Dual effects of nicotine on dopamine neurons
mediated by different nicotinic receptor
Bjo ¨rn Schilstro ¨m, Nina Rawal, Monica Mameli-Engvall, George G. Nomikos
and Torgny H. Svensson
Department of Physiology and Pharmacology, Section of Neuropsychopharmacology, Karolinska Institutet,
171 77 Stockholm, Sweden
Burst firing of dopaminergic neurons has been found to represent a particularly effective means of in-
creasing dopamine release in terminal areas as well as activating immediate early genes in dopamino-
ceptive cells. Spontaneous burst firing is largely controlled by the level of activation of NMDA receptors in
the ventral tegmental area (VTA) as a consequence of glutamate released from afferents arising mainly
in the prefrontal cortex. Nicotine has been found to effectively increase burst firing of dopaminergic
cells. This effect of nicotine may be due to an a7 nicotinic receptor-mediated presynaptic facilitation of
glutamate release in the VTA. By the use of in-vivo single-cell recordings and immunohistochemistry
we here evaluated the role of a7 nicotinic receptors in nicotine-induced burst firing of dopamine cells
in the VTA and the subsequent activation of immediate early genes in dopaminoceptive target areas.
Nicotine (0.5 mg/kg s.c.) was found to increase firing rate and burst firing of dopaminergic neurons. In the
presence of methyllycaconitine (MLA, 6.0 mg/kg i.p.) nicotine only increased firing rate. Moreover, in the
presence of dihydro-b-erythroidine (DHbE, 1.0 mg/kg i.p.), an antagonist at non-a7 nicotinic receptors,
nicotine produced an increase in burst firing without increasing the firing rate. Nicotine also increased
Fos-like immunoreactivity in dopamine target areas, an effect that was antagonized with MLA but not
with DHbE. Our data suggest that nicotine’s augmenting effect on burst firing is, indeed, due to stimu-
lation of a7 nicotinic receptors whereas other nicotinic receptors seem to induce an increase in firing
Received 10 March 2002; Reviewed 27 June 2002; Revised 7 October 2002; Accepted 13 October 2002
Key words: Burst firing, electrophysiology, immediate early gene, nicotine.
The mesolimbic dopamine neurons, which originate
in the ventral tegmental area (VTA) and project to
forebrain structures such as the nucleus accumbens
(NAc), the amygdala and the prefrontal cortex (cf.
Bjo ¨rklund and Lindvall, 1984), has been shown to
play an important role in mediating the reinforcing
effect of natural rewards as well as that of various
drugs of abuse, including nicotine (see e.g. Fibiger and
Phillips, 1986; Koob, 1992; Schultz et al., 1993). Mid-
brain dopamine neurons display two functional firing
modes in vivo, single-spike firing and burst firing
(Grace and Bunney, 1984a,b). Behavioural studies in-
dicate that whereas the single-spike firing mode may
basically serve to provide an ‘enabling’ dopaminergic
tone on target neurons, the burst firing mode may
rather be involved with phasic responsivity of VTA
dopamine neurons to environmental stimuli of atten-
tional and motivational significance, thus providing
a ‘teaching signal’ to the brain (Schultz, 1998). As-
sessment of neurotransmitter release has revealed that
burst firing, compared to single-spike firing, rep-
resents a much more efficient means to increase extra-
cellular concentrations of dopamine (Gonon, 1988), as
well as co-localized peptide neurotransmitters such
Address for correspondence: Professor T. H. Svensson, Department
of Physiology and Pharmacology, Section of Neuropsycho-
pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden.
Tel.: +468-728 79 21Fax: +468-30 84 24
B. Schilstro ¨m, Ernest Gallo Clinic and Research Center, Department of
Neurology, University of California, San Francisco, CA 94110, USA.
N. Rawal, Department of Medical Biochemistry and Biophysics,
Molecular Neurobiology, Karolinska Institutet, 171 77 Stockholm,
G. G. Nomikos, Neuroscience Discovery Research, Lilly Corporate
Center, Eli Lilly and Company, Indianapolis, IN, USA.
International Journal of Neuropsychopharmacology (2003), 6, 1–11. Copyright f 2003 CINP
as neurotensin (Bean and Roth, 1991), in dopamine
target areas. Excitatory amino acids (EAA) are of
fundamental importance for the regulation of firing
mode of dopamine neurons. Thus, spontaneous burst
firing is driven by EAA-containing afferents, originat-
ing to a significant extent in the prefrontal cortex
but also the subthalamic nucleus and the pedunculo-
pontine and laterodorsal tegmental nuclei, activating
N-methyl-D-aspartate (NMDA) receptors in the VTA
(Charle ´ty et al., 1991; Chergui et al., 1993; Grenhoff
et al., 1988). Nicotine may cause a selective acti-
vation of burst firing in VTA dopamine neurons, even
without any significant effect on average firing rate
(Grenhoff et al., 1986; Mereu et al., 1987). We have
previously shown that nicotine increases extracellular
concentrations of glutamate and aspartate in the VTA
(Schilstro ¨m et al., 2000a) and that antagonism of
NMDA receptors in the VTA diminishes nicotine-
induced dopamine release in the NAc as well as
(Schilstro ¨m et al., 1998a, 2000b). Nicotine has also been
shown to activate dopamine neurons in vitro in the
deafferented slice preparation, however, only with
an increase in firing rate, which appears elicited by
direct stimulation of nicotinic receptors on the cell
membrane (Calabresi et al., 1989). Thus, nicotine may
activate dopamine neurons by activation of both pre-
and post-synaptic receptors. The presynaptic facili-
tation of glutamate release has been shown to be
mediated via a7 nicotinic receptors (Mansvelder and
McGehee, 2000; Schilstro ¨m et al., 2000a) whereas
recently published data strongly indicates that the
stimulatory effect of nicotine on the firing rate of
dopamine cells in the midbrain slice preparation
is mediated via b2-subunit-containing nicotinic re-
ceptors (Picciotto et al., 1998). In the present study it
is hypothesized that presynaptic a7 nicotinic receptors
mediate the nicotine-induced increase in burst firing
of dopamine neurons and that other nicotinic re-
ceptors on the dopamine neurons [a majority of which
are so-called high-affinity or heteromeric nicotinic re-
ceptors (Clarke et al., 1985), most likely containing the
a4 and the b2 subunits (Arroyo-Jimenez et al., 1999;
Klink et al., 2001; Picciotto et al., 1998)], may control
firing rate but not necessarily burst firing. The present
study was, therefore, undertaken to examine pharma-
cologically whether putative post-synaptic a4b2* and
presynaptic a7* nicotinic receptors, respectively, may
subserve different functions in the overall stimulatory
effect of systemic nicotine on dopamine cell firing.
For this purpose, we employed single-cell recordings
in vivo of dopamine neurons in the VTA. Nicotine
was given alone as well as after pretreatment with
the a7 selective antagonist methyllycaconitine (MLA)
(Ward et al., 1990) or the nicotinic receptor antagonist
dihydro-b-erythroidine (DHbE) with high affinity for
a4b2* nicotinic receptors (Alkondon and Albuquer-
que, 1993). Furthermore, we tested the effects of the
selective a7 agonist AR-R17779 (Mullen et al., 2000)
and the non-a7-preferring nicotinic receptor agonist
A-85380 (Sullivan et al., 1996), respectively. The post-
synaptic consequences of treatment with nicotine
alone as well as nicotine after pretreatment with MLA
and DHbE, respectively, as assessed by Fos immuno-
histochemistry in the NAc and the medial prefrontal
cortex were also determined.
Materials and methods
All experiments were performed in compliance with
the guidelines of the Ethical Committee of Northern
Extracellular single cell recordings
Male Sprague–Dawley rats (BKI:SD, BK Universal,
Sollentuna, Sweden) weighing 280–350 g at the time of
experiment were anaesthetized with chloral hydrate
(400 mg/kg i.p.) and mounted in a stereotaxic frame
(David Kopf, Tujunga, CA, USA). Anaesthesia was
maintained with additional injections of chloral hy-
drate (approx. 150 mg/kg.h i.p.) and body tempera-
ture was kept at 37 xC with a rectal thermometer
connected to an electrical heating pad. A hole was
drilled in the skull above the VTA, i.e. 3.2¡0.3 mm
anterior of the interaural line and 0.7¡0.2 mm lateral
to the midline (Paxinos and Watson, 1998). When ap-
plicable a catheter was inserted in one of the tail veins
for intravenous administration.
Recording electrodes were pulled in a Narishige
electrode puller from borosilicate glass capillaries with
ively (Clark Electromedical Instruments, Edenbridge,
UK) and filled with 2% Pontamine Sky Blue in 2 M
NaCl. The tips of the electrodes were broken under
microscope to yield an impedance of 2.0–4.0 MV at
135 Hz. The electrode was lowered into the brain using
a hydraulic microdrive (David Kopf) and a reference
electrode was placed in the subcutaneous tissue. Ex-
tracellular action potentials were amplified, discrimi-
nated and monitored on an oscilloscope (TDS 310,
Tektronix) and an audiomonitor (Grass, AM8B/C).
Discriminated spikes were collected on a personal
computer via a CED interface and subsequently ana-
lysed off-line by the CED Spike 2 software. Upon
completion of the experiment a negative current of
5 mA was applied to the electrode for approx. 5 min to
2 B. Schilstro ¨m et al.
mark the recording site. The rat was then killed with
an overdose of anaesthetic, the brain was removed
and placed in a 10% paraformaldehyde/25% sucrose
solution. VTA sections of 50 mm were cut and stained
with Neutral Red and the recording sites were verified
by light microscopy.
In a first set of experiments the contribution of dif-
ferent nicotinic receptor subtypes to nicotine’s effect
on firing activity of dopamine neurons was studied
with the competitive antagonists MLA (6.0 mg/kg i.p.,
RBI) and DHbE (1.0 mg/kg s.c. RBI, Natick, MA,
USA), respectively. Nicotine (0.5 mg/kg free base s.c.,
Sigma Chemical Co., St. Louis, MO, USA) was given
alone after a control period of 3–5 min in one group
and approx. 10 min after pretreatment with each of
the antagonists in another two groups. The putative
effects of MLA (6.0 mg/kg i.p.) and DHbE (1.0 mg/kg
s.c.) on firing rate and burst firing were studied
for a more sustained period of time (20–30 min) in a
In another set of experiments the effects of the nic-
otinic receptor agonists A-85380 and AR-R17779 on
dopamine cell firing were studied. The agonists were
given in incremental doses of 3–24 mg/kg i.v. for
A-85380 and 125–1000 mg/kg for AR-R17779 with an
interval between doses of 3–5 min. AR-R17779 was
also given in the dose range 250–1000 mg/kg i.v. in
two rats, in the dose range 500–1000 mg/kg in one rat
and as a single dose of 1000 mg/kg i.v in the other.
Presumed dopamine neurons were found 7.5–
8.5 mm from the brain surface and were recognized
by their characteristic triphasic action potential wave-
forms of more than 2.0 ms duration, basal firing rates
of 1–10 Hz and frequent occurrence of burst firing
(Wang, 1981). Cells displaying firing rates below 1 Hz
were not used for experiments.
Dopamine cell firing was analysed with respect to
average firing rate and percentage of action potentials
fired in bursts, calculated over consecutive periods of
500 inter-spike time intervals. Since the time period
during which spikes are analysed will depend on fir-
ing frequency, in cells with low firing frequency
(<2.5 Hz) consecutive periods of 250 inter-spike in-
tervals were used to yield better time resolution.
Quantitative analysis in the first set of experiments
was performed on the firing rate expressed as per cent
of baseline during the last analysed interval immedi-
ately before nicotine injection (baseline 100%, being
defined as the average firing rate during the 3–5 min
before injection) and the average firing rate during
a 5-min interval between 5 and 20 min after nicotine
injection (the 5-min interval displaying maximum
effect on firing rate in each cell was selected). Effects
of nicotine were analysed with paired Student’s t test.
Analysis of burst firing was performed on the amount
of burst firing before nicotine injection and the maxi-
mum burst firing during a 5-min period between 5 and
20 min after nicotine injection (the 5-min interval dis-
playing maximum effect on burst firing in each cell
was selected). Since burst firing rates were not nor-
mally distributed, the non-parametric Wilcoxon’s
Matched-Pairs Signed Ranks test was used for stat-
istical analysis. By selecting the maximum effect on
each parameter for statistical analysis the comparison
was, in a few cases, not made from the same 5-min
interval, however, in this way bias towards one of the
parameters was avoided.
In the other set of experiments, dopamine cell firing
was analysed with respect to average firing rate and
per cent of action potentials fired in bursts, calculated
over consecutive periods of 250 inter-spike time inter-
vals. The effect of each dose of the agonists on firing
rate and burst firing was compared to the effect of an
intravenous saline injection immediately preceding
drug injection. Quantitative analysis was made on
firing rates expressed as a per cent of baseline. In order
to include any putative variability due to the intra-
venous injection in the statistical analysis, baseline
was defined as the quotient between the firing rate
before and after saline administration. The results on
firing rate were analysed statistically with Student’s
t test for paired observations. Quantitative analysis
of burst firing was performed on the amount of burst
firing after saline injection and the amount of burst
firing after injection of the respective agonist. Data
were analysed with Wilcoxon’s Matched Pairs Signed
Male Wistar rats (BKI:WR, BK Universal AB, Sollen-
tuna, Sweden) weighing 250–300 g at the time of
arrival were housed in groups of five under standard
laboratory conditions and maintained under a 12 h
light–dark cycle (lights on at 06:00 hours) with un-
limited access to food and water. Rats were habituated
to handling for 10 min a day for 3 d before experiment.
During this time the rats were also injected daily with
1 ml/kg 0.9% NaCl (saline) solution. The final day
before experiment the rats were housed individually
in Plexiglas cages under the conditions described
above. Food and water were not available during the
Nicotinic effects on dopamine cell firing3
A protocol similar to the one for the first set of
electrophysiological experiments was used for the
immunohistochemical experiments. Thus, the effects
of systemic nicotine on FLI were studied and com-
pared with those achieved after pretreatment with
the antagonists MLA and DHbE, respectively. Each rat
received two injections in one of the following combi-
nations: saline–saline, saline–nicotine, MLA–nicotine,
MLA–saline, DHbE–nicotine and DHbE–saline. The
experiments were performed in two sets with four
groups (n=6 in each group) in each set, studying
each antagonist separately. To reduce any putative
differences between the two sets of experiments the
saline–saline and the saline–nicotine groups were
Two hours after the second injection, rats were
deeply anaesthetized with pentobarbital (120 mg/kg
i.p.) and perfused transcardially with 100 ml saline
solution followed by 300 ml 4% paraformaldehyde
(PFA) in phosphate buffer. Brains were removed and
placed in 4% PFA for 2 h postfixation and then kept
in 15% sucrose in phosphate buffered saline (PBS)
overnight. Coronal sections of 30 mm of selected brain
areas were cut on a freezing microtome. Sections
were washed in PBS (2r15 min) and pretreated with
hydrogen peroxide (10 min). After that, the sections
were washed (PBS, 3r20 min) and then incubated
with the polyclonal primary antibody, sheep-a-FOS
(Cambridge Research Biochemicals, Billingham, UK;
OA-11-824), diluted 1/2000 in PBS containing 0.02%
Na azide and 2% normal rabbit serum, for 2 d at
room temperature. After washing (PBS, 3r20 min),
the sections were processed according to the standard
protocol supplied by ABC Vectastain Elite kit (Vector
Laboratories, Burlingame, CA, USA). FLI was visu-
alized through the Ni-DAB reaction. Sections were
mounted on gelatin-coated slides, dehydrated and
coverslipped for microscopic observation. FLI was
determined in the medial prefrontal cortex and the
NAc shell and core. Cells positive for FLI were
counted from a computer screen in a rectangle of
600r445 mm over the medial prefrontal cortex at
r125 magnification. In the NAc shell and core FLI-
positive cells were counted in an area of 230r165 mm
at r272.5 magnification. Cell counting was computer
aided using Biocom’s Histo software.
The mean number of FLI-positive cells in three sec-
tions (both hemispheres) from each rat was calculated.
Statistical differences between the groups were ana-
lysed with one-way ANOVA. A p value of <0.05 was
considered significant and the data were analysed post
hoc with the Least Significant Difference (LSD) test.
Effects of nicotine on rate and firing mode of
VTA dopamine neurons
In 9 out of 10 cells tested, systemic nicotine increased
both firing rate and the amount of burst firing
(Figure 1c). Average basal firing rate was 4.74¡
0.56 Hz and increased after nicotine administration to
34% above baseline (p<0.01, Figure 1a). Median basal
burst firing was 4.65%. Following nicotine adminis-
tration there was an increase in burst firing. The me-
dian increase in burst firing after nicotine was +30.0%
(p<0.01, Figure 1b). The effect of nicotine was evident
within 2 min after injection and the maximum effects
on firing rate and burst firing were reached within
5–20 min after injection (the maximum effects were
therefore selected within this interval; see above).
Generally, cells which were initially firing with a
predominant single-spike firing mode responded to
nicotine first with an increased firing rate and slowly
changed their firing to the bursting mode, whereas
cells which initially displayed a higher amount of
spontaneous burst firing responded with an increased
burst firing with a faster onset.
Effects of MLA and DHbE on rate and firing mode
of VTA dopamine neurons
MLA did not appear to affect the firing of dopamine
cells compared to baseline firing during the 10 min
before nicotine injection. Some cells were kept for
longer periods to reveal if there were any long-term
effects of MLA (n=4). MLA did not overtly affect fre-
quency or burst firing compared to baseline but in in-
dividual cells there were small changes in firing mode.
Two cells responded with a slight decrease in burst
firing in response to MLA and one cell with a small
increase. DHbE also did not appear to affect firing rate
or firing mode of dopamine neurons during the 10 min
before nicotine injection. In three cells studied for
20 min, one cell did not change firing at all, one cell
slightly increased both firing rate and amount of burst
firing and one slightly decreased both firing rate and
Effects of nicotine on firing rate and firing mode of
VTA dopamine neurons in MLA and DHbE
In rats pretreated with MLA the effect of nicotine was
generally less pronounced than in non-pretreated
4 B. Schilstro ¨m et al.
animals. Nine out of 12 cells increased their firing rate
but only 5 of those increased burst firing (Figure 1c).
Average basal firing rate (after MLA injection) was
4.37¡0.34 Hz and increased after nicotine to 27%
above baseline (p<0.05, Figure 1a). Median basal burst
firing was 9.08% and even if some cells increased their
burst firing the median change in burst firing was only
+0.7% (p>0.05, Figure 1b). In rats pretreated with
DHbE the effect of nicotine was also less pronounced
than in non-pretreated animals. However, in these
animals the effects of nicotine were the opposite of
those in MLA-pretreated animals. Thus, only in 5 out
of 14 cells did nicotine induce an increase in firing rate
whereas burst firing was increased in 12 of the 14 cells
registered. Average basal firing rate (after DHbE
injection) was 4.45¡0.50 Hz. Nicotine did not pro-
duce any significant enhancement of firing rate (18%
above baseline, p>0.05, Figure 1a). Following nicotine
administration there was a statistically significant
increase in burst firing. Median change in burst firing
was +6.9% (p<0.001, Figure 1b).
Effects of subtype selective agonists on rate and
firing mode of VTA dopamine neurons
Intravenous administration of incremental doses of
A-85380 resulted in a general increase in firing rate
without any major change in firing mode (Figure 2),
an effect that was antagonized by DHbE (1.0 mg/kg
s.c., n=2, data not shown). Basal firing rate was
4.62¡0.55 Hz before and 4.46¡0.59 Hz (p=0.091,
n=15) after saline injection. Median basal burst firing
was 16.33% before and 13.15% after saline injection
(p=0.502). The firing rate was already increased at the
Firing rate (% of baseline)
Nic MLA + Nic DH?E + Nic
% of spikes in bursts
% of spikes in bursts
% of spikes in bursts
Figure 1. Effects of nicotine in untreated rats (n=10) and in rats pretreated with MLA (n=12) or DHbE (n=14), respectively on
firing rate and burst firing of presumed dopaminergic neurons in the VTA. (a) Nicotine increased firing rate significantly in the
untreated and in the MLA-pretreated rats, respectively. (b) In contrast, nicotine increased burst firing in the untreated and in
the DHbE-pretreated rats, respectively. Firing rate is expressed as per cent of baseline (¡S.E.M.). Burst firing is expressed as
median per cent change in burst firing; *p<0.05, **p<0.01, ***p<0.001 compared to respective control. (c) Distribution of basal
burst firing rates in the individually recorded neurons compared to the maximal effect of nicotine reached within 20 min of
Nicotinic effects on dopamine cell firing5
lowest dose of A-85380, 3 mg/kg, and increased further
with the administration of higher doses (Figure 2). An
accumulated dose of 24 mg/kg appeared to depolarize
the neurons strongly. One neuron reached a frequency
around 10 Hz after this dose and some neurons went
into a state of apparent depolarization blockade. The
results achieved with this high dose are therefore
not shown. Intravenous administration of incremental
doses of the a7 selective agonist AR-R17779 resulted
in changes in firing mode without changes in average
firing rate (Figure 3). The basal firing rate was 4.72¡
0.46 Hz before and 4.60¡0.44 Hz (p=0.061, n=14)
after saline injection. Median basal burst firing was
8.77% before and 8.97% after saline injection (p=
0.080). Burst firing was already significantly increased
at the lowest dose of AR-R17779 and increased further
with the administration of higher doses.
Effects of nicotine on Fos expression in the medial
prefrontal cortex and the NAc shell and core
Upon analysis no significant differences appeared be-
tween the respective saline–saline and saline–nicotine
were therefore pooled and analysed together. One of
the rats in the saline–saline group was excluded due
to a failure during processing of the sections. Nicotine
induced significant increases in Fos expression in all
areas counted compared to saline (Figures 4a,b, 5).
Neither MLA nor DHbE produced any significant
effects on Fos expression when given with saline
(Figure 5). However, in rats pretreated with MLA,
nicotine’s effect on Fos expression in the medial pre-
frontal cortex and in the NAc shell and core was
blocked (Figures 4c, 5). In rats pretreated with DHbE,
Firing rate (% of baseline)
Dose A-85380 (?g/kg i.v.)
Dose A-85380 (?g/kg i.v.)
Figure 2. Cumulative doses of A-85380 (3–12 mg/kg i.v., n=15 at 3 and 6 mg/kg i.v. doses and n=13 at 12 mg/kg dose) produced
significant increases in firing rate at all doses tested compared to control but no effects on burst firing. Firing rate is expressed
as per cent of baseline (¡S.E.M.). Burst firing is expressed as median per cent change in burst firing; *p<0.05, **p<0.01.
Firing rate (% of baseline)
Dose AR-R17779 (?g/kg i.v.)
Dose AR-R17779 (?g/kg i.v.)
Figure 3. AR-R17779 (125–1000 mg/kg i.v., n=10 at 125 mg/kg, n=12 at 250 mg/kg, n=8 at 500 and 1000 mg/kg doses) did
not increase firing rate at any dose tested but significantly increased burst firing at all doses tested. Firing rate is expressed
as per cent of baseline (¡S.E.M.). Burst firing is expressed as median per cent change in burst firing; **p<0.01, ***p<0.001
compared to baseline controls.
6 B. Schilstro ¨m et al.
nicotine tended to increase Fos expression less effec-
tively (Figure 4d). However, when compared to
the effects of nicotine alone in the medial prefrontal
cortex and the NAc shell and core, there were no
statistically significant differences (Figure 5). Thus,
DHbE was not able to block nicotine-induced Fos
The major findings of the present study are: (i) nic-
otine-induced increases in firing rate and burst firing
in dopamine neurons are differentially affected by
pretreatment with the a7 nicotinic receptor antagonist
MLA and the high-affinity nicotinic receptor antagon-
ist DHbE, respectively, (ii) the a7 nicotinic receptor
agonist AR-R17779 selectively increases burst firing
in dopamine neurons, whereas the nicotinic receptor
agonist A-85380 selectively increases basal firing rate
in dopamine neurons, and (iii) that the systemic nic-
otine-induced Fos expression in dopaminergic target
areas, that previously has been shown to be mediated
via dopamine, acting at D1 receptors, is blocked by
MLA but not DHbE.
The effects of systemically administered nicotine
on mesolimbic dopaminergic function are largely
VTA (Nisell et al., 1994; Schilstro ¨m et al., 1998a). Thus,
although all pharmacological agents in this study
were given systemically, the mechanisms of the
effects observed are likely to be found within the
VTA. In our previous studies we observed that an-
tagonism of NMDA receptors in the VTA reduces
both the nicotine-induced dopamine release and the
concomitantly increased Fos expression in the NAc
(Schilstro ¨m et al., 1998a, 2000b). It was subsequently
found that nicotine increases the extracellular con-
centrations of glutamate and aspartate in the VTA
(Schilstro ¨m et al., 2000a), an effect that was blocked by
intrategmental administration of MLA. Moreover,
intrategmental administration of MLA also blocked
nicotine-induced dopamine release in the NAc (Schil-
stro ¨m et al., 1998b). Previously, we have provided
evidence that there exist presynaptic a7* nicotinic re-
ceptors on afferents in the VTA originating in the
prefrontal cortex (Schilstro ¨m et al., 2000a). Altogether,
these data suggest a mechanism where nicotine,
acting at presynaptic a7* nicotinic receptors, may fa-
cilitate the release of EAAs which in turn, through
NMDA receptor activation, stimulates the dopamine
neurons to fire in bursts. Part of this mechanism was
confirmed by Mansvelder and McGehee (2000) who
through receptor mechanismsin the
Figure 4. Representative examples of staining for FLI in coronal sections containing the NAc shell and core at r125
magnification. (a) Fos expression induced by a nicotine injection in a saline-pretreated animal. (b) Fos expression following
two control saline injections. (c) Effects of nicotine on Fos expression in a MLA-pretreated animal. (d) Effects of nicotine in
a DHbE pretreated animal.
Nicotinic effects on dopamine cell firing7
showed that nicotine induces long-term potentiation
of glutamatergic synapses on dopaminergic neurons
via an a7 nicotinic receptor-mediated presynaptic en-
hancement of glutamate release.
Since the effects seen in the present experiments
must essentially reflect pharmacological mechanisms
in the VTA (cf. above), our results strongly support the
hypothesis tested. Accordingly, the nicotine-induced
burst firing appears to be due largely to activation of
a7 nicotinic receptors whereas nicotine’s effect on fir-
ing rate seems largely mediated by non-a7 nicotinic
receptors. The pharmacological tools used in the pres-
ent study were selected to distinguish between a7
and non-a7 nicotinic receptors. Thus, although no firm
conclusions can be drawn as to which receptor sub-
type is controlling the firing rate, previous studies
have shown that the b2 subunit is crucial since nic-
otine is not able to increase the firing rate of dopamine
neurons in b2 knockout mice (Picciotto et al., 1998). In
thea4knockout mousenicotine-mediated currentscan
still be detected although the amplitude and duration
of these currents is smaller than that observed in wild-
type mice (Klink et al., 2001). Moreover, in the same
study combining electrophysiology with single-cell
PCR it was suggested that heteromeric nicotinic recep-
tors expressed by VTA dopamine neurons may either
be a4a5a6(b2)2or a4a5*(b2)2.Functional a7* receptors
have also been shown on dopamine neurons in the
VTA (Pidoplichko et al., 1997), but these do not appear
to be involved in the nicotine-induced increase in their
basal firing rate in vitro (Grillner and Svensson, 2000).
Based on our own and previous findings we propose
that the nicotinic receptor responsible for the nicotine-
induced increase in firing rate may contain a4 sub-
units and b2 subunits but probably not a7 subunits.
The conclusion that different nicotinic receptor
subtypes modulate burst firing and single-spike firing
of VTA dopamine neurons, respectively, derives fur-
ther support from our experimental analysis of the
post-synaptic consequences of these different nicotinic
receptor modulations. It has previously been shown
that burst stimulation of the median forebrain bundle
increases Fos expression in the NAc and other ter-
minal areas of the mesolimbic dopamine system,
whereas regular stimulation with the same number
of pulses does not (cf. Introduction). Accordingly,
we here observed that selective blockade of a7* nic-
otinic receptors with MLA, which effectively reduced
nicotine-induced burst firing, also antagonized the
nicotine-induced Fos expression in the NAc and
the medial prefrontal cortex, whereas blockade of
high-affinity nicotinic receptors with DHbE did not.
Previously, the nicotine-induced Fos expression in
dopamine target neurons as well as the Fos ex-
pression induced by burst stimulation of the median
forebrain bundle has been demonstrated to be due to
activation of dopamine D1 receptors in dopamine tar-
get areas (Chergui et al., 1996; Kiba and Jayaraman,
1994). It has been hypothesized that during burst firing
the high levels of dopamine released saturate the
dopamine reuptake carrier (Chergui et al., 1994) and
that there is an outflow of dopamine into extrasynaptic
sites, where the dopamine D1 receptors appear to be
localized (Caille ´ et al., 1996; Gonon, 1997). Our present
results thus suggest that under otherwise normal
conditions, stimulation of a4b2* nicotinic receptors,
which gives rise to a selective increase in firing rate
may not increase extrasynaptic dopamine to levels
high enough to stimulate dopamine D1 receptors and,
subsequently, induce Fos expression in post-synaptic
neurons. In contrast, stimulation of a7* nicotinic re-
ceptors, which selectively induces burst firing also in-
creased Fos expression. Fos is a transcription factor
that is hypothesized to generally serve as an initial
trigger and regulatory factor for long-term changes in
the expression of a number of genes in post-synaptic
cells. In the behaving monkey, the firing pattern of
midbrain dopamine cells appears to shift from single-
spike firing to burst firing in association with certain
environmental stimuli of attentional and motivational
significance, sending a ‘teaching signal’ to the brain
(see Schultz, 1998). In other words, Fos expression
Figure 5. Effects of nicotine and saline on the number of cells
expressing FLI in rats pretreated with saline, MLA or DHbE
in the medial prefrontal cortex (mPFC) on the left y-axis
and in the nucleus accumbens shell (NAcS) and core (NAcC),
respectively, on the right y-axis. Data are presented as mean
¡(S.E.M.) number of cells counted in both hemispheres
of three sections. Statistical analysis revealed significant
differences between the Sal–Sal vs. Sal–Nic and the Sal–Nic
vs. MLA–Nic groups; *p<0.05, **p<0.01.
8 B. Schilstro ¨m et al.
induced by burst activity in dopamine neurons may
normally serve to induce long-term plasticity in the
post-synaptic areas. Indeed, it was recently shown,
that long-term potentiation in prefrontal cortical
neurons induced by stimulation of the hippocampus is
enhanced by electrical stimulation of the VTA (Gurden
et al., 1999) with a frequency which was previously
shown to evoke dopamine overflow in the prefrontal
to improve cognitive functions, such as working
memory, although the mechanism is not entirely
understood (see Levin and Simon, 1998). Moreover,
working memory seems to be controlled by dopamine
D1 receptors in the prefrontal cortex (see Goldman-
Rakic, 1998; Sawaguchi and Goldman-Rakic, 1991).
Since burst firing of dopamine neurons under physio-
logical conditions seems to be required to stimulate
dopamine D1 receptors, it follows that the cognitive-
enhancing effects of nicotine may be partly related to
its ability to increase burst firing of VTA dopamine
neurons via a7* nicotinic receptor stimulation. In
fact, AR-R17779 has been shown to improve working-
memory performance in rats (Levin et al., 1999).
Clinical studies over the last decade have shown that
cognitive impairment, e.g. working-memory deficits,
represents a major determinant of the outcome of
treatment of schizophrenia (see Green, 1996; Velligan
and Miller, 1999). Moreover, several studies indicate
that some symptoms in schizophrenia, such as de-
ficient sensory (auditory) gating, may be related to
a deficit in central a7* nicotinic receptor expression, a
deficit that, in fact, seems to involve substantial parts
of the forebrain (Guan et al., 1999; see Leonard et al.,
1996). Consequently, a7 receptor agonists may possess
significant therapeutic potential in schizophrenia.
In conclusion, the present study demonstrates that
dopaminergic neuronal activity stimulated by nicotine
is due to activation of more than one nicotinic receptor
subtype. The a7*nicotinic receptor seems tobe respon-
sible for the nicotine-induced burst firing, whereas
another nicotinic receptor subtype, tentatively in the
a4b2* combination, may be involved with the nicotine-
induced increase in basal firing rate. The specific effect
of a7* receptor stimulation on the firing mode of dopa-
mine neurons also appears to have a significant impact
on the plasticity of their target neurons. Thus, long-
lasting adaptive changes in dopaminoceptive neurons
induced by nicotine may require a7* nicotinic receptor
stimulation and, accordingly, these receptors may be
implicated in the development, but not necessarily the
maintenance, of nicotine dependence. In general, by
enhancing the dynamic responsivity of mesocortico-
limbic dopamine neurons to natural attentional and
motivational environmental stimuli, selective a7 re-
ceptor agonists may help to reduce anhedonia and
The Swedish Medical Research Council (grant no.
4747), The Karolinska Institutet and a Pharmacia and
Upjohn Award supported this study. The excellent
technical assistance of Ms Anna Malmerfelt and Mrs
Ann-Chatrine Samuelsson is gratefully acknowledged.
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