acquisition and responding for conditioned …...operant responses which result in its presentation...
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Acquisition and Responding for Conditioned Reinforcement in the Mouse: Effects of Methylphenidate,
and the Role of the Dopamine Transporter
by
James Donald Caleb Browne A thesis submitted in conformity with the requirements
for the degree of Masters of Arts Graduate Department of Psychology, Program in Neuroscience
University of Toronto
© Copyright by J. D. Caleb Browne 2012
ii
Acquisition and Responding for Conditioned Reinforcement in the
Mouse: Effects of Methylphenidate, and the Role of the Dopamine
Transporter
J. D. Caleb Browne
Masters of Arts
Department of Psychology, Program in Neuroscience
University of Toronto
2012
Abstract
This work characterized the ability of mice to respond for conditioned reinforcement, a
phenomenon that can be used to investigate neural substrates of incentive learning. In both
C57Bl/6 and CD1 mice, a reward-associated stimulus acted as a conditioned reinforcer (CR).
Responding was stable over multiple test days, enhanced in CD1 mice by the dopamine
transporter (DAT) blocker methylphenidate, and was extinguished when responding no longer
produced the CR. However, transgenic C57Bl/6 mice overexpressing DAT, which decreased
striatal dopamine by 40% responded normally for CR. Therefore, these results suggest that mice
can be used to study brain mechanisms of incentive motivation. However, the choice of mouse
strain in this paradigm is important as outbred CD1 mice appeared more susceptible to a DAT
blocker compared to the inbred C57Bl/6 strain. These results also suggest that selective
responding for a CR remains intact in a chronically hypodopaminergic state.
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Acknowledgements
I would like to extend my sincerest thanks to Paul Fletcher for his invaluable assistance and
expertise in carrying out the present experiments and immense help in writing this thesis. Also, I
would like to thank Suzanne Erb for her helpful comments in the preparation of this thesis and
Junchul Kim for his time in reading and defending this thesis. I would also like to thank my lab
mates Ashlie Soko, Elizabeth Guy, and Christie Burton for their technical assistance and
discussions about the topics covered in this research. Further, I would like to thank my family for
their continued support and Raena Dempsey for her patience and putting up with my incessant
“working on my thesis” statements.
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Table of Contents
Chapter 1: Introduction 1
1 Incentive motivation and salience attribution 1
2 Role of the dopamine neurons in system in incentive motivation 1
3 Dopamine and conditioned reinforcement 2
4 Drugs of abuse and conditioned reinforcers 3
5 The nucleus accumbens and conditioned reinforcement 4
6 The basolateral amygdala and conditioned reinforcement 4
7 The dorsal striatum and conditioned reinforcement 5
8 The role of dopamine receptors in conditioned reinforcement 6
9 The dopamine transporter and its role in incentive motivation 6
10 Experimental framework and hypotheses 7
10.1 Major goals of these experiments 8
10.2 Hypotheses 9
Chapter 2: Methods 11
1 Subjects 11
2 Apparatus 11
2.1 Conditioned reinforcement 11
2.2 Locomotor activity 12
3 General behavioural procedures 12
3.1 Pre-training 12
3.2 Phase 1: Pavlovian conditioning (training) phase 12
3.3 Phase 2: Operant conditioning (testing) phase 13
4 Experiment 1a: Acquisition, expression, and amplification of responding for a
conditioned reinforcer 14
5 Experiment 1b: Temporal stability and subsequent amplification of responding for a
conditioned reinforcer 14
6 Experiment 1c: Extinction of responding for a conditioned reinforcer 15
6.1 Removal of water restriction as a motivating factor 15
6.2 Extinction training - CS not presented upon CR responding 15
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7 Experiment 2: Locomotor activation by 3.5 and 5 mg/kg doses of MPH 15
8 Experiment 3: Assessment of explicitly unpaired CS and US presentations and
responding for a non-conditioned stimulus in mice 15
9 Experiment 4a: Effect of whole-brain DA transporter overexpression on
responding for a conditioned reinforcer 16
10 Experiment 4b: Effect of MPH and AMPH on responding for a CR in DAT
overexpressing mice 16
11 Statistical Analyses 16
Chapter 3: Results 17
1 Experiment 1a: Acquisition, expression, and amplification of responding for a
conditioned reinforcer 17
1.1 Training 17
1.2 Test of responding for a CR 17
2 Experiment 1b: Temporal stability and subsequent amplification of responding
for a conditioned reinforcer 18
2.1 Extended CR responding 18
2.2 MPH test 2 18
3 Experiment 1c: Extinction of responding for a conditioned reinforcer 19
3.1 Removal from water restriction 19
3.2 Operant responding during extinction conditions 20
4 Experiment 2: Locomotor activation by 3.5 and 5 mg/kg doses of MPH 20
5 Experiment 3: Assessment of explicitly unpaired CS and US presentations and
responding for a non-conditioned stimulus in mice 20
5.1 Training 20
5.2 Test of responding for a CR 21
6 Experiment 4a: Effect of whole-brain DA transporter overexpression on responding
for a conditioned reinforcer 21
6.1 Training 21
6.2 Test of responding for a CR 21
7 Experiment 4b: Effect of MPH and AMPH on responding for a CR in DAT
overexpressing mice 21
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7.1 Effect of MPH 21
7.2 Effect of AMPH 22
Chapter 4: Discussion 23
1 Characterization of the acquisition and expression of conditioned reinforcement
in mice 23
2 Strain differences in conditioned reinforcement 24
3 Operant response type in responding for a CR 24
4 Modulation of responding for a CR by MPH administration in normal mice 25
5 Incentive salience attribution and motivation in DAT upregulated mice 26
6 Effect of MPH and AMPH on responding for a CR in DAT upregulated mice 27
Chapter 5: Conclusions 29
Chapter 6: References 30
Chapter 7: Figure Captions 37
Chapter 8: Figures 40
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List of Figures
Figure 1: Pavlovian Conditioning in C57Bl/6 and CD1 Mice
Figure 2: Effect of MPH on Responding for a CR
Figure 3: Stability of Selective CR Responding Over Time
Figure 4: Second Test of MPH on Responding for a CR
Figure 5: Responding for a CR with Free Water Access
Figure 6: Extinction of CR Responding
Figure 7: Locomotor Activation by MPH
Figure 8: Explicitly Unpaired Pavlovian Conditioning
Figure 9: Test of Responding for an Unpaired CS
Figure 10: Pavlovian Conditioning in DAT-Tg and WT Mice
Figure 11: CR Responding in DAT-Tg and WT Mice
Figure 12: Effect of MPH on Responding in DAT-Tg and WT Mice
Figure 13: Effect of AMPH on Responding in DAT-Tg and WT Mice
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Chapter 1 Introduction
1 Incentive motivation and salience attribution
Incentive motivation promotes goal directed behaviour towards stimuli such as food, sex, and
water, which have intrinsic value in promoting an individual’s fitness. Rewarding stimuli act as
incentives which are viewed as beneficial and elicit or enhance the motivated behaviour required
in attaining them (Bindra, 1968). Evolution has favoured the development of neural circuitry
which amplifies motivation by making stimuli associated with incentives sought after (Robinson
and Berridge, 1993; Schultz, 1998). Through repeated association with a rewarding
unconditioned stimulus (US), Pavlovian learning mechanisms transform benign stimuli into
conditioned stimuli (CS). Congruency in the presence of the CS and US gives an increasingly
accurate prediction of US availability based on the presence of the CS.
The availability of a reward may be signalled by cues associated with it, and without these cues
incentive motivation would be hit or miss; to obtain a valuable reward an animal would simply
have to run into it. Such reward-associated cues require a relevant, up to date account of their
predictive value which is performed by Pavlovian associative memory mechanisms (Robinson
and Berridge, 1993; Berridge and Robinson, 1998). These stimuli do not, however, remain
passively associated with a reward; to guide goal directed behaviour, stimuli must have the
ability to activate motivational neurocircuitry which then initiates behaviour directed at obtaining
the reward. These cues are therefore imbued with incentive salience making them demand
attention and ‘wanted’ in their own right (Robinson and Berridge, 1993; Berridge, 2007). In this
way, the presence of a reward-predictive cue will always be noticed and shift goal directed
behaviour towards it.
2 Role of the dopamine neurons in system in incentive motivation
The mesocorticolimbic dopamine (DA) system constitutes a major component of the circuitry
involved in reward information processing and appears to modulate the motivational significance
of both primary rewards and associated environmental stimuli (Beninger, 1983; Robinson and
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Berridge, 1993; Berridge and Robinson, 1998; Schultz, 1998). Specifically, this DA system
appears to be responsible for the attribution of incentive salience to stimuli as opposed to either
processing their hedonic impact or mediating their association with primary rewards (Berridge et
al., 1989; Berridge and Robinson, 1998; Wyvell and Berridge, 2000). This means that the
involvement of DA in incentive motivation is to mediate the drive to obtain a reward, not
necessarily the perception of ‘liking’ a reward.
The mesocorticolimbic DA system originates in the ventral tegmental area (VTA) of the
midbrain where dopaminergic neurons project to the nucleus accumbens (NAc), amygdala,
hippocampus and medial prefrontal cortex (Ungerstedt, 1971; Kalivas and Volkow, 2005). These
regions receive a pattern of phasic DA output from the VTA as a result of experiencing
motivationally relevant stimuli (White, 1989; Schultz, 1998; McClure et al., 2003; Berridge,
2007; Tsai et al., 2009). The mesocorticolimbic DA system greatly differs from the nigrostriatal
DA system in its projections, originating locus and function. The nigrostriatal system connects
the substantia nigra with the striatum and appears to be largely involved in motor planning and
execution as part of the basal ganglia motor loop (Graybiel et al., 1994). The major source of DA
within the nigrostriatal system is the pars compacta subregion of the substantia nigra, and this
region has particularly dense projections to the dorsal striatum (Haber et al., 2000; Björklund and
Dunnett, 2007).
3 Dopamine and conditioned reinforcement
The attribution of incentive salience to a previously neutral, reward associated stimulus is
thought to be mediated by a DA-dependent mechanism (Robinson and Berridge, 1993). VTA
neurons release phasic bursts of DA to forebrain regions, particularly the NAc, in response to a
stimulus associated with a reward or event that denotes motivational significance (White, 1989;
Schultz, 1998; McClure et al., 2003; Berridge, 2007; Tsai et al., 2009). Although at first robust,
the DA output a reward elicits seems to progressively decrease as it is repeatedly experienced
(Schultz, 1998). However, DA output as a result of experiencing reward-predictive stimuli
remains potentiated, maintaining the salience of those stimuli (Schultz et al., 1993; Phillips et al.,
2003). When these stimuli are presented alone, their incentive salience drives the behavioural
output of an animal to obtain them as if they were the reward itself (Robinson and Berridge,
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1993). Such high motivational value attributed to a reward-predictive CS gives it incentive
properties, so that it may become a reward in its own right. In this way the initially-neutral CS
becomes a conditioned reinforcer (CR) capable of reinforcing new learning and strengthening
operant responses which result in its presentation (Bugelski, 1938; Taylor and Robbins, 1984).
Considering this CR was previously a neutral stimulus which acquired motivational significance
based on its association with a primary reward, responding for this CR is an example of pure
incentive motivation.
Evidence suggests that responding for a CR is mediated predominantly by a DA dependent
mechanism. Administration of substances which amplify DA transmission such as
psychostimulants (e.g. amphetamine, cocaine, methylphenidate and pipradrol) can increase
responding for a CR, enhancing the control a CR exerts over behaviour (Hill, 1970; Robbins,
1978; Beninger and Phillips, 1980). Conversely, the DA receptor antagonist α-flupenthixol
attenuates responding for a CR and prevents the enhancement of responding as a result of
psychostimulants (Robbins et al., 1983; Fletcher and Higgins, 1997). Therefore, the DA system
appears to play a large role in the regulation of responding for a salient, reward associated CR.
4 Drugs of abuse and conditioned reinforcers Rewarding stimuli are highly variable in the environment. The mesocorticolimbic DA system
deals with this in its dynamic nature, allowing fluctuations in reward processing and valuation
(Schultz, 1998). This renders the mesocorticolimbic DA system susceptible to changes in its
physiology which in turn can lead to pathological incentive motivation. Many drugs of abuse act
directly or indirectly on the DA system resulting in elevated DA release (Di Chiara, 1995;
Berridge and Robinson, 1998; Koob and Le Moal, 2001). With repeated drug administration, the
mesocorticolimbic DA system can become sensitized to the behavioural activating and
neurochemical effects of the drug (Koob and Le Moal, 1997; Pierce and Kalivas, 1997). This
sensitization of the DA system can result in permanent neurochemical and neuroanatomical
alterations. Behaviourally, this may manifest in perseveration of drug seeking and drug taking
behaviours due to the amplification of salience attributed to drugs and drug-associated stimuli
(Robinson and Berridge, 1993). As the DA system is progressively sensitized to repeated drug
administration, so is the salience of the drug and drug-associated stimuli; the perceived
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importance of the drug and its associated stimuli is progressively increased and “stamped in” to
the mesocorticolimbic DA system (Robinson and Berridge, 1993).
5 The nucleus accumbens and conditioned reinforcement
The NAc appears to act as a limbic-motor interface where goal-directed behaviour is processed
based on learned motivational significance of stimuli (Mogenson et al., 1980). In terms of
conditioned reinforcement, the NAc has been shown to be a candidate region in mediating not
only the establishment of responding for a CR but also the response-enhancing effect of
stimulants (Parkinson et al., 1999). Intra-NAc infusion of amphetamine (AMPH) potentiates CR
responding (Taylor and Robbins, 1984), while NAc DA depletion attenuates AMPH-induced
elevated CR responding (Robbins and Everitt, 1982). However, the NAc has two functionally
and neuroanatomically distinct subregions which may differentially contribute to incentive
motivation (Voorn et al., 1989; Pontieri et al., 1995): the NAc shell and NAc core. The NAc
shell contains projections to the ventral pallidum and the VTA, while the NAc core projects to
basal ganglia regions such as the globus pallidus and substantia nigra (Meredith et al., 1992). The
NAc shell appears to be involved in modulating the amplified behavioural control of CR
responding observed upon administration of psychostimulant drugs (Parkinson et al., 1999; Ito et
al., 2004). On the other hand, the NAc core appears to play a large role in controlling the basal,
Pavlovian attributed incentive salience value of CRs (and therefore reward-related stimuli in
general) which directs instrumental behaviour (Cardinal et al., 2002). This conclusion is
supported by evidence that NAc core lesions impair the incentive motivation towards CR
responding for both drug- and food-associated CRs (Parkinson et al., 1999; Ito et al., 2004).
6 The basolateral amygdala and conditioned reinforcement
Control over CR responding exerted by the NAc is tightly connected with the basolateral
amygdala (BLA). The BLA is innervated by the VTA and sends glutamatergic projections to the
entire ventral striatum including both the NAc core and shell (Kelley et al., 1982). The BLA is
critical for the formation and retrieval of associations of discrete cues with rewards based on
their incentive properties (Gaffan and Harrison, 1987; Everitt et al., 2003; Ito et al., 2006).
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Lesions of the BLA result in the inability of a CR to facilitate the acquisition of a new response,
a stringent criterion of a CR’s incentive salience (Mackintosh, 1975; Cador et al., 1989). In other
words, animals with damage to the BLA fail to respond for a CR. Reduced CR responding as a
result of BLA lesions has been shown for CRs associated with natural rewards, such as water
(Cador et al., 1989), sucrose (Burns et al., 1993), and sex (Everitt et al., 1989), as well as
psychostimulants (Whitelaw et al., 1996), strongly implicating the BLA in incentive salience
attribution to many types of reward-related stimuli. Such lesions also reduce the ability of
elevated DA transmission in the NAc as a result of psychostimulant administration to potentiate
CR responding (Cador et al., 1989; Burns et al., 1993). This suggests that the incentive salience
of a CR, as measured by responding to obtain it, and its enhancement by psychostimulants
involve an interaction between VTA-NAc and VTA-BLA mediated DAergic mechanisms (Ito
and Canseliet, 2010; Ito and Hayen, 2011). Therefore, the BLA appears to be crucial for learning
about the significance of reward-associated stimuli, as well as for allowing CRs to support and
guide instrumental behaviours.
7 The dorsal striatum and conditioned reinforcement
The powerful control that a CR exerts over instrumental behaviour is relatively resistant to
extinction (Weiss et al., 2001; Vanderschuren et al., 2005). This persistence may be mediated by
the dorsal striatum (DStri) (Everitt and Robbins, 2005), which is involved in the formation of
habit learning and has been implicated in CR control over drug seeking and taking behaviour
exhibited by both rats and humans (Ito et al., 2002; Volkow et al., 2006). Merely experiencing a
CR associated with a drug can lead to a large efflux of DA to the DStri (Ito et al., 2002).
Inactivation of the DStri also affects the ability of CRs to both maintain persistent responding
and reactivate responding following extinction (Di Ciano et al., 2008). Such findings implicate
the DStri in a transition from initial goal-directed behaviour towards CRs to a more habitual,
automatic response as a result of conditioning and time. Thus the DStri may mediate the switch
from action-outcome to stimulus-response control by CRs over behaviour (Everitt et al., 2001;
Vanderschuren et al., 2005).
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8 The role of dopamine receptors in conditioned reinforcement
Dopamine is central to the attribution and maintenance of incentive salience to reward-related
stimuli, particularly psychostimulant drugs of abuse, and regions involved in the mediation of
CR responding are sensitive to changes in DA innervation. G-protein coupled dopamine
receptors in these regions are of the D1-like family (D1 and D5), which stimulate adenylyl
cyclase and subsequently the intracellular cAMP signalling cascade, or the D2-like family (D2,
D3 and D4) which inhibit adenylyl cyclase (Jaber et al., 1996; Anderson and Pierce, 2005). Both
D1 and D2 receptors within the NAc have been shown to mediate the control of responding for
CRs: D1 and D2 receptor antagonists diminish CR responding and potentiated responding due to
psychostimulants, while direct receptor agonists may amplify CR responding (Beninger et al.,
1991; Wolterink et al., 1993).
Interestingly, intra-accumbal D3 receptor antagonism has been shown to disrupt the ability of a
drug associated CR but not a sucrose associated CR to control responding (Di Ciano et al.,
2003). A similar result emerges after D3 receptor antagonism within the BLA for cocaine-
associated CRs (Di Ciano, 2008). Reinstatement of cocaine seeking by CRs following extinction
is also diminished as a result of D3 receptor blockade, but has no effect on sucrose-conditioned
place preference (Vorel et al., 2002). Such evidence of a DA receptor specific to drugs of abuse
as opposed to natural rewards has led to the D3 as a therapeutic target for the treatment of
addiction (Heidbreder et al., 2005).
9 The dopamine transporter and its role in incentive motivation
Appropriate termination of neurotransmission is integral to incentive motivation and neural
communication as a whole. The principle mechanism for this termination in monoamine neurons
is reuptake of neurotransmitter back into the presynaptic neuron by Na+/Cl
—dependent
neurotransmitter transporters (Torres and Amara, 2007). The DA transporter (DAT) exerts
powerful regulatory control over dopaminergic tone by removing DA from the synapse
following bursts of activity. DAT is located most prevalently in the mesocorticolimbic DA
system with the highest density observed in the VTA and SNc DA projection neurons, and
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concurrently their respective ventral and dorsal striatal target regions (Ciliax et al., 1995, 1999).
To maintain temporal precision in neural communication DA is cleared from the synapse by
DAT within 1 second following a phasic burst of activity. If this reuptake transporter is not
present, removal of DA from the synapse is increased 100-fold with kinetics reaching that of
passive diffusion, along with proportional compensatory decreases in DA release (Giros et al.,
1996).
DAT is also the site of action for many drugs of abuse. Cocaine and MPH bind to DAT and
block the reuptake of DA into the presynaptic terminals following phasic burst firing, while
AMPH destabilizes presynaptic DA containing vesicles and reverses DAT, resulting in an
outflow of DA (Giros et al., 1996; Salahpour et al., 2008). Considering that drugs which enhance
responding for a CR such as MPH and AMPH also alter DA kinetics at DAT, this reuptake
transporter may play a large role in the regulation of responding for a CR by controlling the
amount of DA active within the synapse. If higher levels of DAT are present in the synapse,
burst firing of DA neurons signalling motivational incentive salience may be toned down as a
result of more rapid removal from the synapse. This would potentially result in a suppressed
motivational signal manifested in the conditioned reinforcement paradigm as reduced responding
for a CR. The present experiment used mice which overexpressed DAT to examine whether this
form of decreased DAergic tone manifests in decreased motivation to obtain a CR. These mice
exhibit a three-fold increase in DAT which resulted in a 40% decrease in striatal DA while
showing no basic impairments in behaviour. However, DAT overexpressing mice may have a
reduction in basal incentive motivation in that they do not work as hard as wild-type mice to
obtain a milk reward (Salahpour et al., 2008).
10 Experimental framework and hypotheses
The acquisition of a new response task by conditioned reinforcement can be used to understand
the motivational significance of reward-associated stimuli (Mackintosh, 1975). Unlike second-
order schedules of reinforcement, this task can examine the ability of a CR to drive pure
incentive motivation towards obtaining it, and the ability of a drug to enhance this responding
with mutual exclusivity (Sutton and Beninger, 1999). Although this behavioural paradigm has
been used extensively in rats, surprisingly little attention has been given to the study of mice in
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tests of conditioned reinforcement. The use of mice provides not only the possibilities of
pharmacological and physical manipulations of incentive motivation but also behavioural genetic
techniques. Transgenic mice could be used to specifically examine the role of individual receptor
subtypes and neurotransmitter reuptake transporters involved in incentive motivation. Therefore,
extensively characterizing tests of acquisition of, and responding for, conditioned reinforcement
in mice will set the stage for such research and ultimately enhance the understanding of such
behavioural phenomena.
Very few published papers have examined responding for conditioned reinforcement, and the
effects of DAT-acting psychostimulants on this response, in mice. Experiments that have
examined this behaviour have included a relatively limited characterization of this behaviour
which is well established in rats. We felt it important to examine multiple factors to ensure that
this was not a species- or operant-specific behaviour in mice, as other studies have only
examined one strain and one operant response type. For example, Mead and colleagues (2004)
examined lever press responding for a CR using one inbred C57BL/6 strain of mice, and
subsequently examined the ability of AMPH to enhance responding for the CR (Mead et al.,
2004). Therefore work in this field is very limited.
10.1 Five major goals of these experiments
1) Goal one was to fully characterize the ability of mice to learn approach behaviour to a reward
associated CS and to respond for that CS when it acts as a CR. The ability of mice to acquire
and express responding for a CR was also compared to a secondary control group of mice
which, during Pavlovian association training, presentation of the CS is explicitly unpaired
with the US. This allowed us to determine that the apparent incentive salience of the CS
acting as a CR was, in fact, derived from its association with a primary reward. We also
examined the time course of extinction of responding for a CR to ensure this behaviour had
not become completely habitual.
2) Goal two was to examine whether strain influenced responding for CR. Thus, two strains of
mice were compared: the inbred C57Bl/6 and the outbred CD1 strains. The C75BL/6 strain
has previously been used in this behavioural paradigm (Mead et al., 2004), and are the
common background strain used in transgenic mouse studies.
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3) Goal three was to examine whether the type of operant response was important in
determining acquisition and expression of responding for CR. Thus, two operant responses
resulting in CR presentation were compared: lever press and nosepoke. This allowed
comparison of a well established operant (lever press) with an operant that fits within the
behavioural repertoire of mice (nosepoke) (Crawley, 2007).
4) Goal four was to determine whether responding for CR is potentiated following systemic
administration of the psychomotor stimulant methylphenidate (MPH) which acts as a DA
reuptake inhibitor. Early work in rats suggested that DA reuptake blockers provide more
reliable enhancement of CR responding than AMPH when injected systemically (Hill, 1970;
Robbins, 1978; Beninger et al., 1981). This may relate to DAT blocker effects depending on
the initial burst firing of DA neurons as a result of stimulus salience as opposed to AMPH
which can cause DA release into the synapse by reversing DAT regardless of
neurotransmission.
5) Goal five was to utilize genetic techniques to examine the ability of mice exhibiting whole-
brain upregulation of DAT to acquire and express responding for a CR. This genetic
alteration produces a neurochemical profile of less DA being present in the synapse
following excitation, particularly in the striatum. The ability of both MPH and AMPH to alter
responding for a conditioned reinforcer in DAT upregulated mice was also examined. The
distinct mechanisms of action of MPH and AMPH permitted two different approaches to
altering incentive motivation towards the CR.
10.2 Hypotheses
1) Both C57Bl/6 and CD1 strains of mice will learn Pavlovian approach behaviour during
training, express selective responding for a CR on both lever press and nosepoke operants,
and extinguish conditioned reinforcement when the CR is no longer presented upon
responding.
2) Mice receiving explicitly unpaired presentations of CS and US during Pavlovian association
training will not respond for the CS acting as a CR.
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3) The psychomotor stimulant MPH will enhance CR responding of both inbred and outbred
mice strains.
4) C57Bl/6 mice overexpressing DAT will effectively learn Pavlovian approach behaviour, as
associative memory mechanisms will remain relatively unaffected, while exhibiting slightly
reduced responding for a CR on a lever press operant. This is based on the upregulation of
DAT resulting in a 40% decrease in striatal DAergic tone, which has been suggested to
suppress incentive motivation, while leaving glutamatergic associative memory mechanisms
largely unaffected.
5) Administration of MPH to DAT upregulated mice will result in enhanced responding for a
CR to the same levels as wild-type controls, while AMPH will enhance responding for a CR
in both groups, but to a higher level in DAT upregulated mice. MPH overwhelmingly blocks
DAT resulting in enhanced DA in the synapse; regardless of the quantitative difference in
DAT, essentially all transporters will be saturated and blocked by MPH. The amount of DAT
present would presumably play a larger role in the effects of AMPH. Mice with upregulated
DAT would essentially exhibit more potential pores in the presynaptic neuron permitting DA
into the synapse at a higher rate in the presence of AMPH, effectively amplifying this effect
compared to mice with normal levels of DAT
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Chapter 2 Methods
1 Subjects Thirty-six C57Bl/6 and 36 CD-1 mice from Jackson Labs (Maine, USA) were used in this
experiment, along with twelve male and eight female DAT upregulated C57Bl/6 mice of varying
ages obtained from the Salahpour lab at U of T (Salahpour et al., 2008). Mice were pair-housed
in a temperature and humidity controlled room on a 12 hour light dark cycle with lights on at 7
am. All training and testing occurred a minimum of 2 hours after lights on with food available ad
libitum. Water access was restricted for 22 hours for the majority of the study, except where
noted. During water restriction, all mice maintained adequate health and body weight (within
±20% of their average body weight). Mice received habituation to injections with two saline
injections on different days prior to testing with MPH. All procedures were approved by the
Centre for Addiction and Mental Heath Animal Care Committee and adhered to Canadian
Tricouncil guidelines for the humane treatment of experimental animals.
2 Apparatus
2.1 Conditioned reinforcement
Training and testing for responding for a CR was conducted in twelve operant conditioning
boxes (Med Associates, St Albans, VT) measuring 33 by 31 by 29 cm3. The rear stainless-steel
wall of the chamber is curved and contained an array of 5 2.5cm circular apertures located 2.5cm
above the floor and 2.5cm apart. During training, these ports were closed in all boxes using
circular stainless-steel plugs. Following training, these ports were open in the six of twelve boxes
using a nosepoke operant response, and were closed in the other six boxes using a lever press
operant response. The stainless-steel front wall of the chamber contained a horizontally centered
5cm2 reinforcer magazine 2.5cm above the floor. The magazine contained an infrared
photodetector at the entrance and a light mounted on the roof of the magazine. The wall also
contained two retractable levers which were removed during the training phase and inserted
during the testing phase into the six boxes using a lever press operant response. Positioned above
each lever was a yellow stimulus light. A motor-driven dipper was raised to deliver 0.06ml of
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liquid through a hole in the floor of the magazine. Each operant box was illuminated by a
houselight, and was enclosed in a sound-attenuating chamber equipped with a ventilation fan.
The boxes were controlled by an IBM-compatible computer running Med-PC for Windows.
2.2 Locomotor activity
A custom-built activity system containing 16 clear polycarbonate activity chambers was used.
Fourteen infrared photocells spaced 7.5 cm apart and 2 cm above the cage floor lined the cages
along their length.
3 General behavioural procedures
3.1 Pre-training
Prior to any training, mice received at least a week and a half of acclimatization to water
restriction which continued through the entire experiment, unless otherwise specified. This was
done to make the saccharin reward more palatable during Pavlovian association training. During
this period, all mice remained healthy and within 20% of their average bodyweight. It had been
suggested that 22 and 20 hours of water deprivation produce few adverse physiological or
behavioural effects on mice, and that mice adapt more effectively to water rather than food
deprivation (Tucci et al., 2006; Rowland, 2007). Mice also received multiple periods of
saccharin availability to reduce neophobia to saccharin during training.
3.2 Phase 1: Pavlovian conditioning (training) phase
Prior to the training, mice received one day of dipper training which involved up to 60
presentations of a 0.06ml 0.2% saccharin solution to a magazine illuminated upon each
presentation. The next day, water deprived mice were placed into operant boxes where a CS
complex was presented just prior to the delivery of saccharin solution in the dipper 30 times on
an RI60 schedule. The average session length was 40 minutes. The CS was a compound stimulus
consisting of the immediate extinguishing of the houselight, a 5s illumination of both left and
right yellow stimulus lights followed by the sound of the motorized dipper being raised to an
accessible level for 8s to allow adequate approach and consumption time. Both stimulus lights
remained on during the 8s saccharin availability, and were extinguished at the end of this period
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when the motorized dipper descended and the houselight was turned back on. The main
dependent variables recorded were the total number of head entries into the US magazine during
the 5s CS period and the number of entries during the 5s immediately prior to each CS period.
Trials on which mice failed to pick up the US were recorded as “Misses”’; the number of entries
while the dipper was elevated were also recorded (US responses). Following 30 of these CS-US
presentations, mice were promptly removed and placed back into their homecages and given 2
hours of water access after a short delay.
3.3 Phase 2: Operant conditioning (testing) phase
One day prior to testing, mice were placed in the operant chambers with levers or nosepoke ports
presented. Mice were allotted 20 minutes to make 10 responses on the active lever or nosepoke
port which resulted in CS presentation on a random ratio 2 schedule. The CS during the testing
phases was a shortened version of that in the training phase, in that the stimulus lights were
active for 2 seconds and the presentation of the dipper (devoid of saccharin) was 1 second long.
The dipper was only elevated for this amount of time to produce the sound of the dipper
elevating without providing long access to the empty dipper receptacle. Mice were subsequently
removed and placed back into their homecages where they received 2 hours of water access. The
purpose of this day was to habituate the mice to the response lever or nosepoke ports to reduce
confounding novelty on the following test day (Fletcher, 1995).
Following this habituation, responding for a CR was assessed. In the boxes animals received
training in, two levers or nosepoke ports were presented to mice: the CR lever or port and the no
conditioned reinforcer (NCR) lever or port. Responses on the CR lever or port resulted in the
presentation of the motivationally significant light-dipper complex CS, now termed a CR, on a
random ratio 2 schedule. Response on the NCR lever or port had no programmed consequences
and served as a control to examine the specificity of motivationally directed behaviour towards
the CR. After 40 minutes of conditioned reinforcement testing, mice were promptly removed
from the operant chambers and placed into their homecages where they received two hours of
water access after a short delay.
14
4 Experiment 1a: Acquisition, expression, and amplification of responding for a conditioned reinforcer
This experiment assessed the ability of mice to acquire and express conditioned responding for a
saccharin-associated CR in two different strains and across two different operant responses. The
inbred C57Bl/6 mouse strain and outbred CD-1 strain were used to examine cross-strain
generalizability of conditioned reinforcement behaviour and enhancement of responding for a
CR due to MPH administration.
Twenty-four C57Bl/6 and 24 CD-1 mice under 22 hour water restriction received 14 days of
Pavlovian association training between the saccharin US and CS complex. Following the operant
habituation session responding for a CR was examined concurrently with the ability of the DA
reuptake blocker MPH to amplify this behaviour in both strains of mice and on both operant
response types (nosepoke or lever press). Prior to testing, mice received and injection of vehicle,
2.5, 3.5 or 5 mg/kg MPH HCl i.p. (Medisca, Montreal, QC, Canada) according to a Latin square
design to ensure counterbalanced allocation of MPH administration over four test days separated
by at least 72 hours without dosage overlap.
5 Experiment 1b: Temporal stability and subsequent amplification of responding for a conditioned reinforcer
Ten days after the final test day of experiment 1a, the persistence of responding for a CR in the
absence of drug was examined. Each day for 13 days, water restricted mice underwent identical
testing procedures as for experiment 1a without any injections. Following the 13th
day of
baseline testing, mice again received vehicle, 3.5 or 5 mg/kg MPH i.p. just prior to testing
following a Latin square design over three test days separated by 72 hours. The purpose of this
experiment was to ensure responding for a CR was a stable and reliable measure of incentive
motivation in mice, and to assess the effects of MPH following stabilization of this response.
15
6 Experiment 1c: Extinction of responding for a conditioned reinforcer
6.1 Removal of water restriction as a motivating factor
An initial step in response extinction was to remove mice from water restriction and effectively
eliminate the motivating factor of thirst in responding for a saccharin-associated CR. All mice
were given free access to water and drug-free responding for a CR was assessed over a six day
period.
6.2 Extinction training - CS not presented upon CR responding
We then tested for extinction of responding for a CR with continued free access to water. During
a nine day period of extinction training, all procedures remained the same with the exception that
responding on the CR lever or nosepoke port no longer resulted in CR presentation.
7 Experiment 2: Locomotor activation by 3.5 and 5 mg/kg doses of MPH
To confirm that doses of MPH were behaviourally active in both mouse strains this experiment
measured the locomotor response to MPH. Sixteen C57Bl/6 and 16 CD-1 mice chosen at random
from the 48 in the previous experiments were used to assess the locomotor stimulating property
of the MPH doses used in the experiment. Mice were habituated to the locomotor activity boxes
for 1 hour per day over four days. Subsequently, over the course of three test days separated by
72 hours each, mice were placed in locomotor chambers for one hour to stabilize baseline
activity, after which each mouse received vehicle, 3.5 or 5 mg/kg MPH i.p. following a Latin
square design and had their activity monitored for another hour.
8 Experiment 3: Assessment of explicitly unpaired CS and US presentations and responding for a non-conditioned stimulus in mice
Twelve new C57Bl/6 and 12 CD-1 mice under 22 hours of water restriction received 14 days of
training similar to mice in experiment 1, but with an explicitly unpaired CS and US. In this case,
16
extinguishing of the houselight and 5 second illumination of both stimulus lights was never
contingent with the elevation of the dipper and presentation of saccharin.
9 Experiment 4a: Effect of whole-brain DA transporter overexpression on responding for a conditioned reinforcer
Transgenic mice overexpressing DAT (DAT-Tg) were used in this experiment in an attempt to
examine the control DAT exerts over incentive motivation. Water restricted male (wild-type
(mWT), n = 6; mDAT-Tg, n = 5) and female (fWT, n = 4; fDAT-Tg, n = 4) C57Bl/6 mice of
varying ages obtained from the Salahpour lab at U of T underwent the same Pavlovian training
as mice in Experiment 1. Following training, one day of habituation to the lever press operant
was received to eliminate novelty driven responding during testing. Responding for a CR in a
drug-free state was then examined in all mice for three days.
10 Experiment 4b: Effect of MPH and AMPH on responding for a CR in DAT overexpressing mice
Following three baseline tests of responding for a CR, the ability of MPH and AMPH to alter
responding in DAT overexpressing mice was examined. Over the course of two days separated
by 72 hours, mice received either saline or 5 mg/kg MPH in a random order just prior to
assessment of responding for a conditioned CR. Following testing with MPH, the same mice
(with the exception of one fWT mouse; fWT, n = 3) received i.p. administration of saline, 0.2, or
1 mg/kg AMPH sulphate (U.S. Pharmacopeia, Rockville, MD) prior to assessment of responding
for a CR in a random order over the course of three days each separated by 72 hours.
11 Statistical Analyses
SPSS version 15.0 was used to perform three-way repeated measures ANOVAs; paired samples
t-tests were used for post-hoc comparisons between means. Within-subjects variables used in
ANOVAs included: Lever or Port, distinguishing operant response type and CR or NCR operant
responses; Day of training and testing; and Dose of MPH or AMPH. Between-subjects variables
used in ANOVAs included: Strain (C57 or CD1) and Genotype (WT or DAT overexpressing
mice).
17
Chapter 3 Results
1 Experiment 1a: Acquisition, expression, and amplification of responding for a conditioned reinforcer
1.1 Training
Figures 1A and 1C show that over the course of Pavlovian conditioning, both C57Bl/6 and CD-1
mice learned to approach the location of a saccharin reward during CS presentations. Thus, both
strains showed increased responding during CS periods compared to pre-CS periods (CS vs
PreCS: F(1, 45) = 113.213, p < 0.001). This discrimination in responding emerged over time (Day
x CS vs PreCS: F(13, 585) = 64.607, p < 0.001). The lack of a significant main effect of strain (F(1,
45) = 0.007, ns) confirmed that the two strains were not different in their response patterns.
Figures 1B and 1D illustrate that, while early in training mice failed to respond for the US, as
training progressed the number of “missed” trials fell (Day: F(13, 598) = 107.94, p < 0.001), and the
number of responses made during the US presentation increased (Day: F(13, 585) = 68.60, p <
0.001). These measures did not differ between strain (Misses, Strain x Day: F(13, 598) = 0.729, ns;
US, responses Strain x Day: F(13, 585) = 0.194, ns).
1.2 Test of responding for a CR
Figures 2A and 2C show the effect of MPH on lever press responding for a CR in CD1 and
C57Bl/6 strains. Significant main effects of Lever (F(1, 22) = 21.54, p < 0.001) and Strain (F(1, 22) =
6.31, p < 0.05) were found, suggesting that responding on the CR lever was higher than on the
NCR lever, and that CD1 mice made more responses than C57Bl/6 mice. Although the main
effect of Dose was not significant (F(3, 66) = 2.426, p = 0.07), a significant interaction between
Lever and Dose was found (F(3, 66) = 3.66, p < 0.05), which suggested that MPH increased
responding on the CR lever. Selective post hoc tests determined that 3.5 mg/kg of MPH
enhanced lever pressing for a CR only in CD1 mice.
18
Figures 2B and 2D show the effects of MPH on nosepoke responding for a CR in both strains of
mice. A significant main effect of Port (F(1, 22) = 20.12, p < 0.001) indicated that responding on
the CR port was higher than on the NCR port. The total number of responses was similar
between strains (Strain: F(1, 22) = 1.087, ns), and MPH had no effect on responding at any dose
tested (Dose: F(3, 66) = 0.796, ns)
2 Experiment 1b: Temporal stability and subsequent amplification of responding for a conditioned reinforcer
2.1 Extended CR responding
Figures 3A and 3C show lever pressing for a CR in both mouse strains over 13 days of testing. A
significant main effect of Lever (F(1, 22) = 36.42, p < 0.001), and Strain (F(1, 22) = 7.03, p < 0.05)
suggested that responding on the CR lever was higher than on the NCR lever, and that
responding was higher in CD1 than in C57Bl/6 mice. For both strains responding on the CR and
NCR levers remained stable over the 13 days of testing (Day: F(12, 264) = 3.74, ns; Day x Strain:
F(12, 264) = 3.74, ns)
Figures 3B and 3D illustrate nosepoking for a CR in both mouse strains over 13 days of testing.
A significant main effect of Port (F(1, 22) = 56.11, p < 0.001) reflected that responding was higher
on the CR nosepoke port compared to the NCR port. The ANOVA also showed a significant
three way interaction between Port, Test day and Strain (F(12, 264) = 4.91, p < 0.01), which implies
that the interaction between Port and Test day varied between strains. This is most likely
explained by the variability in responding specifically by CD1 mice within the first seven days.
However, over each test day, responding on the CR port was always higher than the NCR port.
2.2 MPH test 2
Figures 4A and 4C show the effects of MPH on lever pressing for CR in both mouse strains in a
second drug test. The ANOVA found significant main effects of Lever (F(1, 22) = 77.70, p <
0.001), Strain (F(1, 22) = 9.62, p < 0.01), and Dose (F(2, 44) = 3.34, p < 0.05). These main effects
reflect the fact that responding was higher on the CR lever compared to the NCR lever, that
MPH increased responding, and the CD1 mice responded at higher levels than C57Bl/6 mice.
The Lever x Dose interaction was significant (F(2, 44) = 6.04, p < 0.01), indicating that MPH
19
increased responding on the CR lever. The overall three-way interaction was not significant;
however post-hoc tests showed that 3.5 and 5 mg/kg MPH increased responding on the CR lever
only in CD1 mice.
Figures 4B and 4D show the effects of MPH on nosepoking for a CR in both mouse strains.
Responses producing the CR were higher than NCR responses (main effect of Port, F(1, 22) =
54.60, p < 0.001), and the total number of responses made was generally higher in CD1
compared to C57Bl/6 mice (main effect of Strain, F(1, 22) = 5.87, p < 0.05). However, MPH
appeared to have no effect on responding at any doses tested (no main effect of dose, F(2, 44) =
0.267, p = ns). None of the interaction terms were significant.
3 Experiment 1c: Extinction of responding for a conditioned reinforcer
3.1 Removal from water restriction
Figures 5A and 5C illustrate lever press responding for a CR in both strains of mice following
removal from water restriction. Responding on the CR lever remained higher than on the NCR
lever in general (Lever: F(1, 22) = 18.62, p < 0.001), and CD1 mice made more responses than
C57Bl/6 mice (Strain: F(1, 22) = 8.61, p < 0.01). However, a Strain x Day two-way interaction (F(5,
110) = 3.6, p < 0.01) suggested the stability of responding differed between strains, likely a result
of the variability seen in CD1 mice during this period. The three way interaction was not
significant.
Figures 5B and 5D illustrate nosepoke responding for a CR in both strains of mice following
removal from water restriction. Responding on the CR nosepoke port remained higher than on
the NCR port (Port: F(1, 22) = 44.62, p < 0.00), and CD1 mice made more responses than C57Bl/6
mice (Strain: F(1, 22) = 7.49, p < 0.05). Although the magnitude of responding did not remain
stable over test days (Day: F(5, 110) = 8.6, p < 0.01), both strains followed a similar pattern of
responding (Strain x Day: F(5, 110) = 1.71, ns). Relative responses on the CR and NCR ports
changed over the course of testing (Port x Day: F(5, 110) = 8.28, p < 0.001), but responding on the
CR port was always higher than the NCR port. The three-way interaction was not significant.
20
3.2 Operant responding during extinction conditions
Figures 6A and 6C show lever pressing during extinction conditions in both strains of mice. No
significant main effect of Lever was observed (F(1, 22) = 1.75, ns), suggesting that responding on
the CR lever did not differ from responding on the NCR lever.
Figures 6B and 6D show nosepoke responding during extinction conditions in both strains of
mice. Responding on the CR nosepoke port greatly decreased over extinction training days (Port
x Day: F(8, 176) = 12.64, p < 0.001).
4 Experiment 2: Locomotor activation by 3.5 and 5 mg/kg doses of MPH
Figure 7 depicts the effect of MPH to enhance locomotor activity as measured by total beam
breaks. CD1 mice were more active than C57Bl/6 mice in general (Strain: F(1, 30) = 14.70, p <
0.01), and MPH enhanced locomotor activity in both strains (Dose: F(2, 60) = 45.43, p < 0.001;
Dose x Strain: F(2, 60) = 2.38, ns). Paired samples t-tests determined that 3.5 and 5 mg/kg MPH
significantly enhanced locomotor activity in both strains (t(15)-values > 5.14, all p-values < 0.05).
5 Experiment 3: Assessment of explicitly unpaired CS and US presentations and responding for a non-conditioned stimulus in mice
5.1 Training
Figures 8A and 8B illustrate that neither C57Bl/6 nor CD1 mice increased their approach to the
reward magazine during CS presentation; in fact, magazine responses during the CS period were
significantly lower than those during the 5s pre-CS period in general (main effect of CS vs
PreCS, F(1, 22) = 82.22, p < 0.001).
Figure 8C and 8D illustrate that as training progressed the number of “missed” saccharin
presentations decreased (Day: F(13, 286) = 11.25, p < 0.001). However this occurred at different
rates between strains in that CD1 mice decreased in number of misses more slowly than C57Bl/6
mice (Strain: F(13, 286) = 4.5, p < 0.05; Strain x Day: F(13, 286) = 2.83, p < 0.01).
21
5.2 Test of responding for a CR
Figures 9A and 9B show lever press responding for a CS that was never paired with the
saccharin US in both strains of mice. No difference was observed between responses made on
the CR and NCR levers (Lever: F(1, 22) = 0.03, ns).
6 Experiment 4a: Effect of whole-brain DA transporter overexpression on responding for a conditioned reinforcer
6.1 Training
Data were pooled between sexes in experiment 4a and 4b as no sex differences were apparent in
either genetic background (data not shown). Figures 10A and 10B show that over the course of
Pavlovian conditioning, both DAT-Tg and WT mice learned to approach the location of a
saccharin reward during CS presentations. DAT-Tg and WT mice did not differ in their response
patterns (Strain: F(1, 18) = 2.55, ns). Thus, both strains showed increased responding during CS
periods compared to pre-CS periods (CS vs PreCS: F(1, 18) = 122.26, p < 0.001). This
discrimination emerged over time (Day x CS vs PreCS: F(13, 234) = 18.48, p < 0.001).
6.2 Test of responding for a CR
Figures 11A and 11B show lever press responding for a CR in a drug-free state in both DAT-Tg
and WT genotypes of mice. Responding was selective for the CR lever relative to the NCR lever
(Lever: F(1, 17) = 75.72, p < 0.001), and no difference in magnitude of CR responding between
DAT-Tg and WT mice was apparent (Genotype: F(1, 17) = 0.44, ns; Genotype x Lever: F(1, 17) =
0.45, ns).
7 Experiment 4b: Effect of MPH and AMPH on responding for a CR in DAT overexpressing mice
7.1 Effect of MPH
Figure 12 illustrates the effect of MPH on lever press responding for a CR in both DAT-Tg and
WT mice. Responding on the CR lever was significantly higher than on the NCR lever (Lever:
F(1, 16) = 193.39, p < 0.001), but this did not differ between DAT-Tg and WT mice (Genotype x
22
Lever: F(1, 16) = 1.53, ns). Administration of MPH had no effect on responding for a CR (Dose:
F(1, 16) = 0.12, ns; Dose x Lever: F(1, 16) = 1.83, ns) in both DAT-Tg and WT mice (Genotype x
Dose X Lever: ns).
7.2 Effect of AMPH
Figure 13 shows the effect of AMPH on responding for a CR in both DAT-Tg and WT mice.
Responding on the CR lever was significantly higher than on the NCR lever (main effect of
Lever, F(1, 16) = 147.10, p < 0.001). A significant main effect of AMPH was also found (F(2, 32) =
19.70, p < 0.001), with AMPH reducing responding at the highest dose. The main effect of
genotype was not significant. The interaction between lever and dose was significant (F(2, 32) =
8.54, p < 0.01), with amphetamine reducing CR responding only. This effect was seen in both
DAT-Tg and WT mice (Genotype x Dose X Lever: ns).
23
Chapter 4 Discussion
This series of experiments provides a detailed characterization of the acquisition of a new
response by conditioned reinforcement in mice, and the augmentation of this behaviour by
changes in DAergic activity in the brain. The results presented here show that mice learn to
approach a CS associated with a reward, and that this CS acquires incentive salience which
drives behaviour towards obtaining it when acting as a CR. Furthermore, selective responding
for a CR is stable over multiple test days, is modulated by MPH administration, and extinguishes
when responding no longer results in CR presentation. Finally, mice chronically overexpressing
DAT exhibit no deficits in learning approach towards a reward associated CS, salience
attribution to this CS, and responding for this CS acting as a CR.
1 Characterization of the acquisition and expression of conditioned reinforcement in mice
The first goal of these experiments was to assess the ability of mice to learn approach behaviour
towards a reward predictive CS and selectively respond to obtain that CS when it acts as a
reward. Both inbred C57Bl/6 and outbred CD1 mouse strains display these facets of conditioned
reinforcement, suggesting that this behaviour is reliable in mice and therefore could be used as
an effective tool in examining brain mechanisms involved in incentive motivation. It is also clear
that the association with a primary reward is critical for imbuing the CS with incentive salience,
subsequently allowing it to act as a CR. Responding for a CR is also strikingly stable over
multiple test days in the two strains and on two types of operant response. With such extended
testing and repetition of this operant behaviour, it is possible that responding may have become
habitual and no longer driven by incentive motivation directed at the CR. However, although not
driven by motivational state (i.e. thirst), responding for the CR during this extended testing
period is still driven by its incentive motivational properties. When the CR was omitted
following lever and nosepoke responses that normally produced it, selective responding on those
levers and ports was abolished within three days of testing. This latter finding shows that
responding directed towards the CR was not habitual, and did indeed depend on the outcome of
the behavioural response. Overall, these results suggest that mice effectively attribute a reward
24
associated CS with incentive salience enabling it to act as a CR, and this salience driven response
pattern is stable over an extended period of testing.
2 Strain differences in conditioned reinforcement
Both inbred C57Bl/6 and outbred CD1 mice show reliable responding for a CR which is resistant
to decrement as a result of repeated testing, but extinguishes when the operant response no
longer produces the CR. Although some variability in response patterns were present such as
CD1 mice making more responses on average, both strains exhibited significantly higher
responding on the manipulandum delivering the CR as opposed to the one with no programmed
consequence (NCR).
The higher and more variable responding exhibited by CD1 mice may stem from a higher basal
activity level within the DA system and differential susceptibility to DAergic drugs between
strains. CD1 mice showed higher CR responding throughout the study compared to C57Bl/6
mice, and MPH administration resulting in a more pronounced effect on responding for a CR in
CD1 mice. Although few studies have directly compared C57Bl/6 and CD1 strains of mice in
terms of DA system activity, one study suggests that the inbred C57Bl/6 mice may have mild
decreases in DA system function relative to outbred CD1 mice. It was also suggested that CD1
mice exhibit decreases in DA system activity but with more variability, presumably based on
their genetic diversity (Prasad and Richfield, 2008). These present experiments do, however,
suggest that while both strains of mice learn about, and respond for, conditioned reinforcement,
pharmacological manipulations, at least of the DAergic system, produce a stronger effect in CD1
relative to C57Bl/6 mice. Therefore, CD1 mice may be a better choice of mouse strain in which
to demonstrate drug effects on the incentive motivational properties of reward-related cues in
mice.
3 Operant response type in responding for a CR
Two operant response types were also examined in these experiments: lever pressing and
nosepoking for a CR. While lever pressing is a well established type of response in this and other
reinforcement paradigms, nosepoking has been suggested to be a more natural behaviour in mice
(Crawley, 2007). Therefore, mice may have showed more responding for a CR following a
25
nosepoke response rather than a lever press; however, responding in fact appeared to be strongest
for lever pressing. To understand this effect, it is important to note that the nosepoke operant in
experiment 1 was not merely a substitution of levers for nosepoke ports; the operant response
elements were in different physical locations.
Nosepoke ports were located at the opposite end of the chamber from the reward magazine and
stimulus lights, while levers flanked the reward magazine. Therefore, mice engaging in either
nosepoking or lever pressing likely differed in several ways including head direction, visibility of
the visual component of the CR during the actual operant behaviour (head inside a hole versus
facing the CS), and distance from the sound of the dipper. In the lever press condition, mice are
oriented towards two major aspects of the CR that nosepoking mice are not: the stimulus lights
and the general direction of the sound that the dipper mechanism originates from. In the future, a
more thorough assessment of nosepoking relative to lever pressing for a CR should involve
substituting levers with nosepoke ports in the exact same location.
Both nosepoking and lever pressing served as effective operant behaviours for assessing
responding with conditioned reinforcement. Although the nosepoke response seemed more
variable than lever pressing over the course of experiment 1 (e.g. figure 3, particularly CD1, and
figure 5), responding on the CR port was always higher than responding on the NCR port.
However, this variability prompted the use of the more stable lever press operant in further
experiments. Thus, these experiments show that lever press responding for a CR is an effective
tool for examining cue-controlled incentive motivation in mice.
4 Modulation of responding for a CR by MPH administration in normal mice
The psychomotor stimulant MPH failed to increase responding in Experiment 1a. This result was
surprising given that this class of drug reliably increases CR responding in rats (Robbins, 1976;
Beninger and Phillips, 1980; Taylor and Robbins, 1984; Ito et al., 2000; Fletcher et al
unpublished observations). However, MPH has previously been shown to not significantly
enhance responding for a CR unlike other psychostimulants (Robbins, 1978). In the present
experiment mice were administered saline or MPH in a Latin square design over four days. This
meant that many mice had MPH “on board” during, and experienced injections just prior to, their
26
very first assessment of this new behaviour. This may have resulted in a higher variability in
behaviour over these test days not reflective of true levels of responding for a CR. However, a
second assessment of the effect of MPH on responding for a CR following a period of
stabilization occurred in experiment 1b where MPH greatly enhanced lever pressing for a CR in
CD1 mice but not C57Bl/6 mice. As described in previously, this may relate to differences in DA
system of CD1 mice compared to C57Bl/6 mice. Nosepoke responding for a CR, however, did
not appear to be affected by any dose of MPH. Based on the confounding physical differences
between lever press and nosepoke responding in these experiments it is difficult to specify the
exact reason for this.
5 Incentive salience attribution and motivation in DAT upregulated mice
One reason for characterizing the conditioned reinforcement paradigm in mice was the potential
utility of using behavioural genetic techniques to examine underlying mechanisms of incentive
motivation and salience attribution. Given that MPH, which elevates extracellular DA (Schweri
et al., 1985), increased responding for CR we hypothesized that reduced DA activity would
impair this response. We tested this hypothesis using a transgenic mouse that has a three-fold
increase in DAT and consequent 40% decrease in DAergic tone in the striatum. These mice have
previously shown an increase in susceptibility to AMPH reward and possible reductions in basal
motivation to obtain a reward relative to wild-type animals (Salahpour et al., 2008). However, no
detriments were expected in forming a CS-US association as measured by approach to the CS
during US presentation, as this conditioned approach does not appear to require phasic DA
output (Berridge, 2007; Parker et al., 2010). However, lower DAergic tone was expected to
result in decreased incentive salience of this CS, which would suppress the motivation to respond
for it when acting as a CR. For example DA receptor antagonists which functionally block DA
activity reduce responding for CR (Beninger et al., 1980; Ito et al., 2000). Therefore, DAT
upregulated mice were expected to exhibit less responding for a CR compared to wild-type
control mice.
C57Bl/6 mice overexpressing DAT showed no impairments in learning conditioned approach to
a reward associated CS, which is consistent with reports in rats showing that impaired DA
27
function does not block approach to a reward-related CS (Taylor and Robbins, 1984; Parker et
al., 2010). Unexpectedly, however, these mice despite their hypodopaminergic state showed no
impairments in responding for that CS acting as a CR. In comparison to the previous study by
Salahpour and colleagues (2008), these results suggest that mice chronically expressing whole-
brain upregulation of DAT do not exhibit major impairments in incentive salience attribution or
incentive motivation towards a cue associated with a natural reward.
One reason for this lack of effect could be a compensatory presynaptic mechanism of
upregulating the amount of DA released from vesicles to counteract the more rapid uptake of DA
which has developed as a result of chronic DAT overexpression. DAT knockout mice exhibit a
similar, but opposite form of compensatory mechanism, in that vesicular DA release is
downregulated 73% in homozygous and 34% in heterozygous mice (Giros et al., 1996). A
second possibility is a postsynaptic compensatory mechanism such as D1 receptor upregulation,
which may work in concert with presynaptic compensatory mechanisms to alleviate deleterious
effects of DAT upregulation. Phasic DAergic neurotransmission activates D1 receptors in the
NAc and DAT knockout mice exhibit a 55% downregulation in D1 receptor mRNA in the basal
ganglia (Giros et al., 1996; Goto and Grace, 2005). Therefore, these compensatory mechanisms
may facilitate signalling to normal levels in DAT upregulated mice by competing with the
enhanced uptake of DA back into presynaptic terminals, ultimately rescuing the effects of DAT
upregulation.
6 Effect of MPH and AMPH on responding for a CR in DAT upregulated mice
In an attempt to highlight the role of DAT in modulating DAergic tone and therefore the
incentive salience of a CR, MPH and AMPH were administered to DAT upregulated mice. These
two psychostimulants have two disparate mechanisms of action at the DAT. AMPH reverses
DAT following breakdown of presynaptic DA containing vesicles, making the AMPH effect
dependent on DAT quantity and not necessarily dependent on phasic DA output, while MPH
blocks reuptake of DA following burst firing (Schweri et al., 1985; Giros et al., 1996). DAT
upregulated mice have previously shown major increases in locomotor activity and a leftward
28
shift in the dose required for induction of conditioned place preference compared to wild-type
mice following administration of AMPH, but not following MPH (Salahpour et al., 2008).
Administration of 5 mg/kg MPH to DAT upregulated mice found only a slight, albeit not
significant increase in responding for a CR. However, administration of this dose of MPH also
showed no effect compared to saline in this set of wild type mice, which was unexpected based
on the results obtained from experiment 1b. The sample size of each group was very small in this
experiment and variability within these groups in terms of mouse age and sex was high; perhaps
with a larger sample size in each condition, the true effect of MPH administration may be more
apparent.
Administration of AMPH had very interesting effects on all mice. A relatively high 1 mg/kg dose
of AMPH abolished general operant responding in both DAT upregulated and wild-type mice, in
that both CR and NCR lever responding dropped greatly compared to saline. Interestingly, this
decrease in responding on both the CR lever NCR lever was proportional between DAT
upregulated and wild-type mice. This may reflect an AMPH induced shift of salience away from
performing the operant task in general which would decrease responding for a CR. However, a
low 0.2 mg/kg dose of AMPH appeared to have no major effect on responding for a CR or on the
NCR lever, besides a slight decrease in responding on the CR lever in DAT upregulated mice
which did not reach significance.
Therefore, administration of either MPH or AMPH appeared to have no differential effect on
mice with upregulated DAT compared to their WT controls. This may be a result of pre- or
postsynaptic compensatory mechanisms in DAT upregulated mice which rescue basal DAergic
tone in these mice and prevents psychostimulants from having any major effect on operant
responding relative to wild-type mice. However, an important limitation of this pilot experiment
was a small sample size in each group and variability in the mice used in terms of age and sex.
Utilizing a larger sample size of these transgenic mice and a limited age group may provide more
reliable results in the future.
29
Chapter 5 Conclusions
Although Mead and colleagues (2004), among others, have used the conditioned reinforcement
paradigm in mice previously, a thorough inspection of its viability has not been performed. The
present set of experiments has shown that using mice in this behavioural task is, in fact, reliable
and valid. Inbred and outbred strains of mice: 1) learn conditioned approach to a stimulus
predicting reward, 2) will perform different operant responses for the CS when it acts as a CR, 3)
show remarkable stability in responding for this CR over multiple weeks of testing, 4) exhibit at
least slight enhancements in responding for a CR as a result of MPH induced enhancements in
DAergic tone, and 5) extinguish responding for the CR when the operant response no longer
results in its presentation. This experiment also shows that the motivation to obtain the CR is
driven by its incentive salience as a result of being associated with a natural reward. It is
important to note that the data presented suggest that strains of mice may differ in their
magnitude of CR responding and responsivity to DAergic drugs. Therefore the strain of mouse
used in this paradigm should be carefully selected based on the manipulation of responding for a
CR.
The primary reason for examining the conditioned reinforcement paradigm in mice was to utilize
transgenic techniques in analyzing the underlying mechanisms of incentive motivation and
salience attribution. Mice chronically expressing a three-fold increase in DAT and a consequent
40% decrease in striatal DA showed no differences from wild-type animals in either conditioned
approach or conditioned reinforcement. This suggests that, perhaps by some long-term
compensatory mechanisms, these mice do not exhibit impairments in basic incentive motivation.
Administration of 5 mg/kg MPH, or 0.2 or 1 mg/kg AMPH did not significantly alter responding
relative to wild-type animals. However, the true effects of psychostimulant administration may
not have been captured due to the variability in sex and age of mice used in this experiment.
Therefore, acquisition of a new response with conditioned reinforcement is a reliable behavioural
paradigm in mice, and can be used in concert with transgenic techniques to further investigate
underlying mechanisms of incentive motivation.
30
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Chapter 7
Figure Captions
Figure 1: C57Bl/6 (n = 24) and CD1 (n = 24) mice both learn Pavlovian conditioned approach to a
reward location directed by a CS. A and C: Head entries made into the reward magazine upon CS
presentation increase over training compared to those made during a 5-second period just prior to CS
onset. B and D: Number of magazine entries upon saccharin presentation (US presentation, during the last
8s of the CS) increased while number of missed reward presentations decreased over training in both
strains.
Figure 2: Initial assessment of the effect of MPH on lever pressing and nosepoking for a CR in C57Bl/6
and CD1 mice. The number of CR and NCR responses was compared across saline, 2.5, 3.5 and 5 mg/kg
MPH. In both strains and operant response types, mice selectively responded for the CR. A and C: MPH
did not enhance lever pressing for a CR in C57Bl/6 mice (n = 12), while 3.5 mg/kg MPH enhanced CR
responding in CD1 mice (n = 12) relative to saline. B and D: No dose of MPH enhanced nosepoke
responding for a CR in either C57Bl/6 (n = 12) or CD1 (n = 12) mice. * Indicates p < 0.05 relative to
saline.
Figure 3: Extended assessment of responding for conditioned reinforcement in C57Bl/6 and CD1 mice
over 13 days of testing. A-D: In both strains, lever pressing and nosepoke (in each condition, n = 12)
responding for the CR remained significantly higher than NCR responding on all days.
Figure 4: Second assessment of the effect of MPH on lever pressing and nosepoking for a CR in C57Bl/6
and CD1 mice. The number of CR and NCR responses was compared across saline, 3.5 and 5 mg/kg
MPH. A and C: Both 3.5 and 5 mg/kg MPH slightly increased lever pressing for a CR in C57Bl/6 mice (n
= 12) and significantly enhanced CR responding in CD1 mice (n = 12). B and D: Neither dose of MPH
enhanced nosepoke responding for a CR in C57Bl/6 (n = 12) or CD1 (n = 12) mice. * Indicates p < 0.05
relative to saline.
38
Figure 5: Phase 1 of response extinction: comparison of CR and NCR responding on lever press and
nosepoke operants over six days following removal from water restriction. A and C: Responding on the
CR lever remained significantly higher than on the NCR lever in C57Bl/6 mice (n = 12) following
removal from water restriction, while this appeared to result in more erratic responding by CD1 mice (n =
12). B and D: Nosepoke responding for a CR was resistant to removal from water restriction in both
C57Bl/6 (n = 12) and CD1 (n = 12) mice.
Figure 6: Phase 2 of response extinction: true extinction of conditioned reinforcement was achieved by
omitting CR presentations after responding. The number of CR and NCR responses were compared over
nine tests during which CR lever pressing or nosepoking had no programmed consequence. A and C: In
C57Bl/6 mice (n = 12), selective CR lever pressing decreased to NCR levels within three days of
extinction conditions, while CR lever pressing in CD1 mice (n = 12) dropped to NCR levels on the first
day of extinction. B and D: Nosepoking for a CR greatly decreased and approached NCR levels under
extinction conditions in both C57Bl/6 (n = 12) and CD1 (n = 12) strains, but remained significantly
higher during the nine-day period.
Figure 7: Locomotor stimulating effects of MPH. Locomotor activity was measured as the number of
beams broken in a 60-minute period after treatment with saline, 3.5, and 5 mg/kg MPH. Both C57Bl/6 (n
= 16) and CD1 (n = 16) mice exhibited significantly enhanced motor activation by 3.5 mg/kg of MPH,
which did not differ significantly from a 5 mg/kg dose. * Indicates p < 0.05 relative to saline.
Figure 8: Mice did not learn Pavlovian conditioned approach towards the CS used in experiment 1 when
it is explicitly unpaired with the saccharin reward. A and C: C57Bl/6 (n = 12) and CD1 (n = 12) mice did
not increase in number of head entries to a reward magazine during non-associated stimulus onset (when
reward is unavailable). B and D: Number of missed reward presentations declined in both strains of mice,
although at a less rapid pace in CD1 mice; mice increasingly obtain the rewards over training, the
presence of which are unsignalled.
Figure 9: In unpaired mice, responses on the CR lever were compared with the NCR lever over four days
of drug-free assessment of conditioned reinforcement. Neither C57Bl/6 (n = 12) nor CD1 (n = 12) showed
a preference for the lever that resulted in presentation of the non-associated stimulus.
39
Figure 10: Mice overexpressing DAT exhibit normal Pavlovian conditioned approach to a reward-
predictive CS. Wild-type (WT, A; n = 10) and DAT overexpressing (DAT-Tg, B; n = 10) both increase
reward magazine entries during CS onset over the course of training relative to a 5s pre-CS period.
Figure 11: Mice overexpressing DAT exhibit normal responding for conditioned reinforcement.
Responding on the CR and NCR lever over three test days was compared between backgrounds of mice.
Both WT (A; n = 10) and DAT-Tg (B; n = 9) mice exhibited selective responding on the CR lever relative
to the NCR lever. The number of CR responses made on each day was also similar between WT and
DAT-Tg mice.
Figure 12: Effects of MPH on responding for a CR in DAT overexpressing mice. Responses made on the
CR and NCR levers were compared between WT and DAT-Tg mice following either saline or 5 mg/kg
MPH treatment. MPH did not significantly enhance responding for a CR in either WT (A; n = 9) or DAT-
Tg (B; n = 9) mice.
Figure 13: Effects of AMPH on responding for a CR in DAT overexpressing mice. Responses made on
the CR and NCR levers were compared between WT (A; n = 9) and DAT-Tg (B; n = 9) mice following
saline, 0.2, and 1 mg/kg AMPH. Administration of 1 mg/kg AMPH significantly attenuated responding
on both CR and NCR levers relative to saline and 0.2 mg/kg AMPH in both WT and DAT-Tg mice, while
0.2 mg/kg AMPH had no effect in either group of mice. * Indicates p < 0.05 relative to saline.
40
Chapter 8
Figures
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Figure 2
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Figure 7
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Figure 9
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Figure 11
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Figure 12
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Figure 13