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INDIVIDUAL DIFFERENCES IN REWARD SEEKING AND INHIBITION: RELATIONSHIP WITH
MONOAMINE TRANSPORTERS FUNCTION
1
Darna ,
2
Marusich ,
2
Bardo ,
1
Dwoskin
M.
J.A.
M. T.
L. P.
1 Department of Pharmaceutical Sciences, College of Pharmacy, and
2 Department of Psychology, University of Kentucky, Lexington, Kentucky, USA
Introduction
Aim of the project
Identification of precise neural mechanisms that may explain the association of individual
differences in reward seeking, inhibition, and drug reward.
Methods
Animals: Sprague-Dawley 45-day-old male rats were used in all experiments.
Behavioral measures of reward seeking and inhibition: Rats were assessed in 6 different
behavioral tasks over the course of 60 days in a counterbalanced order. Behavioral
procedures for reward seeking were: (1) activity in inescapable novelty; (2) novelty place
preference; and (3) sucrose reinforcement on a progressive ratio (PR) schedule.
Behavioral procedures for inhibition were: (1) passive avoidance; (2) cued go/no-go; and
(3) delay discounting. Following the completion of all six behavioral tests, rats were
implanted with an intravenous catheter into the jugular vein for drug self-administration.
Rats were given access to i.v. d-amphetamine (0.03 mg/kg/infusion) available by pressing
a lever in an operant chamber. The number of lever presses required for an infusion
increased from one to five (FR 1-FR 5) over the course of 23 sessions. Subjects were then
given access to different unit doses of d-amphetamine (0.0056, 0.01, 0.056, 0.1
mg/kg/infusion) and saline vehicle for self-administration over the course of another 19
sessions. Subsequently, rats were killed by rapid decapitation for neurochemical analysis.
[3H]DA Uptake: Brain regions, mPFC and OFC, were homogenized in ice-cold 0.32 M
sucrose solution with a Teflon pestle homogenizer. Homogenates were centrifuged at
2,000g for 10 min at 4oC. Supernatants were centrifuged at 20,000g for 17 min at 4oC.
Maximal velocity (Vmax ) and affinity (Km ) of [3H]DA uptake were determined using
saturation analysis. These synaptosomes were preincubated in assay buffer for 5 min at
34oC, and then incubated for 5 min at 34oC with 7 concentrations (0.1 nM - 1µM) of
[3H]DA in a 250 µl total volume for both regions. Kinetic analysis of [3H]DA uptake by
dopamine transporter (DAT) was assessed in the presence of desipramine (5 nM) and
paroxetine (5 nM) to prevent [3H]DA uptake into norepinephrine- and serotonincontaining nerve terminals, respectively. Nonspecific uptake was determined in the
presence of 10 µM nomifensine.
Kinetic analysis of DAT uptake assay in synaptosomes of
mPFC and OFC
mPFC
Vmax 11.12.2
Km 0.07 0.05 µM
15
Total
pmol/min/mg
[3H] DA Uptake
pmol/min/mg
NS
Specific
10
5
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
Vmax 6.01.1
Km 0.05 0.03
µM
10
Total
NS
160
Specific
140
5
0
0.00
0.25
DA, M
0.50
0.75
1.00
1.25
DA, M
Vmax: maximal velocity of DA uptake
Km: affinity, concentration of DA taken up at ½ Vmax
[3H]DA uptake into synaptosomes of mPFC and OFC from
individual rats previously tested in 6 behavioral assays
followed by self-administration of d-amphetamine
Rat Number
and date of Exp
mPFC
Vmax
3.2
2.1
1.9
1.8
2.1
6.7
5.6
2.5
4.4
5.3
5.0
3.3
5.7
2.6
3.2
5.8
2.6
3.1
2.3
3.1
3.5
1.50
100
80
60
40
20
0
0
5
10
15
20
25
Vmax of [3H]DA Uptake in OFC
Conclusions
OFC
Km (nM)
(pmol/min/mg)
001 (050609)
003 (050709)
010 (051209)
011 (051309)
006 (051909)
007 (052009)
009 (052209)
012 (052609)
015 (070709)
016 (070709)
022 (070809)
024 (070809)
014 (070909)
019 (070909)
021 (071009)
023 (071009)
029 (010510)
031 (010610)
032 (010610)
033 (010710)
035 (010710)
r = 0.4543
p < .05
120
Breakpoint
pmol/min/mg
15
Positive correlation of DA uptake in OFC and PR
breakpoint
OFC
20
[ 3H] DA Uptake
pmol/min/mg
Vulnerability to drug abuse is related to a sensation seeking personality trait, which
represents two component constructs that can be designated as reward seeking and
inhibition. The mesocorticolimbic dopamine (DA) system has been implicated in reward
seeking, whereas inhibition is subserved by frontal cortical regions involving both DA and
serotonin (5-HT) systems. Anatomical studies have shown that medial prefrontal cortex
(mPFC) is implicated in both reward seeking and inhibition, whereas orbitofrontal cortex
(OFC) is implicated in inhibition. Evidence suggests that individual differences in sensation
seeking are mediated, at least in part, by differences in brain DA function. Extracellular
DA concentrations represent the net effect of both release and uptake processes,
indicating that the DA transporter (DAT) may be an important molecular mechanism for
individual differences in drug abuse vulnerability. In the current study, the role of DAT was
evaluated to study the potential role of DA systems in explaining the association of
individual differences in reward seeking, inhibition, and drug reward in mPFC and OFC
obtained from individual rats.
Results
Vmax
Km (nM)
(pmol/min/mg)
66
199
147
279
81
125
86
143
61
79
85
164
32
51
79
100
165
486
137
116
238
6.1
0.6
5.4
1.7
2.8
6.1
8.8
3.7
4.5
4.4
11.3
3.3
22.3
2.3
3.5
6.0
1.4
5.2
3.0
4.7
3.7
77
73
60
1458
93
115
600
57
48
33
58
29
45
51
490
52
154
132
165
50
93
 A significant positive correlation was obtained between DAT function in
OFC and progressive ratio breakpoint, indicating extracellular levels of DA
in these regions are related to food reward.
To the extent that PR breakpoint is an index of reward seeking, these
results suggest that enhanced DAT function in OFC may increase reward
seeking. This may occur because rats attempt to compensate for the
enhanced DAT function, which leads to greater DA clearance, by
increasing their effort to obtain sucrose reward.
Future directions
 An ongoing study with larger number of animals will address further
the role of DA system in relation of predictor and outcome variables in
drug reward.
 Serotonin transporter (SERT) function will be evaluated to study the
potential role of 5-HT systems in explaining the association of individual
differences in reward seeking, inhibition, and drug reward.
 Cellular localization using the biotylination assay will also be performed
if transporter function in a specific regions is shown to be associated with
one of the predictor variables.
Acknowledgement
Supported by P50 DA05312 (CDART grant)