclei in the medial prefrontal cortex or in the dorsolateral striatum. The increase in accumbal Fos-like immunoreactivity was attenuated by pretreatmen[ with mecamylamine (I.0 mg/kg, s.c.). Our data demonstrate that locomotor activating effects similar to those evoked by systemically' administered nicotine, including behavioral sensitization, can be produced by intra-tegmental nicotine administration. Moreover, such local VTA administration of the drug was found to significantly affect neurons within DA target areas. Our findings support the notion that the effects of ~ystemically administered nicotine in mesolimbic target areas are largely dependent on stimulation of nicotinic receptors in the VTA.

,~evword~': Behavioral sensitization: Dopamine: Fos immunohis(ochemistry: Mesolimbic: Nicotine: Ventral tegmental area

I. Introduct ion

Nicotine is a psychostimulant drug with reinforcing and dependence-producing actions in animals as well as in humans. In rats nicotine has been shown to increase locomotor activity and, similarly to findings with other dependence-producing psychostimulants, this behavioral activa- lion is significantly enhanced during chronic pretreatment

with the drug {2,8,29,31,42]. Several studies demonstrate that stimulation of the mesolimbic dopamine (DAY system is of critical importance for the reinforcing and stimulatory properties of nicotine {5,10,18,33,43]. Thus, lesions of the

" Cnrresponding author. Fax: - 46 (8) 30-8424. t Present address: Laboratory of Pharmacology, Department of Basic

Sciences. School ol Medicine. University of Crete, P.O. Box 1393. tteraklinn, Crete. Greece.

mesolimbic DA system have been flmnd to attenuate nicotine self-administration, as well as the locomotor stimulant

effect of nicotine in rats [6,12]. Moreover, nicotine increases both the firing rate and burst activity of midbrain DA neurons [7,16,22,25] . Systemic injection of nicotine also increases release [19], synthesis and metabolism of DA [1,17] preferentially in the nucleus accunqbens (NAt), i.e., a major projection area for lhe mesolimbic DA sys-

tem. There is ample evidence that nicotine exerts its stimula-

tory action on brain DA activity, at least in part, via a direct action on the DA cells. Accordingly, nicotinic receptors have been demonstrated both on the cell bodies and on the ten-ninals of midbrain DA neurons [9,41] and nicotine can clearly affect DA activity at both locations. Thus, nicotine has been found to produce an increased activity of cells in the ventral tegmental area (VTA), i.e., the cell body region of the mesolimbic DA system, via local

()006-8993/96/Sl5.00 ('opyright ~i 1906 Elsevier Science B.V. All rights reserved. I ' l l S()006 ~9o ' I ( ' t 6100432 5

134 G. Pa#tagM et al. / Brain Re.s'can'h 730 ( 1996J /J,?- 142

nicotinic receptor activation [3]. Nicotine has also been found to stimulate DA release from accumbal synapto- somes [40] and when directly administered into the NAc the drug has been found to increase extracellular concentrations of DA [26,32]. However, several studies indicate that stimulation of DA neurons at the somatodendritic, rather than at the nerve terminal level is of major importance for the behavioral stimulation by nicotine. Thus. the drug, either in low doses in drug-naive rats or at high dose levels in nicotine-pretreated animals, exerts a more pronounced effect on locomotor activity when it is applied into the VTA than when it is administered into the NAc [21,37]. Furthermore, Museo and Wise [30] found that repeated injections of the nicotinic receptor agonist cyti- sine into the VTA cause progressively increased locomotor stimulation. Also, microinfusions of the nicotinic receptor antagonist dihydro-[B-erythroidine into the VTA, but not into the NAc, were found to decrease nicotine self-administration [I 1]. In accordance with these behavioral results, local administration of the non-competitive, nicotinic receptor antagonist mecamylamine into the VTA, but not into the NAc, was found to antagonize systemic nicotine- induced DA release within the NAc [32].

Although the effect of nicotine on midbrain DA neurons has attracted considerable scientific attention, the postsynaptic consequences of this presynaptic DA activation are less well characterized. One way to evaluate the effect of DA in postsynaptic neurons is to examine their expression of immediate early genes, such as c-jos, by means of in situ hybridization of their mRNAs or by immunohistochemistry of the protein they encode. Several immediate early genes can apparently serve as markers for neuronal activity and may, accordingly, be activated by various stimuli, including drugs (see Ref. [28] for review). Thus, the expression of c-/os and other immediate early genes has been found to be increased in DA target areas by several drugs which increase the extracellular concentra- tion of DA such as cocaine, amphetamine and morphine [15,23,27,46]. Indeed, systemic administration of nicotine has also been shown to increase Fos-like immunoreactivity in several brain regions, including some which receive a dopaminergic innervation from the VTA [24,34,38].

In the present study we examined in the rat the effect of local nicotine injections into the VTA on locomotor activity as well as on postsynaptic neuronal function, as as- sessed by Fos immunohistochemistry. Nicotine was injected bilaterally into the VTA via permanently implanted guide cannulae in drug-naive as well as mecamylamine- pretreated animals. In order to evaluate the effect of intra- tegmental nicotine injections on target areas for the mesolimbic DA system, in drug-naive or mecamylamine- pretreated animals, the number of Fos-positive nuclei was counted in the nucleus accumbens, the medial prefrontal cortex and the dorsolateral striatum. Finally, the behavioral effect of repeated intra-tegmental injections of nicotine was investigated in order to examine whether this treat-

ment would cause behavioral sensitization, as previously observed with chronic, systemic nicotine administration.

2. Materials and methods

2.1. Animals and surgeo:

Male Wistar rats weighing 280-330 g at the time of surgery were housed in groups with free access to food and water in a temperature and humidity controlled room under a 12:12 h light/dark cycle. On the day of surgery, the animals were anesthetized with pentobarbital (60 mg/kg, i.p.) and stereotaxically implanted bilaterally with guide cannulae (25 gauge) into the VTA. Coordinates were 5.2 mm posterior to bregma, 0.7 mm lateral to the midline, and 7.4 mm below the skull surface according to Paxinos and Watson [35]. The tips of the guide cannulae were located 1.0 mm above the actual injection sites. The guide cannulae were then fixed to the skull with anchoring screws and dental cement. Stainless steel plugs prevented the guide cannulae from clogging. Following implantation and for the entire duration of the experiments, the animals were housed individually. All behavioral experiments started 7-10 days after surgery.

2.2. Measurement o[" locomotor actiz,i O,

The animals were monitored in two identical square open field arenas (680 × 680 × 450 mm), each equipped with two rows of eight photocells along two adjacent sides, placed 40 and 125 mm above the floor. The open field was enclosed in a ventilated, sound-attenuating box kept in the dark. Measurements were performed between 10.00-17.00 h. Locomotor activity was tested as described in detail by Ericson et al. [14]. Interruptions of photocell beams were collected by means of a microcomputer and allowed recording of peripheral motor activity (or motor activity at the edges of the box; total number of interruptions of the photobeams in the lower row located closest to the walls), rearing (total number of photobeam interruptions in the upper row), forward locomotion (successive photobeam interruptions in the lower row when the animal is moving in the same direction) and total locomotor activity (total number of photobeam interruptions in the lower row). In the present study, total locomotor activity, peripheral activity and forward locomotion measurements showed a similar profile in response to nicotine. We graphically presented, however, only the forward locomotion data, since this parameter is more representative of the pure ambula- tory activity of the rat.

2.3. Fos immunohis tochemis to ,

Fos immunohistochemistry was performed as described by Robertson and Fibiger [39] and Chergui et al. [4]. Two

G. Panagis el al./Brain Research 730 (1996) 133 142 t35

hours after intra-tegmental drug administration, the animals were deeply anesthetized with pentobarbital (100 mg /kg , i.p.) and transcardially perfused with saline (150 ml) lbllowed by 300 ml of 4 ~ paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), Subsequently, the brains were removed, postfixed for 2 h in paraformaldehyde and placed overnight in 15% sucrose in PBS. The brains were then cut in 30-bun sections on a microtome.

Sections were washed in PBS (2 X 15 rain) and placed m 0.3% H~O~ in PBS and then washed in PBS (3 × 20 rain) before being incubated tbr 48 h in PBS containing !).3% Triton X, 0.02 Na azide, 2% normal rabbit antibody and Fos primar~ antibody diluted 1:500 (sheep polyclonal antibody, Cambridge Research Biochemicals OA-11-824). Fhe sections were then washed in PBS (3 X 20 rain), and incubated lbr I h with a biotinylated rabbit anti-sheep secondary antibody (Vector Laboratories) diluted 1:200. M'ter washing in PBS (3 × 20 rain) and then being incubated for 1 h in PBS containing avidin biotinylated horseradish peroxidase complex (1:100, Vector Labs), the sections were washed again in PBS and then rinsed lot 10 rain in 0.2 M Na acetate buffer. The reaction was visual- ized using a glucose oxidase-3,3-diaminobenzidine nickel method terminated by washing in PBS.

Sections ,acre mounted on gelatin-coated slides, dehy- drated and covet'slipped for microscopic observation. The number of Fos-positive nuclei was counted at 250 × magnification within a 500 × 500 g m grid over the NAc, the medial prefrontal cortex, and the dorsolateral striatum. Cells were counted bilaterally in three sections from each legion in all brains.

2.4. Drues and drug administration

( - ) - N i c o t i n e - { +)-tartrate (Sigma) was dissolved in saline and the pH was adjusted to 7.2-7.4 with NaOH. All doses of nicotine used were expressed as free base. Mecamylanline hvdrochloride (Sigma) was dissolved in saline.

For intra-tegmental injections, an injection cannula, constructed from a fused silica capillary tube (150 bun o.d., 75 p~nl i.d.) was inserted into the guide cannula, protruding I mm into the VTA, and was connected via polyethylene tubing to a 5 btl Hamilton microsyringe. A w~lume of 0.5 ill nicotine solution or saline was injecled into the hand-restrained animal during 30 s. The injeclion cannula was then left in place for 30 s after the injection. in order to allow sufficient diffusion of the drug. Mecamy- lamine or saline was injected subcutaneously 31) rain prior to the intra-tegmental injections.

2.5. Histoh~,g, ical e.ramination of iJ{fi,clioH ~ile,s

After completion of the behavioral experiments, the animals were sacrificed, their brains removed and fixed in a solution of 5% lornmldehyde in 259; sucrose. Each brain was sliced on a microtome (50 i,~mk stained with neutral red for microscopic examination. Only Ihe rats in which tracks fronl the cannulae were verified to be located in the VTA were included in this study (see Fig. I lkw a representative photomicrograph from the brain of an animal lhal received five bilateral, intra-tegmental rejections).

I ig. 1. Represcnlaliw' photomicrograph illustrating the localization ol intra-tegmental rejection sites. {pper arrow indicates tile tip ol Ille guide c:mnula and hwvcr arrow hldkalc~ the tip of the injection c'amlula, which protruded 1 mm into lhe ventral legnlental area.

136 (;. P(Itl(II@~ ~'1 o/. / Brain Re.watch 730 (1(,)9:~) 133 142

2.6. Statisti~'al analysis

Behavioral data were evaluated by using the raw values from either 10 min recording periods or the total counts over 60 rain. Data were analyzed by one-, two- or three-way analysis of variance (ANOVA) followed by Newman-Ke- uls test for multiple comparisons.

For immunohistochemical data, the mean value of Fos- positive nuclei was calculated and the data were evaluated by one-way ANOVA followed by Newman-Keuls test. A two-tailed P < 0.05 was considered significant.

2.7. Experimental procedure

2.7.1. Experiment 1 The effect of nicotine administered acutely in various

doses into the VTA on locomotor activity in 12 drug-naive rats was examined. Two days prior to the start of the experiments, the animals were habituated to the testing apparatus for 1 h daily. During the test days, the animals were transferred to the test room and placed in holding cages for 60 rain, in order to become accustomed to this environment. Before the intra-tegmental injection of nicotine or saline, the animals were placed for 15 min in the locomotor boxes. This approach enabled us to measure primarily pure locomotor activity and to a lesser extent exploratory behavior, since the animals were able to habit- uate very well. Each animal received four doses of nicotine (0.02, 0.2, 2 and 8 txg per side) as well as vehicle. The sequence of injections was counterbalanced with respect to order at a rate of one injection every 3 days. The test session was initiated immediately after the injection and lasted for l h.

2.7.2. Experiment 2 The effect of mecamylamine on nicotine-induced loco-

motor activity was examined. Twelve rats received a sub- cutaneous injection of either mecamylamine or saline followed 30 rain later by bilateral intra-tegmental injections of either nicotine (8 b~g per side) or saline, as described in Expt. 1. Thus, all rats were subjected to four treatments in a randomized order with 3 days between each session. Test sessions started immediately after the intra-tegmental injections and lasted for I h.

2.7.3. Experiment 3 The effects of acute intra-tegmental injections of nico-

tine on Fos-like immunoreactivity in the nucleus accumbens, the medial prefrontal cortex and the dorsolateral striatum were examined. Three groups of rats were bilaterally injected in their home cages first with saline (1.0 ml /kg , s.c.) and 30 min later with either nicotine (2 btg/side, n = 3; or 8 bLg/side, n = 6) or saline (n = 4) into the VTA and two groups of rats received a subcuta- neous injection of mecamylamine followed 30 min later by an intra-tegmental injection of either nicotine (8 >g / s ide ,

n = 5) or saline (n = 3). Fos immunohistochemistry was then performed in all brains, as described above.

2.7.4. Exl~eriment 4 This experiment was designed to determine whether

repeated administration of nicotine directly into the VTA is sufficient to bring about a sensitization of locomotor activity, similar to that seen after repeated systemic nico- fine administration. Two days prior to the start of the experiments, the animals were habituated to the testing environment for 1 h daily. The animals were then bilaterally iniected six times with 2.0 #,g of nicotine (n = 7) or saline (n - 7) per side, at a rate of one injection every 2 days. This dose of nicotine was chosen since it was the highest dose tested that did not significantly increase locomotor activity when given acutely. Immediately after each bilateral injection, the animals were placed in the activity apparatus and locomotion was measured tbr I h.

3. Results

3.1. Effects qf acute intra-tegmental nicotine adminislra- tion (m locomotor actit,itv (Expt. 1)

An overall ANOVA showed that nicotine produced a significant increase in forward locomotion (F4:4 = 2.97, P = 0.028) compared to saline, for the entire 60 rain-period of measurement (Fig. 2). Post-hoc comparisons revealed that this effect was statistically significant only for the highest dose of nicotine (8 Ixg/side: P = 0.015). Locomo- tor activity peaked within the first 10 min period and then declined to levels not significantly higher than in the saline treated animals for the remainder of the experiment. Dur-

5 0 0 o

Q. " 4 0 0 L L,. el)

t,.- • - 300 E II)

..o 2 0 0 o 0-, o "- 100 IX.

0 0,02 0.2 2.0 8.0

Dose of nicotine (pg/0.5pl/side)

Fig. 2. Effects of bilateral, intra tegmental injections of saline or of

various doses of nicotine (nic: Ixg /0 .5 tx l / s ide , n = 12) on lorward

locomotion measuremenls in drug--naive rats. Data are presented as mean

( + S.E.M.) photobeam interruptions over 60 rain fol lowing drug administration. / ' < 0.05 for nicoline \% saline.

G. Panagis et al. / Bruin Research 730 (1996) 133 142 137

o 200 o-, O .

L.. =,..

150

E 100

O

o 50 . £ :

o sal • nic • nic • nic • nic

i . . . . .

r [ [ i i i

10 20 30 40 50 60

0 ,02 0.2 2.0 8.0

500 O

= 400 L- L - q )

.~_ 300

E

200 O

O • " 100

Ix.

sal-sal mec-sal

i k +

sal-nic mec-nic

T i m e (min}

t"ig. 3. Effects or bilateral, intra-tegmental injections of saline (sal) or of various doses oI nicotine (nic: in beg/0.5 bLI/side, n = 12), on forward locomotion. Mean (+S.E.M.) photobeam interruptions are shown for 6 ,uccessive 10-rain periods, beginning immediately after injection. * P < ~).05 lot nicotine (8.1)) \s. saline.

ing the first ten rain o f measurement , the highest dose of

nicotine (8 l a g / s i d e ) s ignif icant ly increased forward loco-

motion in compar i son to saline treated animals ( P = 0.038:

Fig, 3). Total locomotor activity and peripheral act ivi ty

increased in response to various doses of nicotine, simi-

larly to forward locomot ion (overal l A N O V A s : F4,54 =

3.08, P = 0.024 and F4,54 = 2.58, P = 0.047 for the total

locomotor activity and the peripheral act ivi ty data, respec-

t ively: data not shown). On the other hand, rearing was not

s ignif icant ly alt~'cted by nicotine adminis tered at any dose

q &,,.~ = 1.75, P - 0.15: data not shown).

D r u g t r e a t m e n t

Fig. 4. Effects of pretreatment with mecamylamine (mec; 1.0 mg/kg, s.c.. n 12) or saline (sal; 1.0 ml/kg, s.c., , = 12) 30 nfin prior to bilateral, intra-tegmental injections of nicotine (8.0 l.tg/0.5 ~LI/side) or saline on forward locomotion measurements. Data are presented as mean ( _+ S.E.M,) photobeam interruptions over 60 rain fl~llowing drug administration. " ' ' P <0.001, sal+nic vs. sal+sal: ~ + ' P <().001, sat+nit v s . n l e c + n i c .

number of Fos-posi t ive nuclei compared to the number

observed in sal ine-treated animals (Fig. 5: see also Fig. 6

for representat ive photomicrographs) . This effect was sig-

nif icantly reduced by pretreatment with mecamylamine (I

m g / k g , s.c.), 30 rain prior to the intra-tegmental nicotine

inject ions (Fig. 5). Inject ions o f mecanwhtmine alone, at

?.2. E{/})cts (dmecamylamine ml nicotine-induced Iocomo- ,'or actiNtv (kLvpt. 2)

M e c a m y l a m i n e (1 m g / k g : s.c.), adminis tered 30 min

before bilateral intra-tegmental inject ions of nicot ine (8

i xg / s i de ) , comple te ly blocked the effect of nicot ine on

rorward locomot ion during the 60 rain tes t ing-per iod ( P =

0.001: Fig. 4). Total locomotor act ivi ty and peripheral

activity were affected in a manner s imilar to forward

locomot ion measurements ( P = 0.004 and P = 0.039 for

lhe total locomotor activity and peripheral act ivi ty data,

respect ively; data not shown). As in Expt. 1, rearing was

not s ignif icant ly affected by intra- tegmental nicotine injec-

tions (data not shown). Mecamylamine , at the present

dose, did not by itself affect any behavioral parameter

when comparcd to the levels of activity observed after

~aline (Fig. 4) .

3.,?. Eff~,c/,s' :?/' intra-tegmental injections o[ nicotine on ,-fi')s e.rl~r('ssion in dopaminer~,ic terminal fields (Expt. 3)

In the nucleus accumbens , bilateral, in t ra- tegmental in-

jec t ions of nicotine (8 l a g / s i d e ) s ignif icant ly increased the

", 150 c ,.k

= k + O

• ~, 120 .e~, A

@1 Q

90 ~

E ×,¢x] . _ 60

O .~- 30 i O 1

sal + sal + sal + mec + mec + sal nic 2.0 nie 8.0 sal nie 8.0

D r u g t r e a t m e n t

Fig. 5. Effects of bilateral, mtra-tegmental iniections of saline (sad or nicotine (nic 2.0 and 8.0 p,g/side) administered alone or 30 rain after mecamylamme (I.0 mg/kg, s.c.) or saline (I.O ml/kg, s.c.) on the number of Fosdike immunoreactive cells in the nucleus accumbens. Dala are presented as mean (±S.E.M.) total number of Fos-positive nuclei within a 500×500 t ln] grid over the nucleus accumbens al a 250X

magnification. A l l experiments were periormed, i.e. the animals were

perfused. 2 h after the bilateral, intra-tegmental injections o f saline or

mcotme. P < 0 . 0 1 . s a ] + n i c 8 . 0 w . sa l+sa l : ~ P < I).()5. sa l+n ic 8.0

vs. m c c + n i c 8.().

138 (;. t ' ~ ~tbL~ ~'I ~tl. / Bra i l l R~2.'~C¢II'ch 7.:,(i ¢ 1996) 1.'~3 142

Fig. 6. Pho tomicrographs illustrating Fos- immunoreac t ixe cells in the nucleus accumbens of rats injected bilaterally into the ventral tegmcntal area ,a ith

saline (A). or nicotine adminis tered alone at a dose of 2.0 Ixg / s ide (B), or 8.0 t x g / s i d e (C), or nicotine (8 ixg / s ide ) 30 rain after systemic administrat ion

of mecamylamine al a dose of 1.0 m g / k g , s.c. (D). Scale bar = 100 izm.

the present dose, did not significantly alter the number of accumbal los-posi t ive nuclei compared to saline-treated animals (Fig. 5). ~= .o 16oo

lntra-tegmental injections of nicotine did not signifi- " cantly affect the number of Fos-posit ive nuclei in either ~ 1200

the medial prefrontal cortex or the dorsolateral striatum .~

(data not shown). E 8 0 0

q)

o

o 4 0 0 #_

3.4. Effects of repeated intra-tegmental injections of nicotine on locomotor actit, ity (Expt. 4)

Repeated, daily bilateral intra-tegmental injections of nicotine every two days, at a subthreshold dose (2.{) Ixg / s ide) for locomotor activity, produced a progressive increase in locomotion compared to the effect of the first injection, as well as compared to the activity measured in animals treated with an equal number of saline injections (Fig. 7). This effect became statistically significant after the 5th ( P = 0.020) and 6th ( P = 0.0006) injection (Fig. 7). Furthermore, after six injections, the nicotine-induced locomotor stimulation was significantly elevated up to 50 min after an intra-tegmental injection, compared to the

@+

nie 2.C

s a l

i i i i r i

1 2 3 4 5 6

Days of treatment

Fig. 7. Effects of repeated nicotine (2.0 l ,*g/0.5 ix l / s ide , *; 7) or saline

(0.5 I~l /s ide, n = 7) injections (one every 2 days) bilaterally into the

ventral tegmenlal area on forward locomotion of rats over 6 days of treatment. Data are presented as mean (_+S.E.M.) photobeam interruptions over 60 rain following drug administration. P < 0.05. ..... t' < 0.001, nic 2.0 vs. s a l o n day 6; + P < { ) . 0 5 , + + P < 0 . 0 1 . nic 2.0 on da>

6 vs. nic 2.0 on day I.

G. Panagiv et al. / Brain Research 730 (1990J 133 142 139

o 4 0 0

t . t

• 300

E m 200 q}

0

100 g_

o sal -day 1 • sal -day 6 [] nic-day 1 • nic-day 6

. I - +

10 20 30 40 50 60

Time (rain)

Fig. 8. Effects of bilateral, intra-tegmental injections o f nicotine (nic; 2.0

p,g/() .5 p , l / s idc , n = 7) or saline (sal: 0.5 p , I / s ide , n = 7) on forward

locomotion, d ining the first (day I ) or the sixth (day 6) day of treatment.

Data are presented as mean ( + S . E . M . ) photobeam interruptions. ' P <"

I).05. " P < 0.01, uic day 6 vs. sal-day 6: P < 0 . 0 5 , ++ P < 0 . 0 1 ,

n i t -day 6 ' , s n ic -da \ I.

effect of the first injection as well as compared to animals treated with saline ( P = 0.004-0.03 l; Fig. 8). Total locomotor activity and peripheral activity were affected in a manner similar to forward locomotion measurements ( P = 0.0001 and P = 0.0002 for the total locomotor activity and peripheral activity data, respectively, after the 6th injeclion; data not shown). On the other hand, rearing was not significantly affected by repeated administration of nico- fine (data not shown).

4. Discussion

The major finding of the present study is that bilateral injections of nicotine locally into the VTA cause both locomotor stimulation and, at the same time, an increased number of Fos-positive nuclei in the NAc. We also demonstrate that repeated, daily intra-tegmental injections of nicotine, in a dose which does not affect locomotion acutely, produce a gradual increase in locomotor activity,, i.e. behavioral sensitization.

The increased locomotor activity observed in the present study after acute intra-tegmental injections of nicotine is in accordance with previous reports [21,37]. Moreover, lhe pattern of response, i.e. a rapid increase of rather short duration, is similar to the response seen after both systemic and intra-tegmental injections of nicotine in previous studies. Reavill and Stolerman [37] reported an enhanced locomotor activity alter intra-tegmental injections of nicotine at a dose of 8 t~g/side in rats that had been administered nicotine systemically for six days, a treatment which is known to enhance the locomotor effects of systemic nicoline. As argued by these authors, the 8 I_Lg dose of nicotine used would have amounted to about 0.05 m g / k g if given systemically, which is close to the threshold for locomotor

stimulation. The present data indicate that this amount of nicotine injected bilaterally in the VTA is behaviorally effective also in drug-naive animals. It should be noted that, in our study, the animals were very well habituated to the test environment which is reflected in the relatively small decrease in activity levels in saline-treated animals over the recorded period (see Fig. 2). Consequently, this low baseline activity level may allow the disclosure of the stimulatory effec! exerted by acutely administered intra- tegmental nicotine on locomotion in the present study. Indeed, Leikola-Pelho and Jackson [21] reported a significant stimulatory action of intra-tegmental injections of nicotine, although at a much lower dose (0.02 p~g/side) than the dose that proved to be effective in our hands. The reason for this discrepancy is not clear. Nicotine, however, has been shown to attenuate the reduction in activity, caused by exposure to an aversive environment, at doses lower than those needed to ew)ke general psychostimula- tion [44]. Moreover, Leikola-Pelho and Jackson do not appear to have used habituated animals in their behavioral assessments. It is possible, therefore, that without exten- sive habituation, the test apparatus constitutes an aversive environment per se in which nicotine can antagonize the locomotor suppression at lower dose level than that needed to elicit locomotor stimulation in the present study. Never- theless, it is striking that both the aforementioned studies [21,37], as well as our own, in which nicotine or cyticine is injected in the VTA, show, due to an as yet unspecified mechanism, relatively fiat dose-effect curves as regards drug-induced stimulation of locomotor activity.

The capacity of systemically administered mecamylamine to block the locomotor stimulant effect of intra-tegmental nicotine indicates that this antagonism is due to nicotinic receptor stimulation within the VTA. Although it was not conclusively established that the observed belmv- ioral effect of intra-tegmental nicotine injections is, indeed, due to increased dopaminergic activity, this notion seems exceedingly likely, since the behavioral stimulation elicited by systemic nicotine administration has been shown to be critically dependent on mesolimbic dopaminergic activity (cf. Sectkm 1 ). Furthermore, nicotine seems to increase lhe firing of DA neurons in the VTA via stimulation of somatodendritic nicotinic receptors [3]. Finally, the present results show that intra-tegmental injections of nicotine produce a pattern of locomotor activation that greatly resembles the qualitative response to systemic nicotine. Thus, our findings strongly support the notion that activation of the mesolimbic dopaminergic system at the somatodendritic level is of maior importance for the behavioral stimulant action of nicotine.

Systemic injections of nicotine have previously been shown to increase Fos-like immunoreactivity in the NAc, the medial prefrontal cortex and the dorsolateral strialum [20]. in the present study, a behaviorally effective dose of nicotine (8 l, t g / s i d e ) injected into the VTA also increased Fos-like immunoreactivity in the NAc. The attenuation of

140 G. Pam*~is el al. / Brain Research 730 ( 1996J I_¢3-142

this effect by pretreatment with mecamylamine suggests that nicotine exerts its biochemical effect in DA target areas by stimulating nicotinic receptors within the VTA. Clearly, a number of previous studies suggest the involvement of DA in this effect. Thus, systemic nicotine induced expression of Fos-like immunoreactivity in the NAc has been found to be attenuated by pretreatment with the DA Dl-receptor antagonist SCH 23390 [20]. In addition, local administration of nicotine into the VTA increases extracellular concentrations of DA in the NAc [32]. Furthermore, burst stimulation of the median forbrain bundle, which increases release of DA in target areas, has recently been observed to increase Fos-like immunoreactivity in the ipsilateral NAc, an effect which could be blocked by pretreatment with SCH 23390 [4]. Indeed, systemic administration of nicotine has been found to potently increase burst activity in VTA-DA neurons [16]. Interestingly, whereas lesioning of dopaminergic afferents to the NAc causes a considerable reduction in nicotinic receptor labelling in this region [9], the accumbal neurons express very low levels of mRNA for different subunits of nicotinic receptors [45]. Consequently, the nicotine-elicited effects on accumbal neuronal activity are probably mediated via accumbal afferents. Hence, our results clearly suggest that intra-tegmental injections of nicotine affect neurons within the NAc largely through facilitation of its dopaminergic input. Systemic nicotine has previously been shown to increase Fos-immunoreactivity in the cingulate gyrus [20]. However, we did not observe any major effect in the medial prefrontal cortex of acute intra-tegmental injections of nicotine, although a clear tendency towards an increase was detected. The mechanisms behind the presently observed difference in responsiveness to nicotine between NAc and the medial prefrontal cortex are not clear. The fact, however, that the number of Fos-positive nuclei in the control group was rather high may have contributed to conceal an actual effect of nicotine in our experiments, especially in view of previous observations that stress associated with injections in hand-restrained animals markedly increase the number of Fos-positive nuclei in the prefrontal cortex [36]. Interestingly, we have recently found that chronic, systemic treatment with nicotine causes an enhanced nicotine-induced increase in Fos-immunoreactivity in DA target areas and that this effect is attenuated by pretreatment with SCH 23390 (Nisell et al., unpublished observations). Thus, systemic nicotine-induced Fos-expression in the brain appears to be mediated, at least in part, via D~-receptor activation. Since chronic, systemic nicotine administration has been shown to enhance the nicotine-induced DA release preferentially in the medial prefrontal cortex [31], the possibility remains that repeated intra-tegmental injections may produce a more pronounced effect on prefrontal, cortical Fos-immunoreactivity, than single injections. Also, as far as the striatum is concerned we found a tendency towards increased levels of Fos-immunoreactivity in response to intra-tegmental injections of

nicotine. When the position of the injection site was analyzed in relation to the level of Fos-immunoreactivity in the ipsilateral striatum for each animal, only injections that were performed within the lateral parts of the VTA were found to increase the number of Fos-positive nuclei in the dorsolateral striatum. Hence, the slightly increased Fos-immunoreactivity in this region may reflect an over- flow of nicotine into the adjacent pars compacta of the substantia nigra, which provides its major DA input.

Repeated, systemic injections of nicotine have previously been shown to increase the stimulatory effects of the drug on locomotor activity in rats [2,8,29,31,42]. Our results demonstrate that such behavioral sensitization can take place also after repeated, intra-tegmental injections of nicotine. Previous data show that sensitization of the locomotor response to systemically administered nicotine after five daily injections is also associated with an enhance- ment of the nicotine-induced DA release in the NAc [2]. On the other hand, prolonged treatment with systemic nicotine over 12 days appears to result in an increase in DA release preferentially in the medial prefrontal cortex, whereas nicotinds stimulatory action on DA release in the NAc remains unaffected [13,31]. Although the present data do not reveal how repeated, intra-tegmental injections of nicotine specifically affect dopaminergic activity in the different terminal regions, our results strongly support the notion that behavioral sensitization to nicotine primarily involves actions of the drug within the VTA. Sensitization to the behavioral effects of systemically administered nico- fine has been observed in both context-dependent [8] and context-independent protocols [2,29,42]. Ill the present study, the observed sensitization to the locomotor activating effects of nicotine may well be context-dependent, since the animals received repeated treatment in the test- environment and not in their home-cages.

In summary, our results demonstrate that the locomotor stimulant action of acute systemically administered nicotine, as well as the behavioral sensitization observed with chronic treatment, can be produced by local applications of nicotine within the VTA. Moreover, when associated with behavioral stimulation such local intra-tegmental nicotine administration also increases the number of Fos-positive nuclei in the NAc, indicating altered postsynaptic neuronal function. These findings support the notion that the effects of systemically administered nicotine in mesolimbic target areas are largely dependent on stimulation of nicotinic receptors in the ventral tegmental area.

Acknowledgements

This work was supported by grants from the Swedish Medical Research Council (Projects 4747 and 11026), the Karolinska Institutet, the Swedish Society of Medicine, the Council for Tobacco Research, USA, Inc., AB LEOS in Helsingborg Research Foundation and Svenska Tobaks

G. Panagis et al. / Brain Research Z?O (1996) 133 142 141

AB. G.P. was supported by an ERASMUS fellowship. The excellent technical assistance of Mrs Anna Malmerfelt is gratefully acknowledged.

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