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Transcript
BRAIN
RESEARCH
ELSEVIER
Brain Research 730 (1996) 133-142
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. Introduction
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 activalion 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
" Cnrrespondingauthor. 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 system.
There is ample evidence that nicotine exerts its stimulatory 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('t6100432
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 synaptosomes [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 cytisine 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 nicotineinduced 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 concentration 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 assessed by Fos immunohistochemistry. Nicotine was injected bilaterally into the VTA via permanently implanted
guide cannulae in drug-naive as well as mecamylaminepretreated animals. In order to evaluate the effect of intrategmental nicotine injections on target areas for the
mesolimbic DA system, in drug-naive or mecamylaminepretreated 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. M e a s u r e m e n t o[" l o c o m o t o r 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 ambulatory activity of the rat.
2.3. Fos i m m u n o h i s t o c h e m i s t o ,
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
hours after intra-tegmental drug administration, the animals were deeply anesthetized with pentobarbital (100
m g / k g , 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 visualized using a glucose oxidase-3,3-diaminobenzidine nickel
method terminated by washing in PBS.
Sections ,acre mounted on gelatin-coated slides, dehydrated 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.
t35
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. Mecamylamine or saline was injected subcutaneously 31) rain prior
to the intra-tegmental injections.
2.5. Histoh~,g,ical e.ramination o f 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' photomicrographillustrating the localization ol intra-tegmental rejection sites. {pper arrow indicates tile tip ol Ille guide c:mnulaand
hwvcr arrow hldkalc~ the tip of the injection c'amlula, which protruded 1 mm into lhe ventral legnlental area.
136
(;. P(Itl([email protected]~~'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-Keuls test for multiple comparisons.
For immunohistochemical data, the mean value of Fospositive nuclei was calculated and the data were evaluated
by one-way A N O V A 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 habituate 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.
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 nicofine 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 adminislration (m locomotor actit,itv (Expt. 1)
An overall A N O V A 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). Locomotor 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-
2.7.2. Experiment 2
The effect of mecamylamine on nicotine-induced locomotor activity was examined. Twelve rats received a subcutaneous 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 nicotine 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
m l / k g , 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 subcutaneous injection of mecamylamine followed 30 min later by
an intra-tegmental injection of either nicotine (8 > g / s i d e ,
500
o
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L,.
400
el)
t,.-
•E
300
II)
..o
o
0-,
o
"IX.
200
100
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: I x g / 0 . 5 t x l / s i d e , n = 12) on lorward
locomotion measuremenls in drug--naive rats. Data are presented as mean
( + S.E.M.) photobeam interruptions over 60 rain following drug administration.
/' < 0.05 f o r nicoline \% saline.
G. Panagis et al. / Bruin Research 730 (1996) 133 142
137
ik+
o
o
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•
•
•
200
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=,..
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sal
nic 0 , 0 2
nic 0.2
nic 2.0
nic 8.0
500
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100
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10
20
30
40
50
60
Time
100
sal-sal
(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.
mec-sal
Drug
sal-nic
mec-nic
treatment
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
vs. nlec + nic.
ing the first ten rain o f measurement, the highest dose of
nicotine (8 l a g / s i d e ) significantly increased forward locomotion in c o m p a r i s o n to saline treated animals ( P = 0.038:
Fig, 3). Total l o c o m o t o r activity and peripheral activity
increased in response to various doses of nicotine, similarly to forward l o c o m o t i o n (overall A N O V A s : F4,54 =
3.08, P = 0.024 and F4,54 = 2.58, P = 0.047 for the total
l o c o m o t o r activity and the peripheral activity data, respectively: data not shown). On the other hand, rearing was not
significantly alt~'cted by nicotine administered at any dose
q &,,.~ = 1.75, P - 0.15: data not shown).
?.2. E{/})cts (dmecamylamine ml nicotine-induced Iocomo,'or actiNtv (kLvpt. 2)
n u m b e r of Fos-positive nuclei c o m p a r e d to the number
o b s e r v e d in saline-treated animals (Fig. 5: see also Fig. 6
for representative photomicrographs). This effect was significantly reduced by pretreatment with m e c a m y l a m i n e (I
m g / k g , s.c.), 30 rain prior to the intra-tegmental nicotine
injections (Fig. 5). Injections o f m e c a n w h t m i n e alone, at
",
150
c
,.k
=k+
O
M e c a m y l a m i n e (1 m g / k g : s.c.), administered 30 min
before bilateral intra-tegmental injections of nicotine (8
i x g / s i d e ) , c o m p l e t e l y blocked the effect of nicotine on
rorward l o c o m o t i o n during the 60 rain testing-period ( P =
0.001: Fig. 4). Total l o c o m o t o r activity and peripheral
activity were affected in a m a n n e r similar to forward
locomotion m e a s u r e m e n t s ( P = 0.004 and P = 0.039 for
lhe total l o c o m o t o r activity and peripheral activity data,
respectively; data not shown). As in Expt. 1, rearing was
not significantly affected by intra-tegmental nicotine injections (data not shown). M e c a m y l a m i n e , at the present
dose, did not by itself affect any behavioral parameter
when c o m p a r c d to the levels of activity o b s e r v e d 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 a c c u m b e n s , bilateral, intra-tegmental inj e c t i o n s of nicotine (8 l a g / s i d e ) significantly increased the
•~,
120
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@1
Q
90
E
._
~
×,¢x]
60
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.~i
O
30
1
sal +
sal
sal +
sal + m e c +
nic 2.0 nie 8.0
sal
Drug
mec +
nie 8.0
treatment
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 5 0 0 × 5 0 0 tln] 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<0.01.sa]+nic8.0w.
s a l + s a l : ~ P < I).()5. s a l + n i c 8.0
vs. m c c + n i c 8.().
138
(;. t' ~ ~tbL~ ~'I ~tl. / Braill R~2.'~C¢II'ch 7.:,(i ¢ 1996) 1.'~3 142
Fig. 6. P h o t o m i c r o g r a p h s illustrating F o s - i m m u n o r e a c t i x e cells in the nucleus a c c u m b e n s of rats injected bilaterally into the ventral tegmcntal area ,a ith
saline (A). or nicotine administered alone at a dose of 2.0 I x g / s i d e (B), or 8.0 t x g / s i d e (C), or nicotine (8 i x g / s i d e ) 30 rain after systemic administration
of m e c a m y l a m i n e 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-positive nuclei compared to saline-treated
animals (Fig. 5).
lntra-tegmental injections of nicotine did not significantly affect the number of Fos-positive nuclei in either
the medial prefrontal cortex or the dorsolateral striatum
(data not shown).
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.{)
I x g / s i d e ) 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
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nie 2.C
800
o
o
#_
400
sal
i
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1
2
3
4
r
5
i
6
Days of treatment
Fig. 7. Effects of repeated nicotine (2.0 l,*g/0.5 i x l / s i d e , *; 7) or saline
(0.5 I~l/side, 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 d a y I.
G. Panagiv et al. / Brain Research 730 (1990J 133 142
o
400
t.t
sal-day 1
sal-day 6
nic-day 1
nic-day 6
.I-+
•
300
E
m
200
q}
o
•
[]
•
0
g_
100
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 i d c , n = 7) or saline (sal: 0.5 p , I / s i d e , n = 7) on f o r w a r d
locomotion, dining the first (day I ) or the sixth (day 6) day of treatment.
Data are presented as mean ( + S . E . M . ) p h o t o b e a m 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 - d a y 6 ' , s n i c - d a \ 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 nicofine (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_Lgdose 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
139
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 intrategmental 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 psychostimulation [44]. Moreover, Leikola-Pelho and Jackson do not
appear to have used habituated animals in their behavioral
assessments. It is possible, therefore, that without extensive 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. Nevertheless, 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 belmvioral 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 overflow 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 enhancement 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 nicofine 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 testenvironment 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
AB. G.P. was supported by an ERASMUS fellowship. The
excellent technical assistance of Mrs Anna Malmerfelt is
gratefully acknowledged.
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