Title:
The effect of nicotine induced behavioral sensitization on dopamine D1 receptor
pharmacology: An in vivo and ex vivo study in the rat
Authors:
W. Goutier1,3, J.J. O’Connor2,3, J.P. Lowry3, A.C. McCreary1,*
Author Affiliations:
1
Abbott Healthcare Products B.V., C.J. van Houtenlaan 36, 1381 CP Weesp, the
Netherlands (formerly Solvay Pharmaceuticals B.V.),
2
UCD School of Biomolecular and
Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, Belfield
Dublin 4, Ireland, and
3
Department of Chemistry, National University of Ireland Maynooth,
Maynooth, Co. Kildare, Ireland.
*Corresponding Author:
Correspondence should be addressed to Dr. Andrew C. McCreary, present address: Brains
On-Line, P.O. Box 4030, 9701 EA, Groningen, the Netherlands. Tel.: +31(0)503171440 Fax.:
+31 (0)50-317-1449 E-mail address: andymccreary@yahoo.co.uk (A.C. McCreary).
1
Abstract
Behavioral sensitization is a phenomenon which can develop following repeated intermittent
administration of a range of psychostimulants, and other compounds, and may model
neuroplastic changes seen in addictive processes and neuropsychiatric disease. The aim of
the present study was to investigate the effect of dopamine D1 ligands on nicotine-induced
behavioral sensitization and their molecular consequences in the striatum. Wistar rats were
chronically (5 days) treated with vehicle or nicotine (0.4mg/kg;s.c.) and locomotor activity
was measured. Following a 5 day withdrawal period, rats were pretreated with vehicle or the
D1 antagonist SCH-23390 (0.03mg/kg;i.p.) and challenged with nicotine. Either 45 min or 24
h post-challenge, the striatum was isolated and ex vivo receptor binding and cAMP
accumulation (using LC-MS/MS) were assessed. Chronic nicotine administration induced the
development and expression of locomotor sensitization, of which the latter was blocked by
SCH-23390. Nicotine-induced sensitization had no effect on forskolin stimulated cAMP
accumulation but increased the efficacy of dopamine for the D1 receptor and decreased the
potency of D1 agonists. These effects were antagonized by in vivo pre-challenge with SCH23390. No effect on D1 receptor binding was observed. Moreover, time dependent effects
were observed between tissue taken 45 min and 24 h post-challenge. The present findings
provide a connection between behavioral sensitization and intracellular cAMP accumulation
through the dopamine D1 receptor. Together these data suggest that changes in D1 signaling
in the dorsal striatum may play an important role in the underlying mechanisms of nicotineinduced behavioral sensitization.
Keywords:
Dopamine D1; Nicotine; Behavioral Sensitization; cAMP; SCH-23390; Receptor Binding; Rat
2
1. Introduction
Nicotine can produce a wide array of behavioral effects, and while similarities have
been shown between nicotine and a number of psychostimulants nicotine possesses a distict
mechanism of action compared with psychostimulants. For example, cocaine and
amphetamine act directly on dopaminergic neurotransmission by blocking uptake and/or
stimulating dopamine release, by contrast nicotine binds to nicotinic acetylcholine receptors
(nAChRs) and indirectly affects dopaminergic neurons (see review by Mash and Staley,
1997).
Dopamine (DA) is an important neurotransmitter and mediator that controls many
aspects of cognitive, emotional, motor and endocrine function and has reward and
reinforcing properties. Alteration of dopaminergic neurotransmission in the mesolimbic,
mesocortical and nigrostriatal pathways has been suggested to contribute to the
pathogenesis of many neuropsychiatric disorders, e.g. schizophrenia, tardive dyskinesia,
psychoses, Parkinson’s disease, and substance misuse (Marsden, 2006).
Many behavioral effects of nicotine in animals and man are already widely understood.
Altering dopaminergic neurotransmission can result in different behavioral effects depending
on the administration regimen. Repeated intermittent administration of a wide range of
psychostimulants or other compounds can produce behavioral sensitization, a gradual
increase in drug induced locomotor activity or other behaviors (e.g. conditioned place
preferences). The first reports describing behavioral sensitization were those of Tatum and
colleagues (1929) and Downs and Eddy (1932). Behavioral sensitization in animals may
share common mechanisms with the psychotic state in humans, models stimulant induced
psychoses (Segal and Schuckit, 1983; Post et al., 1984; Robinson and Becker, 1986), and
has been extensively linked to drug addiction (Robinson and Berridge, 1993).
Dopamine acts on different central dopamine receptor subtypes, which can be divided
into five subtypes, D1 to D5 (Kebabian and Calne, 1979; Neve and Neve, 1997) that cluster
3
into two families: the D1 and D5 and D2 to D4 (Garau et al., 1978; Monsma et al., 1990;
Bunzow et al., 1998). These receptors are found, predominantly, in the caudate–putamen,
nucleus accumbens, olfactory tubercle, cortex and hippocampus of the brain (Angulo et al.,
1991; Fremeau et al., 1991). From a molecular perspective, dopamine D1 receptors are Gprotein coupled receptors (GPCRs) and activate intracellular adenylate cyclase and cyclic
adenosine monophosphate or cAMP (Kebabian and Calne, 1979), which play a major role in
cell signaling and is involved in the mechanisms underlying psychostimulant induced
behavioral sensitization (Schroeder et al., 2004).
In spite of many previous studies, nicotine induced behavioral sensitization is still not
well understood and the role of the dopamine D1 receptor has not been investigated.
Therefore, the present study investigated the effect of the dopamine D1 antagonist SCH23390 on the expression of nicotine induced sensitization. We hypothesize that a dopamine
D1-like antagonist such as SCH-23390 will inhibit adenylate cyclase activity and
subsequently attenuate the expression of nicotine induced sensitization. In the present study
we used a multidisciplinary approach and extended behavioral studies with the addition of ex
vivo cAMP accumulation experiments to investigate the effect of sensitization and pretreatment with prototypical dopamine D1 receptor ligands at a cellular level in the caudateputamen of the rat.
4
2. Experimental procedures
2.1.
Drugs
(-)-Nicotine ((S)-(-)-1-Methyl-2-(3-pyridyl)pyrrolidine dihydrate (+)-ditartrate salt) and saline
were purchased from Acros Organics (Belgium). Dopamine, SCH-23390 ((R)-(+)-7-Chloro-8hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride), SKF-82958
((±)-6-Chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
hydrobromide),
SKF-38393
((±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol
hydrochloride), (+)-butaclamol, forskolin (colforsin), LY-741626,
adenosine-cyclic-3’,5’-
monophosphate, and adenosine-triphosphate were purchased from Sigma Aldrich (the
Netherlands). 1-Methyl-3-isobutylxanthine was purchased from Calbiochem (Canada). For
the in vivo experiments, (-)-Nicotine dihydrate ditartrate (1.228 mg/mL) was dissolved in 0.9%
sterile saline and adjusted to pH 5.0 with NaOH/HCl. The final dose for administration was
0.4 mg/kg (-)-nicotine (measured as the freebase). (-)-Nicotine and vehicle were
administered subcutaneously (s.c.) in volumes of 1 mL/kg body weight. SCH-23390 was
dissolved in saline and adjusted to pH 6.6 with NaOH/HCl. SCH-23390 and corresponding
vehicle were given by intraperitoneal (i.p.) administration in volumes of 2 mL/kg. For the ex
vivo experiments, standard solutions (0.1 mM) were made by dissolving the compounds in
HPLC grade water (dopamine) or dimethylsulfoxide (all other tested compounds) prior to
each experiment. Subsequent serial dilutions in Krebs-Ringer buffer were performed to
obtain the desired test concentration, as stated in the Results.
2.2.
Subjects
Male Wistar rats (HsdCpb:WU, Harlan, Horst, the Netherlands) weighing between 250-270 g
upon delivery were used. Animals were housed 2 rats/cage in a temperature (21±1°C) and
humidity (40-50%) controlled environment, and were habituated for 1 week prior to
experimentation. The rats had ad libitum access to food (RM1 (E) pellets, SDS Special Diet
5
Service, England) and tap water, except during experimental sessions. Lighting was
maintained under a 12 h light-dark cycle (lights on 06:00-18:00 h). All experimental care and
procedures were performed between 08:00 and 16:00 h and complied with the National
Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by
the local Institutional Animal Care and Use Committee (“Animal Ethical Committee” of Abbott
Healthcare Products B.V., number 0207-025) and were in accordance with all local laws.
2.3.
Locomotor activity measurements
Animals were matched for body weight and randomly assigned to treatment groups.
Locomotor activity measurements were performed using 24 transparent Plexiglas cages
(21x36x18 cm) and were placed between 7 horizontal photobeams. Photobeam-breaks were
recorded as ambulatory movement (locomotor activity) using a commercially available
activity system (Photobeam Activity System, San Diego Instruments, USA), in 5 min time
epochs and quantified using “Photobeam Activity System” software.
2.4.
Experimental Design (in vivo)
All animals underwent the following protocol: One day prior to each experiment (day 0) all
rats were randomly assigned to five experimental groups and habituated to the test
environment for 60 min followed by a saline injection and 60 min locomotor activity recording
to familiarize the animals to handling and testing stress. There was no significant treatment
effect following vehicle administration ([F(2,20)=1.175; P=0.3293]) and was representative
for all habituation data recorded during these studies.
On the first test day (day 1) all rats of the five experimental groups were habituated to
the test apparatus for 60 min, before administration of either vehicle (s.c., 2 groups) or
nicotine (0.4 mg/kg; s.c., 3 groups). The rats were directly placed back in the test apparatus
and locomotor activity was measured for a further 45 min. This first time dose of nicotine was
considered the acute dose. The administration procedure was repeated on days 2, 3 and 4
but animals were returned to their home cages directly. On day 5, the procedure was
6
repeated and locomotor activity measurements were performed for 45 min, as described
above. See Fig 1. for the experimental design.
Following a protracted withdrawal period of 4 days (day 9), conditioning effects to the
testing apparatus were assessed by a challenge with saline and additionally served as an
acclimatization phase. On the following day (day 10), in order to test the effects of SCH23390 on the expression of nicotine induced sensitization, rats were placed in the test
apparatus and 30 min later received vehicle or SCH-23390 (0.03 mg/kg; i.p.). The rats were
returned to the test apparatus and locomotor activity was measured for 30 min. After 30 min
the rats received a vehicle (s.c., 1 vehicle group) or nicotine challenge (0.4 mg/kg; s.c., other
4 groups) and locomotor activity was measured for another 45 min. See Fig. 1. for the
experimental design.
2.5
Tissue preparation (receptor binding)
The rats were decapitated at 24 h after the last in vivo challenge, without anesthetic, and the
dorsal striatum of both hemispheres were dissected (according to Paxinos and Watson, 1998;
see Fig. 2) as quickly as possible and directly frozen on dry-ice. The samples were stored at
-80°C until membrane preparation. Frozen rat striatal samples were weight and
homogenized in ice-cold Tris buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA) using GentleMax
tissue homogenizer (Miltenyi Biotec, Germany). The membranes were precipitated by
centrifugation at 10,000xg for 10 min at 4°C and washed by re-homogenization using the
GentleMax. This was followed by centrifugation at 50,000xg for 10 min at 4°C. The final
pellet was re-homogenized in incubation buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA,
containing 120 mM NaCl, 5 mM KCl, with or without 1.5 mM CaCl2 and 4 mM MgCl2,
purchased from Sigma Aldrich) to obtain the required membrane concentration. The tissue
was kept at room temperature for 30 min before starting the incubation.
2.6
Receptor binding protocol (ex vivo)
7
The whole assay was performed using MultiScreen®HTS 96-well filterplates (1.0 μm Glass
Fiber Type B Filter, Millipore, USA) using only a small volume (i.e. 200 μL format) and
required less tissue and reagents whilst still showing high specific binding (i.e. >70% in this
study). [3H]-SCH-23390 (N-methyl-3H, spec.act. 2.7047 TBq/mmol) was purchased from
Perkin Elmer (USA) and the actual radioligand concentration was determined. Compounds
were diluted in incubation buffer and were tested in triplicate. To each well was added 20 μL
test compound, 20 μL [3H]-SCH-23390 (0.8 nM), and the incubation was started upon
addition of 160 μL tissue homogenate (5 mg/mL striatal membranes). Non-specific binding
was defined by the binding in the presence of 1 μM (+)-butaclamol. The plates were shaken
using a well-plate shaker (TeleShake-Variomag, Thermo, USA) and incubated for 2 h in a
well-plate incubator at 25ºC. The incubations were terminated by rapid filtration through the
filterplates using a vacuum unit (Millipore), followed by washing the filterplates rapidly three
times with 100 μL ice-cold 50 mM Tris-HCl/1 mM EDTA buffer. After air drying the filterplates,
each well received 35 μL scintillation cocktail (MicroScint-OTM, Perkin-Elmer) and the
filterplates were sealed. After 24 h equilibration, radioactivity was determined using a beta
scintillation counter (MicroBeta, Perkin-Elmer).
2.7
Tissue preparation (cAMP assay)
45 min or 24 h following the last nicotine challenge, the rats were decapitated using a
guillotine, without anesthetics, and the dorsal striatum of both hemispheres were dissected
as a whole (according to Paxinos and Watson, 1998; see Fig. 2) as quickly as possible.
Directly after preparation, the tissue was kept in ice-cold Krebs-Ringer Buffer for maximal 30
min. 3 mM K+ Krebs-Ringer buffer (KRB, Gibco-Invitrogen, the Netherlands) contained (in
mM): 121 NaCl, 2 KCl, 25 NaHCO3, 1.2 MgSO4, 11 D-(+)-glucose, 1.2 KH2PO4, with
modifications as used during the whole experiment: 1.2 CaCl2 and 22 D(+)-glucose. The
buffers were freshly prepared on each test day, kept at 37°C and gassed with 5% CO2/95%
O2 for at least 30 min before use.
8
The dissected brain areas from animals within the same treatment group were pooled
(i.e. from 4-6 rats at a time) and sliced together. The freshly prepared tissue was then sliced
in two directions (coronal, 90° apart) using a McIlwain tissue chopper (300 μm setting), and
subsequently washed with three 5 mL aliquots of ice-cold oxygenated KRB and allowed to
settle. Then the slices were washed 3x times with warm (37°C) oxygenated KRB followed by
60 min incubation in a 37°C water bath with a 5% CO2/95% O2 atmosphere.
Variation within the in vivo treated groups made it impossible to use tissue from only a
single rat for an n=1 ex vivo experiment. To overcome this problem the dissected brain areas
from at least four animals within the same treatment group were pooled. This significantly
reduced intra-experimental group variation. For illustration, an ‘n=1 experiment’ comprised all
concentrations of the test compound conducted in quadruplo within 1 individual ex vivo
experiment using tissue from 4-8 rats treated within 1 individual in vivo experiment.
2.8.
cAMP accumulation assay (ex vivo)
Adenylate cyclase activity in the striatal slices was monitored by the measurement of
intracellular cAMP and ATP levels as previous described for in vitro cell cultures (Goutier et
al., 2010) with some modifications. Briefly, the striatal tissue (both hemispheres) of 4 animals
from the same treatment group was dissected and pooled. The pooled striatal tissue was
sliced together and re-suspended in 10 mL KRB, washed 3 times by precipitation, and
divided over two 96-well filterplates using large orifice pipette tips. The buffer was removed
using vacuum-filtration (Millipore) and the slices were washed with 100 μL of assay-buffer (i.e.
KRB with 1 μM Forskolin and 1 mM IBMX). Following removal of the assay-buffer, 100 μL of
the diluted test compounds were added and the filterplates were incubated in a water bath
(37°C) with a 5% CO2/95% O2 atmosphere. After 30 min. the incubation was stopped by
vacuum-filtration, the slices were quickly washed with 100 μL HPLC-grade water and were
lysed by the addition of 100 μL ice-cold lysis-buffer (50:50 HPLC-grade water and
acetonitrile). After 30 min. of incubation at 4°C the plates were vacuum-filtered collecting the
9
fractions in normal 96-well plates, and heat-sealed (Thermosealer, Abgene) to prevent
evaporation of the solvent.
2.9.
Sample analysis of cAMP and ATP using LCMS
The samples were analyzed using LC-MS/MS as described in Goutier et al. (2010), with the
following modifications: The nucleotides cAMP and ATP were analyzed using a triplequadrupole Premier XE mass-spectrometer (Waters, Breda, the Netherlands) in the positive
electrospray ionization mode (ESI-MS/MS). A single ion monitoring (SIM) chromatograph
was used to only detect the selected mass in the analysis: cAMP m/z 330>136, and ATP m/z
508>410.
2.10. Data analyses
The in vivo behavioral data were analyzed and are presented as mean total ambulant
movements (beam breaks) over a period of 30 min (pre-treatment) or 45 min (challenge)
after injection ± standard error of the mean (s.e.m.). Data were taken from all animals
regardless of their behavioral response. The main effects of drug treatments were analyzed
using one-way analysis of variance (ANOVA). Bonferroni’s post hoc multiple comparison test
was used where appropriate, but only following significant ANOVA. Graphs and statistical
analyses were performed using Prism graphing software (GraphPad, USA). Differences
between day 1 vs. day 5 were analyzed using two-way ANOVA with a within-subjects factor
of day and between-subjects factor of treatment. All data with P<0.05 were considered
statistically significant.
Regarding the cAMP accumulation assay, each filterplate contained two controls, i.e.
basal and forskolin (1 µM), tested in quadruplo. A single experiment comprised up to twenty
96-well plates and at least 1 plate contained a full concentration-response curve of SKF38393 as the reference compound (positive control). In order to examine the
pharmacological activity of the test compounds, each concentration of the test compound
was tested in quadruplo (i.e. 4 wells within the same 96-well plate). At the end of each
10
experiment, a mean was calculated from the quadruplo values and outliers (>2x the standard
deviation, s.d.) were removed. The calculated mean from this individual experiment was
considered as n=1. For illustration, if stated n=3 this value is taken from 3 means of 3
individual ex vivo experiments after 3 individual in vivo experiments.
Data from the ex vivo experiments represents mean±s.e.m. of at least three
independent experiments and were conducted in quadruplicate. For each tissue sample, the
cAMP concentration was corrected for the ATP concentration by dividing cAMP levels by
total amount of nucleotides: cAMP/(cAMP+ATP)*100. These ratios were then expressed as a
percentage of their respective control. Graphs, pEC50 and Emax values were calculated using
Prism graphing software (GraphPad, USA) and concentration response curves were fitted
using non-linear regression (i.e. Hill three-parameter sigmoidal equation). The maximal
forskolin induced stimulated conversion was taken as the maximum value and the maximal
inhibition (at 10 µM of the reference compound) as the minimum and these values were fixed
during the fitting process.
11
3. Results
3.1
Effect of SCH-23390 on the expression of nicotine induced sensitization
Prior to each injection, a habituation period was reserved to achieve stable basal
locomotor activity (Fig. 3A). On day 1, acute nicotine administration (0.4 mg/kg; s.c.)
increased locomotor activity in naïve rats and returned back to basal after ca. 45 min.
Administration of nicotine significantly increased locomotor activity in naïve rats (one-way
ANOVA, [F(3,27)=15.47; P<0.0001]). On day 5, following chronic intermittent nicotine
administration, a subsequent nicotine challenge resulted in an increased response
([F(3,27)=55.75; P<0.0001], Fig. 3B). When comparing day 1 and day 5, statistical analysis
using a two-way ANOVA showed a significant effect of time ([F(1,54)=24.25; P< 0.0001]), for
drug ([F(3,54)=64.13; P<0.0001]) and for the interaction between the two factors
([F(3,54)=5.505; P=0.0023], Fig. 3C), suggesting the animals developed sensitization to
nicotine.
Following a withdrawal period of 4 days (i.e. day 9), rats were given a vehicle
challenge. While a weak significant treatment effect was observed ([F(3,28)=5.459;
P=0.0044], data not shown), post hoc analysis showed no significant effect vs. vehicle
suggesting that the observed nicotine induced sensitization is drug induced rather than
context-dependent sensitization. The following day (day 10), the effect of SCH-23390 on
nicotine induced sensitization was examined. Three groups of animals were pre-treated with
vehicle (s.c.) and one nicotine sensitized group was pre-treated with SCH-23390 (0.03 mg/kg;
i.p.). The acute administration of SCH-23390 in nicotine sensitized rats did not show any
significant treatment effect; [F(3,28)=1.527; P=0.2293] (Fig. 4A and B), which suggests that
SCH-23390 did not cross-sensitize with nicotine, and that it was devoid of effect on
locomotor activity when tested alone. Subsequently, rats received a vehicle (s.c.) or nicotine
challenge (0.4 mg/kg; s.c.) and a significant treatment effect was observed ([F(3,28)=50.23;
P<0.0001], Fig. 4C). Bonferroni’s post hoc analysis showed that the group receiving nicotine
12
for the first time (i.e. nicotine control-group) showed enhanced locomotor activity (P<0.001)
and that the animals which received nicotine days 1-5 showed an enhanced or sensitized
response (P<0.001, compared to vehicle). Furthermore, it was shown that SCH-23390 (0.03
mg/kg) pre-treatment blocked the expression nicotine induced sensitization (P<0.01,
compared to chronic nicotine, Fig. 4D).
3.2
Effect of chronic nicotine induced behavioral sensitization on ex vivo receptor
binding
To investigate putative changes in receptor binding affinity at the dopamine D1
receptor following chronic nicotine treatment, the affinities of a selection of dopaminergic
ligands was tested in competition experiments against [3H]-SCH-23390. The endogenous,
and non-selective agonist dopamine alone, the selective dopamine D1 receptor agonists
SKF-38393 and SKF-82958, and the selective dopamine D1 receptor antagonist SCH-23390,
concentration dependently antagonized the [3H]-SCH-23390 binding (Fig. 5A to D,
respectively). There was no effect observed of chronic nicotine treatment on the potency or
efficacy of these ligands for dopamine D1 receptor binding. In each case a two-site model
was considered, however, a single binding site equation showed a significantly better fit
according to the F-test (P<0.05).
3.3
Effect of forskolin and dopamine on cAMP accumulation following nicotine
induced sensitization
Forskolin was tested in a concentration range from 1 nM to 0.1 mM to determine its
potency on cAMP accumulation. Forskolin significantly increased cAMP formation in a
concentration dependent manner (Fig. 6A). Following prior in vivo treatment, the results
showed no change in potency following nicotine sensitization compared to vehicle or
sensitization and pre-challenge with SCH-23390. Moreover, prior in vivo treatment had no
effect on basal cAMP levels ([F(3,19)=1.408; P=0.2712], data not shown).
13
Dopamine was tested in a concentration range up to 0.1 mM and showed a small but
concentration dependent increase in cAMP accumulation in striatal slices. Dopamine
produced a maximum stimulation of cAMP production of 167±28% (at 0.1 mM) over basal
levels in the striatum of vehicle treated animals (Fig. 6B). Specific antagonism of the
dopamine D2 receptor subtype by co-incubation of dopamine and LY-741626, increased the
potency of dopamine: dopamine pEC50 4.4±0.2 vs. dopamine + LY-741626 co-incubated
pEC50 5.6±0.4, Fig. 6C). In contrast to the results observed with the dopamine D2 antagonist,
co-incubation of dopamine with the dopamine D1 antagonist SCH-23390 completely
abolished the effect of dopamine and as a result prevented stimulation of cAMP
accumulation (pEC50<5.0, Fig. 6D). This is in agreement with the in vivo behavioral
sensitization observations which are also blocked by SCH-23390 (present data).
In addition, using tissue of animals sensitized to nicotine, it was found that behavioral
sensitization did not affect dopamine stimulated cAMP accumulation alone (Fig. 6B).
However, the efficacy (Emax) of dopamine was significantly increased following sensitization
when tested in the presence of LY-741626 (Emax vehicle 162% vs. chronic nicotine 213%, Fig.
6C) but not SCH-23390 (pEC50<5.0, Fig. 6D). Furthermore, the in vivo pre-challenge with
SCH-23390 prior to the nicotine challenge, strongly reduced the efficacy (Emax) of dopamine
(Fig. 6B) and dopamine in the presence of LY-741626 (Fig. 6C). It had no effect on
dopamine in the presence of SCH-23390 (pEC50<5.0, Fig. 6D).
To investigate the temporal effect (time-response) of the post-challenge period before
dissection of the brain tissue, the striatum of animals from the same treatment group were
dissected 24 h after the nicotine challenge. Forskolin stimulated cAMP accumulation in a
concentration dependent manner, comparable to the results seen with tissue taken 45 min
post-challenge and no difference in time-response effect was observed for either treatment
group (Table 2). In contrast to the results observed after 45 min post-challenge, no effect of
dopamine on cAMP accumulation was observed (pEC50<5.0 for vehicle and nicotine
sensitized groups, Table 2).
14
Furthermore, the effect of nicotine on cAMP accumulation was tested ex vivo on the
striatum of non-treated, vehicle and nicotine sensitized animals in a concentration range from
0.01 to 10 µM. Nicotine did not affected cAMP accumulation, pEC50 values <5.0 (n=3, data
not shown).
3.4
Effect of dopamine D1 ligands on cAMP accumulation following nicotine
induced sensitization
The dopamine D1 agonists SKF-38393 and SKF-82958 showed a concentration
dependent increase in cAMP accumulation using striatal slices from vehicle treated animals
(Fig. 7A and B, respectively). When SKF-82958 was tested at higher concentrations (i.e. 10100 µM) the simultaneously measured ATP levels were strongly decreased suggesting
cytotoxicity. The effect of the dopamine D1 agonists was also tested in tissue from sensitized
animals (tissue taken at 45 min post-challenge). The potency of both dopamine D1 agonists
was reduced after acute and chronic nicotine administration (Fig. 7A and B), i.e. SKF-38393
showed a shift in potency of cAMP accumulation in vehicle (pEC50 7.2±0.2) versus acute
(pEC50 6.7±0.2) and versus chronic (pEC50 6.0±0.2) nicotine treated animals. SKF-82958
showed a shift in potency; vehicle (pEC50 7.7±0.2), acute (pEC50 7.3±0.4) and chronic
nicotine (pEC50 6.2±0.2).
Furthermore, the in vivo pre-challenge with SCH-23390 increased the potency of
SKF-38393 on cAMP accumulation and completely reversed or antagonized the effect of the
nicotine sensitization on cAMP accumulation (Fig. 7A), on tissue taken 45 min post-challenge.
The same effect was observed with SKF-82958, although, demonstrating a smaller
magnitude and lower maximal effect (Fig. 7B). Incubation with SCH-23390 ex vivo had no
effect on cAMP accumulation regardless of the prior in vivo treatment (Fig. 7C) and
antagonized the effect of SKF-38393 (1 µM, Fig. 7D). When the striatum was dissected 24 h
post-challenge, both dopamine D1 agonists produced a concentration dependent stimulation
of cAMP accumulation similar to those observed 45 min post-challenge (Table 3.). In contrast
to results obtained with tissue taken at 45 min post-challenge, nicotine induced behavioral
15
sensitization and in vivo pre-treatment with SCH-23390 did not affect the potency or efficacy
of SKF-38393 on cAMP accumulation when tissue was taken 24 h post-challenge (Table 3.).
Nicotine sensitization attenuated the potency of SKF-82958 and its maximal effect; vehicle,
202%; acute nicotine, 165%; nicotine sensitized, 179% (Table 3.). In agreement with the
results obtained using tissue taken at 45 min post-challenge, SCH-23390 alone and in the
presence of SKF-38393 (1 µM) were not affected by behavioral sensitization. Furthermore,
the presence of 10 µM nicotine did not affect the potency or efficacy of SKF-38393 in rat
striatal slices of non-treated animals (data not shown).
16
4. Discussion
The present study investigated the effect of SCH-23390 on the expression of nicotine
induced behavioral sensitization and the consequences of sensitization on cAMP
accumulation (ex vivo). This study showed, for the first time, that in vivo pre-treatment with
SCH-23390 blocked the expression of nicotine induced behavioral sensitization which was
directly related to dopamine D1 stimulated cAMP accumulation.
Previous studies have shown an important role of dopamine in behavioral
sensitization (Ujike et al., 1989) and a more specifically, in the context of this manuscript, a
role for dopamine D1 receptors (Vezina, 1996). Therefore, the dopamine D1 receptor
antagonist SCH-23390 blocked the expression of psychostimulant induced sensitization, e.g.
cocaine (McCreary and Marsden, 1993; Kuribara and Uchihashi, 1993) and 3,4methylenedioxymethylamphetamine (MDMA, Ramos et al., 2004). Despite previous studies,
nicotine induced behavioral sensitization is less well understood and the role of the
dopamine D1 receptor has not been investigated, to the best of our knowledge. The present
study showed that pre-treatment with SCH 23390 attenuated the expression of nicotine
sensitized activity. The dose of SCH-23390 used in the present study (0.03 mg/kg) was
based on prior studies from our laboratory, which demonstrated dose dependent attenuation
of the expression of nicotine induced sensitization by SCH-23390 (unpublished data, not
shown). This dose was also found the most effective dose on dopamine D1 receptor
occupancy following systemic administration of SCH-23390, evident as an inverted U-shaped
dose-dependent effect (Neisewander et al., 1998). Although it could be argued that an
evaluation of the SCH-23390 dose response, in the presence of nicotine, would have
established a more thorough understanding of the pharmacological interactions our study
supports the view that dopamine D1 receptors mediate both behavioral and neurochemical
processes associated with nicotine treatment. The ability of SCH-23390 to block these
effects of nicotine were not dependent on the non-specific suppression of behavioral activity
17
since SCH-23390 did not alter the baseline response in rats previously exposed to nicotine;
data were similar to those from rats pretreated with saline. This is in agreement with previous
studies showing no effect of SCH-23390 on baseline response in naïve animals (O’Neill et al.,
1991; McCreary and Marsden, 1993; Daniela et al., 2004). Our findings coincide with a
previous finding that SCH-23390 blocked acute nicotine-induced locomotor activity (O’Neill et
al., 1991). Although the present study was limited by the absence of a SCH-23390+nicotine
control group in naive rats, which could be argued to complicate the interpretation of the
sensitization results. Although this pattern of data precludes the conclusion that dopamine
D1 (or rather D1-like) receptors were involved in the sensitized stimulus effects of nicotine
per se, our data do suggest that SCH-23390 blocked the expression of these behaviors.
Previous studies in our laboratory have shown that intermittent administration of
nicotine produced locomotor sensitization with a sensitized response within 5 days (de Bruin
et al., 2011; Goutier et al., 2013) and therefore the present study used this period of time to
induce sensitization. The results shown are representatives for the in vivo experiments
performed, and that were conducted to obtain sufficient tissue for the ex vivo studies
described in this paper.
Behavioral studies indicate that neuronal nAChRs participate in complex brain
functions such as attention, memory, and cognition, whereas clinical data suggest their
involvement in the pathogenesis of neuropsychiatric disorders such as schizophrenia,
depression and Parkinson's diseases (Mihailescu and Drucker-Colín, 2000). Therefore,
nicotine sensitization is suggested to model aspects of these disorders. This complex brain
signaling involves multiple brain areas and it has been demonstrated that the striatum is a
more densely interconnected region and functions as a ‘hub region’, containing medial spiny
neuron GABAergic projection neurons and cholinergic interneurons expressing dopamine D1
receptors, and therefore important in regulating basal ganglia activity. Therefore, the present
study focused on the striatum as one of the ‘nodes’ putatively involved in neuropsychiatric
disease.
18
The effect of systemic nicotine administration on dopaminergic neurotransmission
have been extensively described, for example, Lizumi and colleagues (1997) showed upregulation on tyrosine hydroxylase immunoreaction following nicotine administration, Di
Chiara (2000) reported that rats receiving nicotine showed a highly selective increase in the
release of dopamine in the striatum, and subsequent effects on striatal dopamine D1 and D2
receptor binding (Fung et al., 1996) and mRNA up-regulation (Bahk et al., 2002). The
present study showed that nicotine induced behavioral sensitization did not affect the
receptor binding affinity of dopamine, SKF-38393, SKF-82958, or SCH-23390 for the
dopamine D1 receptor under the test conditions described above, i.e. this study used tissue
which was taken at 24 h post-challenge, although it could be argued that the functional effect
on the dopamine receptors may only be short lasting.
Few studies have investigated the functional, molecular consequences of behavioral
sensitization on secondary messenger systems. For example, Schroeder and colleagues
showed that activation of adenylate cyclase enhanced the acquisition of cocaine induced
sensitization, but was not sufficient to induce behavioral sensitization on its own (Schroeder
et al., 2004). Adenylate cyclase is an intracellular enzyme which catalyzes the production of
cyclic adenosine monophosphate (cAMP), an important intracellular second messenger and
plays a major role in cell signaling. In this study, cAMP accumulation was calculated as a
ratio of the measured ATP and cAMP. Using this ratio, ATP levels were used to correct for
cell viability and variability in number of cells per sample (Goutier et al., 2010).
The present study demonstrated low potency for dopamine on cAMP accumulation
(i.e. low mM range) relative to its high receptor binding affinities (nM/μM), which might be
explained by the breakdown (oxidation) of dopamine during the incubation (above). The
dopamine D1-like and D2-like receptors are respectively stimulatory and inhibitory coupled to
adenylate cyclase. It was hypothesized that dopamine as an endogenous ligand would have
equal affinity for both dopamine D1-like and D2-like receptor subtypes and the net effect of
dopamine on cAMP accumulation is zero. However, Mottola and colleagues showed that
dopamine decreased forskolin stimulated cAMP accumulation (Mottola et al., 2002)
19
suggesting preferential affinity for the dopamine D2-like receptor. In contrast, the present
study
demonstrated
that
dopamine
stimulated
cAMP
accumulation
(Fig.
6B).
Wehypothesized that antagonizing the dopamine D2 receptor would prevent the suspected
inhibition and keep the dopamine D1 receptor available for dopamine stimulation. The
present data showed that dopamine co-incubated with the selective dopamine D2 antagonist
LY-741626 revealed the dopamine D1 component of dopamine (Fig. 6C) which is in
agreement with previous data (Barnett et al., 1987). When rats were sensitized to nicotine,
an increase in potency to stimulate ex vivo cAMP accumulation was found. This result is in
contrast with previous data (Barnett et al., 1987) which showed a decrease in dopamine
stimulated adenylate cyclase activity after chronic amphetamine administration; herein
dopamine was used as an index for dopamine D1 receptor activation. However, the present
results of the dopamine-induced D1 receptor activation were observed in the presence of D2
receptor blockade and a dopamine D1/D2 receptor “(de-)activation synergy” could explain the
discrepancy between the two studies.
The acute and chronic nicotine sensitization regime had no effect on adenylate
cyclase activity, following forskolin (a direct activator of adenylate cyclase) stimulation. The
effects seen with dopamine are most likely receptor mediated, and not due to changes in
adenylate cyclase activity per se. It is in agreement with findings described by Schroeder and
colleagues (Schroeder et al., 2004) that the effect of forskolin on behavioral sensitization is
the result of downstream process and/or longer lasting phenomenon. Acute administration of
a high dose of psychostimulants (amphetamine 5 mg/kg or methylphenidate 50 mg/kg) but
not with lower doses, resulted in the desensitization of dopamine stimulated adenylate
cyclase activity (Barnett et al., 1986). This effect occurred within 25 min but recovered rapidly,
within 90 min. It might be possible that the decrease of dopamine D1 agonist stimulated
cAMP accumulation, seen in the present study after 45 min but not after 24 h post-challenge,
might be a result of desensitization of the dopamine D1 receptor, although further studies will
be needed to clarify this. Moreover, in contrast to data recorded with tissue taken 45 min
post-challenge, dopamine did not have any effect in tissue taken at 24 h. This suggests there
20
may be different processes taking place at different time points. Therefore, as suggested
from the 45 min data, there is a change in the dopamine D1 mediated pathways, i.e. increase
in potency and efficacy (when tested in the presence of LY-741626). However, the 24 h data
suggested a decrease in potency, as dopamine failed to increase cAMP accumulation.
Considering that cAMP is a relatively fast acting second messenger it is conceivable that
over time (45 min to 24 h) other downstream signaling molecules may have been activated,
thereby express a longer-lasting effect. This notion is supported by the action of rolipram, a
cAMP phosphodiesterase inhibitor, on methamphetamine-induced sensitization and
concomitantly increased cAMP accumulation (Iyo et al., 1996). Future studies should
investigate tissue taken at additional time points between the 45 min and 24 h post-challenge
in order to fully evaluate the temporality of the effect.
In this study SKF-38393 and SKF-82958 were considered selective dopamine D1
receptor agonists and SCH-23390 as a selective dopamine D1 receptor antagonist (O’Boyle
and Waddington, 1984), although, previous in vitro studies have shown that SCH-23390 also
expresses high affinity for the serotonin 5HT2 receptor subtype (Millan et al., 2001). However,
the doses required to induce a similar response in vivo are >10-fold higher than those
required to induce a dopamine D1 mediated response and in the scope of the present work
are therefore considered selective (for review see Bourne, 2001). The functional role of
dopamine D1 ligands remains controversial, mainly due to the lack of selective and fully
efficacious dopamine D1 receptor ligands, the unfavorable pharmacokinetic profile and
adverse effects (see Salmi et al., 2004 for review). Therefore, the potential clinical utility of
dopamine D1 receptor ligands remains elusive.
From a translational perspective behavioral sensitization can occur in human, but
whether sensitization to nicotine or how it is expressed in humans remains unclear, as is its
role in human smoking cessation/addiction (see for discussion DiFranza and Wellman, 2007).
Therefore, it is prudent to conduct further non-clinical and clinical (sensitization) studies to
further assess the putative pharmacotherapeutic potential of dopamine D1 ligands in
neuropsychiatric disorders (see Zhang et al., 2009 for review).
21
In summary, the present study describes a multidisciplinary approach providing
evidence for the pharmacological inhibition of the expression of nicotine induced behavioral
sensitization by SCH-23390. The data also provide ex vivo biochemical evidence that
dopamine D1 receptors can modulate behavioral sensitization and cAMP mediated cell
signaling. Due to the complexity of brain signaling it is likely that other neurotransmitter
systems (e.g. serotonergic, noradrenergic, and cholinergic) may play a role as well.
Nonetheless, in agreement with previous studies it is conceivable that dopamine and the
dopamine D1 receptor plays an important role in the mechanism underlying behavioral
sensitization to nicotine.
22
References
Angulo, J.A., Coirini, H., Ledoux, M., Schumacher, M, 1991. Regulation by dopaminergic
neurotransmission of dopamine D2 mRNA and receptor level in the striatum and nucleus
accumbens of the rat. Mol. Brain Res. 11,161-166.
Bahk, J.K., Li, S., Park, M.S., Kim, M.O., 2002. Dopamine D1 and D2 receptor mRNA upregulation in the caudate-putamen and nucleus accumbens of rat brains by smoking. Prog.
Neuro-Psychopharmacol. Biol. Psych. 26,1095-1104.
Barnett, J.V., Kuczenski, R., 1986. Desensitization of rat striatal dopamine-stimulated
adenylate cyclase after acute amphetamine administration. J. Pharmacol. Exp. Ther.
237,820-825.
Barnett, J.V., Segal, D.S., Kuczenski, R., 1987. Repeated Amphetamine Pretreatment Alters
the Responsiveness of Striatal Dopamine-Stimulated Adenylate Cyclase to AmphetamineInduced Desensitization. J. Pharmacol. Exp. Ther. 242,40-47.
Bourne, J.A., 2001. SCH 23390: the first selective dopamine D1-like receptor antagonist.
CNS Drug Rev. 7,399-414.
Bunzow, J.R., Van Tol, H.H.M., Grandy, D.K., Albert, P., Salon, J., Chstie, M.C., Machida,
C.A., Neve, K.A., Civelli, O., 1998. Cloning and expression of a rat dopamine D2 receptor
cDNA. Nature 336,783-787.
Daniela, E., Brennan, K., Gittings, D., Hely, L., Schenk, S., 2004. (+) Effect of SCH 23390 on
(+/-)-3,4-methylenedioxymethamphetamine hyperactivity and self-administration in rats.
Pharmacol. Biochem. Behav. 77,745-750.
23
De Bruin, N.M.W.J., Kloeze, B.M., McCreary, A.C., 2011. The 5-HT6 serotonin receptor
antagonist SB-271046 attenuates the development and expression of nicotine-induced
locomotor sensitization in Wistar rats. Neuropharmacology 61,451-457.
Di Chiara, G., 2000. Role of dopamine in the behavioural actions of nicotine related to
addiction. Eur. J. Pharmacol. 393,295–314.
DiFranza, J.R., Wellman, R.J., 2007. Sensitization to nicotine: how the animal literature might
inform future human research. Nicotine Tob. Res. 9,9-20.
Downs, A.W., Eddy, N.B., 1932. The effect of repeated doses of cocaine on the rat. J.
Pharmacol. Exp. Ther. 46,199-200.
Fremeau Jr, R.T., Duncan, G.E., Fornaretto, M.G., Dearry, A., Gingrich, J.A., Breese, G.R.,
Caron, M.G., 1991. Localization of D1 dopamine receptor mRNA in brain supports a role in
cognitive, affective, and neuroendocrine aspects of dopaminergic neurotransmission. Proc.
Natl. Acad. Sci. 88,3772-3776.
Fung, Y.K., Schmid, M.J., Anderson, T.M., Lau, Y.S., 1996. Effect of nicotine withdrawal on
central dopaminergic systems. Pharmacol. Biochem. Behav. 53, 635-640.
Garau, L., Govoni, S., Stefanini, E., Trabucchi, M., Spano, P.F., 1978. Dopamine receptors:
pharmacological and anatomical evidences indicate that two distinct dopamine receptor
populations are present in rat striatum. Life Sci. 23,1745-1750.
24
Goutier, W., Spaans, P.A., van der Neut, M.A., McCreary, A.C., Reinders, J.H., 2010.
Development and application of an LC-MS/MS method for measuring the effect of (partial)
agonists on cAMP accumulation in vitro. J. Neurosci. Methods. 188,24-31.
Goutier, W., Kloeze, B.M., McCreary, A.C., 2013. The Effect of Varenicline on the
Development and Expression of Nicotine-induced Behavioral Sensitization and CrossSensitization in Rats. Addiction Biology, in press.
Iyo, M., Bi, Y., Hashimoto, K., Inada, T., Fukui, S., 1996. Prevention of methamphetamineinduced behavioral sensitization in rats by a cyclic AMP phosphodiesterase inhibitor, rolipram.
Eur. J. Pharmacol. 312,163-170.
Kebabian, J.W., Calne, D.B., 1979. Multiple receptors for dopamine. Nature 277,93-96.
Kuribara, H., Uchihashi, Y., 1993. Dopamine antagonists can inhibit methamphetamine
sensitization, but not cocaine sensitization, when assessed by ambulatory activity in mice. J.
Pharm. Pharmacol. 45,1042-1045.
Lizumi, H., Kawashima, Y., Tsuchida, H., Utsumi, H., Nakajima, S., Fukui, K., 1997. Effect of
nicotine-administration on tyrosine hydroxylase containing neuron in the rat forebrain;
immunohistochemical study with semiquantitative morphometric analysis. Nihon Arukoru
Yakubutsu Lgakkai Zasshi 32,503-510.
Marsden, C.A., 2006. Dopamine: the rewarding years. Brit. J. Pharmacol. 147,S136-S144.
Mash, D.C., Staley, J.K., 1997. Neurochemistry of drug abuse. In: Drug Abuse Handbook
(Karch SB, ed.). CRC Press.
25
McCreary, A.C., Marsden, C.A., 1993. Cocaine-induced behaviour: Dopamine D1 receptor
antagonism by SCH 23390 prevents expression of conditioned sensitisation following
repeated administration of cocaine. Neuropharmacol. 32,387-391.
Mihailescu,
S., Drucker-Colín,
R., 2000. Nicotine,
brain nicotinic receptors, and
neuropsychiatric disorders. Arch. Med. Res. 31,131-144.
Millan, M.J., Newman-Tancredi, A., Quentric, Y., Cussac, D., 2001. The "selective" dopamine
D1 receptor antagonist, SCH23390, is a potent and high efficacy agonist at cloned human
serotonin2C receptors. Psychopharmacology (Berl) 156,58-62.
Monsma Jr, F.J., Mahan, L.C., McVittie, L.D., Gerfen, C.R., Sibley, D.R., 1990. Molecular
cloning and expression of a D1 dopamine receptor linked to adenylate cyclase activation.
Proc. Natl. Acad. Sci. 87,6723– 6727.
Mottola, D.M., Kilts, J.D., Lewis, M.M., Connery, H.S., Walker, Q.D., Jones, S.R., Booth, R.G.,
Hyslop, D.K., Piercey, M., Wightman, R.M., Lawler, C.P., Nichols, D.E., Mailman, R.B., 2002.
Functional selectivity of dopamine receptor agonists. I. Selective activation of postsynaptic
dopamine D2 receptors linked to adenylate cyclase. J. Pharmacol. Exp. Ther. 301,1166-1178.
Neisewander, J.L., Fuchs, R.A., O'Dell, L.E., Khroyan, T.V., 1998. Effects of SCH-23390 on
dopamine D1 receptor occupancy and locomotion produced by intraaccumbens cocaine
infusion. Synapse 30,194-204.
Neve, K.A., Neve, R.L., 1997. Molecular biology of dopamine receptors. in The Dopamine
Receptors. Neve KA, Neve RL (Eds.). Humana Press, Totawa, NJ, USA. 27–76.
26
O’Boyle, K.M., Waddington, J.L., 1984. Selective and stereospecific interactions of R-SK&F
38393 with [3H]piflutixol but not [3H]spiperone binding to striatal D1 and D2 dopamine
receptors: Comparisons with SCH 23390. Eur. J. Pharmacol. 98,433-436.
O’Neill, C., Nolan, B.J., Macari, A., O’Boyle, K.M., O’Connor, J.J., 2007. Adenosine A1
receptor-mediated inhibition of dopamine release from rat striatal slices is modulated by D1
dopamine receptors. Eur. J. Neurosci. 26,3421-3428.
Paxinos, G., Watson, C., 1998. The rat brain in stereotaxic coordinates. Fourth Edition.
Academic Press Ltd, London.
Post, R.M., Robinson, D.R., Ballenger, J.C., 1984. Conditioning, sensitisation and kindling. In
Neurbiology of Mood Disorders. RM Post and JC Ballenger (Eds.). Williams and Wilkins.
Baltimore, USA. 167-168.
Ramos, M., Goñi-Allo, B., Aguirre, N., 2004. Studies on the role of dopamine D1 receptors in
the development and expression of MDMA-induced behavioral sensitization in rats.
Psychopharmacology 177,100-110.
Robinson, T.E., Becker, J.B., 1986. Enduring changes in brain and behaviour produced by
chronic amphetamine administration: a review and evaluation of animal models of
amphetamine psychosis. Brain Res. 396,157-198.
Robinson, T.E., Berridge, K.C., 1993. The neural basis of drug craving: An incentivesensitization theory of addiction. Brain Res. Brain Res. Rev. 18,247-291.
Salmi, P., Isacson, R., Kull, B., 2004. Dihydrexidine--the first full dopamine D1 receptor
agonist. CNS Drug Rev. 10,230-242.
27
Schroeder, J.A., Hummel, M., Unterwald, E., 2004. Repeated intracerebroventricular
forskolin administration enhances behavioral sensitization to cocaine. Behav. Brain Res.
153,255-260.
Segal, D.S., Schuckit, M.A., 1983. Animal models of stimulant induced psychosis. In: I.
Creese (Ed.). Stimulants: Neurochemical, Behavioral, and Clinical Perspectives. Raven. New
York, USA, 131-168.
Tatum, A.L., Seevers, M.H., Collins, K.H., 1929. Morphine addiction and its physiological
interpretation based on experimental evidences. J. Pharmacol. 36,447-475.
Ujike, H., Onoue, T., Akiyama, K., Hamamura, T., Otsuki, S., 1989. Effects of selective D-1
and D2 dopamine antagonists on development of methamphetamine-induced behavioral
sensitization. Psychopharmacology (Berl) 98,89-92.
Vezina, P., 1996. D1 dopamine receptor activation is necessary for the induction of
sensitization by amphetamine in the ventral tegmental area. J. Neurosci. 16,2411-2420.
Zhang, J., Xiong, B., Zhen, X., Zhang, A., 2009. Dopamine D1 receptor ligands: where are
we now and where are we going. Med. Res. Rev. 29,272-294.
28
Legends
Figure 1. – Treatment scheme and termination of the in vivo experiments. Schematic of the
treatment protocol used to induce nicotine sensitization and study the effect of SCH-23390
on the expression of sensitization. Experimental time line represents locomotor activity
measurement. Grey areas indicate drug administration, i.e. nicotine (0.4 mg/kg; s.c.) or SCH23390 (0.03 mg/kg; i.p.). The in vivo experiments were terminated after either 45 min or 24 h
post-challenge and brain tissue was dissected for subsequent ex vivo experiments.
Figure 2. - Schematic of dissected brain area (CPu). The pair of vertical lines in the sagittal
schematic (top left) indicate the anterior and posterior limits of the series of coronal
schematics, coordinates were +1.7 - +0.2 mm anterior relative to Bregma (Paxinos and
Watson, 1998). CPu indicates caudate putamen, the region of interest in the coronal sections,
and corresponds to the dorsal striatum. Diagrams were modified from Paxinos and Watson
(1998).
Figure 3. – Development (acquisition) of nicotine induced behavioral sensitization. A.) Graph
showing a 5 min locomotor activity time course following acute nicotine (0.04 mg/kg; s.c.). B.)
Graph presenting a time course of a nicotine challenge following chronic intermittent nicotine
administration showing behavioral sensitization. C.) Bar-graph presenting Day 1 vs. 5 (twoway ANOVA). VEH, vehicle; NIC, nicotine (0.4 mg/kg, s.c.). Post hoc analysis using
Bonferroni’s method, *** P<0.001. Data presented are mean±s.e.m. of n=8 animals per
group.
29
Figure 4. – The effect of SCH-23390 on the expression of nicotine induced sensitization. A.)
Graph presenting time course and B.) graph presenting bar-graph of the effect of acute SCH23390 (0.03 mg/kg; i.p.) following a withdrawal period from behavioral sensitization. C.)
Graph presenting time course and D.) bar-graph showing that SCH-23390 blocked the
expression of nicotine induced sensitization. VEH, vehicle; NIC, nicotine (0.4 mg/kg, s.c.);
SCH, SCH-23390 (0.03 mg/kg, i.p.). One-way ANOVA followed by Bonferroni’s post hoc
analysis, ** P<0.01, *** P<0.001. Data presented are mean±s.e.m. of n=8 animals per group.
Figure 5. – Effect of dopaminergic ligands on dopamine D1 receptor binding. Membrane
preparations from vehicle treated or nicotine sensitized rats were incubated with [3H]-SCH23390 and increase concentration of Dopamine (A), SKF-38393 (B), SKF-82958 (C), SCH23390 (D). Without Mg2+ or Ca2+ (see methods). For pIC50 values see Table 1. Data
represent mean±s.e.m. of n=1-4 independently performed experiments.
Figure 6. – Effect of forskolin and dopamine on cAMP accumulation. The effect of forskolin (a
diterpene derivative and direct activator of adenylate cyclase) and dopamine (endogenous
ligand) on cAMP accumulation in striatal slices from vehicle (■), chronic nicotine (sensitized,
▲) treated rats, and animals sensitized and pre-challenged with SCH-23390 (●). The effect
of forskolin (A), dopamine (B), dopamine in the presence of 1 µM LY-741626 (C) and
dopamine in the presence of 1 µM SCH-23390 (D) were assessed. Tissue was taken 45 min
post-challenge. See Table 2. for pEC50 and Emax values, n-numbers and data for acute
nicotine treatment.
Figure 7. - Effect of dopamine D1 receptor agonists on cAMP accumulation. The effect of
SKF-38393 (A.), SKF-82958 (B.), SCH-23390 (C.) and SCH-23390 in the presence of 1 μM
SKF-38393 (D.) on cAMP accumulation in striatal slices from animals chronically treated with
vehicle (■), sensitized to nicotine (▲), or nicotine sensitization blocked by SCH-23390 (●).
30
Tissue was taken 45 min post-challenge. See Table 3. for pEC50 values, n-numbers and data
for acute nicotine treatment.
31
Illustrations and Tables
Figure 1.
32
Figure 2.
33
Figure 3.
DEVELOPMENT
A
Mean locomotor activity
(beambreaks/ 5 min)
Acute Nicotine
(Day 1)
250
200
150
100
50
0
-50-40-30-20-10 0 10 20 30 40
Time (min)
B
Mean locomotor activity
(beambreaks/ 5 min)
Chronic Nicotine
(Day 5)
250
200
150
100
50
0
-50-40-30-20-10 0 10 20 30 40
Time (min)
C
Mean locomotor activity
(beambreaks/ 45 min)
Day 1 vs. 5
Day 1
Day 5
600
***
***
400
200
0
Day 1-5
VEH
◊ Vehicle
VEH
NIC
NIC.
□ Vehicle
34
▲ Nicotine
▼ Nicotine
35
Figure 4.
EXPRESSION
A
B
SCH-23390 pre-challenge
(Day 10)
Mean locomotor activity
(beambreaks/ 30 min)
Mean locomotor activity
(beambreaks/ 5 min)
SCH-23390 pre-challenge
(Day 10)
250
200
150
100
50
0
-50
-40
-30
-20
-10
0
Vehicle
SCH-23390 (0.03 mg/kg)
C
400
200
0
Day 1-5 VEH
Day 10 VEH
Time (min)
Vehicle
Vehicle
600
VEH
VEH
NIC
VEH
NIC.
SCH
(0.03)
D
Nicotine challenge
(Day 10)
250
Mean locomotor activity
(beambreaks/ 45 min)
Mean locomotor activity
(beambreaks/ 5 min)
Nicotine challenge
(Day 10)
200
150
100
50
0
0
10
20
30
40
Time (min)
Vehicle
Nicotine
Nicotine
Nicotine
***
**
600
400
200
0
Day 1-5
VEH
Day 10 { VEH
VEH
36
***
VEH
NIC
NIC.
VEH
VEH
SCH
------------ NIC ------------
Figure 5.
A
B
120
120
100
% of control
% of control
100
80
60
40
20
Vehicle
Chronic Nicotine
80
60
40
20
0
Vehicle
Chronic Nicotine
0
-11 -10
-9
-8
-7
-6
-5
-4
-3
-2
-14 -13 -12 -11 -10 -9
Log [Dopamine] (M)
-8
-7
-6
-5
-4
Log [SKF-38393] (M)
D
120
120
100
100
% of control
% of control
C
80
60
40
20
0
Vehicle
Chronic Nicotine
-13 -12 -11 -10 -9
-8
-7
Vehicle
Chronic Nicotine
80
60
40
20
0
-6
-5
-4
-3
-13 -12 -11 -10 -9
Log [SKF-82958] (M)
-8
-7
-6
Log [SCH-23390] (M)
37
-5
-4
-3
Figure 6.
A
B
2500
% of control
% of control
3000
2000
1500
1000
500
0
-10 -9 -8 -7 -6 -5 -4
Log [forskolin] (M)
-3
-10
-9
-8 -7 -6 -5 -4
Log [Dopamine] (M)
-3
D
220
200
180
160
140
120
100
% of control
% of control
C
220
200
180
160
140
120
100
-10 -9 -8 -7 -6 -5 -4 -3
Log [Dopamine + 1 µM LY-741626] (M)
■ Vehicle
▲ Nicotine sensitized
220
200
180
160
140
120
100
-10 -9 -8 -7 -6 -5 -4 -3
Log [Dopamine + 1 µM SCH-23390] (M)
● Nicotine sensitized + SCH-23390 pre-treatment
38
Figure 7.
B
240
220
200
180
160
140
120
100
-10
% of control
% of control
A
-9
-8
-7
-6
-5
Log [SKF-38393] (M)
240
220
200
180
160
140
120
100
-4
C
-10
-9
-8
-7
-6
Log [SKF-82958] (M)
-5
D
% of control
(SKF-38393, 1uM)
% of control
120
100
80
60
40
20
0
-12 -11 -10 -9 -8 -7 -6 -5
Log [SCH-23390] (M)
■ Vehicle
▲ Nicotine sensitized
120
100
80
60
40
20
0
-12 -11 -10 -9 -8 -7 -6 -5 -4
Log [SCH-23390 + 1 µM SKF-38393] (M)
-4
● Nicotine sensitized + SCH-23390 pre-treatment
39
Table 1.
Table 1. Affinity of dopaminergic ligands for dopamine D1 receptor
[3H]-SCH-23390 receptor binding in rat dorsal striatal membranes. Values are pIC50 mean
(n=1-4).
VEH-VEH
NIC-NIC
Dopamine
4.3
4.4
SKF-38393
6.4
6.4
SKF-82958
7.3
7.3
SCH-23390
9.4
9.3
24 h post-challenge
40
Table 2.
Table 2. - Effect of forskolin and dopamine on cAMP accumulation in rat striatal slices
Values are pEC50 mean±s.d. n-number is given between parentheses and Emax values (%)
between brackets.
VEH-VEH
VEH-NIC
NIC-NIC
NIC-SCH+NIC
Forskolin*
4.8 ± 0.2 (4)
4.5 ± 0.2 (4)
4.6 ± 0.2 (4)
4.8 ± 0.1 (4)
Dopamine*
4.4 ± 0.2 (6)
3.8 ± 0.1 (4)
4.7 ± 0.2 (6)
3.8 ± 0.1 (4)
Dopamine +
5.6 ± 0.4 (4)
5.9 ± 0.3 (4)
5.8 ± 0.3 (4)
4.3 ± 0.4 (4)
[162]
[172]
[213]
[166]
<4.0 (4)
<4.0 (3)
<4.0 (4)
<4.0 (4)
Forskolin*
4.5 ± 0.2 (5)
4.5 ± 0.3 (4)
4.5 ± 0.2 (4)
4.5 ± 0.2 (3)
Dopamine*
<5.0 (3)
n/a
<5.0 (3)
n/a
45 min post-challenge
LY-741626 (1µM)
Dopamine +
SCH-23390 (1µM)
24 h post-challenge
n/a, not available; VEH, vehicle; NIC, nicotine; SCH, SCH-23390. *Note: The highest
concentration tested (100 µM) did not produce a maximal effect or plateau; therefore it is
theoretically not possible to calculate a pEC50 value. However, the pEC50 values were
calculated using an estimated maximal value at 0.1 mM, calculated by the fitting software
(PRISM).
41
Table 3.
Table 3. - Effect of D1 ligands on cAMP accumulation in rat striatal slices
Values are pEC50 mean±s.d. n-number is given between parentheses and Emax
values (%) between brackets.
VEH-VEH
VEH-NIC
NIC-NIC
NIC-SCH+NIC
SKF-82958
7.7 ± 0.2 (4)
7.3 ± 0.4 (3)
6.2 ± 0.2 (4)
7.6 ± 0.3 (3)
SKF-38393
7.2 ± 0.2 (5)
6.7 ± 0.2 (3)
6.0 ± 0.2 (5)
6.4 ± 0.2 (4)
SCH-23390
<5.0 (3)
<5.0 (3)
<5.0 (3)
<5.0 (3)
SCH-23390 +
8.2 ± 0.3 (5)
8.4 ± 0.2 (5)
7.9 ± 0.5 (5)
7.9 ± 0.2 (4)
SKF-82958
7.7 ± 0.4 (7)
7.2 ± 0.5 (2)
7.4 ± 0.5 (7)
n/a
SKF-38393
6.9 ± 0.1 (8)
7.0 ± 0.3 (4)
7.0 ± 0.2 (6)
6.9 ± 0.3 (3)
SCH-23390
<5.0 (3)
n/a
<5.0 (3)
n/a
SCH-23390 +
8.4 ± 0.2 (4)
8.3 ± 0.1 (3)
8.1 ± 0.3 (4)
8.1 ± 0.3 (3)
45 min post-challenge
SKF-38393 (1µM)
24 h post-challenge
SKF-38393 (1µM)
n/a, not available; VEH, vehicle; NIC, nicotine; SCH, SCH-23390.
42