approach and avoidance processing: investigating a ... · in motivation in response to conditioned...

Approach and Avoidance Processing: Investigating a Rostrocaudal Gradient in the Nucleus Accumbens Core

by

Laurie Hamel

A thesis submitted in conformity with the requirements for the degree of Master of Arts

Department of Psychology University of Toronto

© Copyright by Laurie Hamel 2015

Approach and Avoidance Processing: Investigating a

Rostrocaudal Gradient in the Nucleus Accumbens Core

Laurie Hamel

Master of Arts

Department of Psychology University of Toronto

2015

Abstract

The nucleus accumbens is a site of integration of positively and negatively valenced information.

While a rostrocaudal topographical gradient in valence processing has been found in the

accumbens shell, a potential gradient has not fully been explored in the core in relation to its role

in motivation in response to conditioned cues. In the current study, rats were trained to associate

visual cues with appetitive, aversive and neutral outcomes. In a test of motivational bias, the

aversive and appetitive cues were superimposed in a maze arm and rats’ exploratory bias was

measured for this arm vs. a neutral cued arm. Animals receiving GABA receptor agonists in the

caudal region displayed a bias in the direction of aversion, whereas those undergoing inactivation

of the rostral core displayed an ambivalence similar to controls, with an additional behavioural

difference of augmented chewing. This suggests a rostrocaudal differentiation in valence

processing in the core.

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Acknowledgments I would like to thank my supervisor, Dr. Rutsuko Ito, for the opportunities she has given me, for

her guidance, and for always being kind and encouraging. I thank my committee members, Dr.

Suzanne Erb and Dr. Takehara-Nishiuchi, for their feedback and for the interesting questions

they have elicited. I want to thank Dr. Anett Schumacher for all of the skills she has taught me

and for always being cheerfully available to help. Finally I would like to thank my labmate

David Nguyen for all of his intellectual and moral support.

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Table of Contents

1 Introduction .......................................................................................................................................... 1

2 Methods ............................................................................................................................................. 12

2.1 Subjects ..................................................................................................................................... 12

2.2 Surgery....................................................................................................................................... 13

2.3 Conditioned Cue Preference Task ........................................................................................ 13

2.4 Training Procedures ................................................................................................................. 14

2.5 Drugs and Infusions ................................................................................................................. 15

2.6 Testing Procedures .................................................................................................................. 15

2.7 Data Analysis ............................................................................................................................ 17

2.8 Histology .................................................................................................................................... 17

3 Results ............................................................................................................................................... 17

3.1 Histology .................................................................................................................................... 17

3.2 Training ...................................................................................................................................... 18

3.3 Conflict test ................................................................................................................................ 18

3.4 Novelty Preference Test .......................................................................................................... 20

4 Discussion ......................................................................................................................................... 20

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List of Figures

Figure 1 49

Figure 2 49

Figure 3 50

Figure 4 50

Figure 5 51

v

List of Diagrams Diagram 1 52

Diagram 2 52

Diagram 3 53

Diagram 4 53

Diagram 5 54

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1 Running Head: APPROACH-AVOIDANCE PROCESSING 1 Introduction

The most fundamental decisions that an organism must make in order to ensure survival and

reproduction involve the resolution of approach versus avoidance directed toward environmental

signals. Examples of critical behavioural challenges which have persisted over evolutionary

history include foraging for food and water and seeking mates while under the threat of predation

and competition. The main psychological functions that must be performed in service of these

goals include assigning a motivational value, or “valence”, to stimuli based on innate knowledge

and learned experiences, weighting potentially conflicting contingencies, and orchestrating

appropriate behavioural responses in response to a constantly changing internal and external

environment (Cosmides & Tooby, 2013; Tooby & Cosmides, 1990).

In humans, approach and avoidance decisions incorporating ideological values and long-term

goals can be much more complex than those made by other animals, but the brain systems that

evolved to process motivationally significant information are highly conserved across

phylogeny. As such, both rational and sometimes irrational decision-making is performed using

circuitry derived from the heritage of motivated decisions made by our mammalian ancestors

(Levine, 2009; MacLean, 1990). Given that imbalances in the brain’s processing of appetitive

versus aversive information are thought to underlie diverse conditions encompassing addiction,

fear disorders, dysregulated eating, depression and schizophrenia, a deeper understanding of the

neurobiological mechanisms mediating these functions is an overarching goal of many

researchers (Aupperle & Paulus, 2010; Grace, 2010; Robinson & Berridge, 2000).

In mammals, emotional and motivational functions are instantiated in a network of brain

structures including the hypothalamus, neocortical regions, and limbic sites such as the

hippocampus, amygdala and nucleus accumbens (Nieh, Kim, Namburi, & Tye, 2013). The

accumbens is a structure of particular interest given that it is a site of confluence in the

processing of valenced information from many other regions. Mogenson, Jones, and Yim (1980)

corralled evidence and developed the influential idea that the nucleus accumbens is a motor-

limbic interface functionally linking motivation and action. The origin of this idea was attributed

to Graybiel (1976), who noted that the accumbens, by virtue of its position receiving inputs from

limbic structures processing drives and emotions and projecting to basal ganglia structures

2 APPROACH-AVOIDANCE PROCESSING

implicated in the initiation of actions, was ideally situated for this role. The accumbens’ major

dopaminergic innervation from the ventral tegmental area of the midbrain (Moore & Bloom,

1978) was also an important factor in this model, since dopamine had already been implicated in

mediating motivated behaviour (Kelly, Seviour, & Iversen, 1975; Ungerstedt, 1971).

The accumbens and its dopamine innervation have continued to be implicated in reward and

motivation for natural reinforcers such as food and sex, as well as drugs of abuse, which may be

viewed as hijacking the natural reward system (Kelley & Berridge, 2002). The breadth of reward

function subserved by the accumbens is illustrated by studies of food-seeking. The accumbens

has been implicated in mediating the palatability and affective responses to food, the

motivational and approach functions of consumption, as well as in instrumental learning about its

acquisition (Kelley, 2004). Drugs of abuse from diverse categories, including stimulants,

opioids, ethanol and nicotine share the property of triggering dopamine release preferentially in

the nucleus accumbens (Di Chiara, Imperato, & Mulas, 1987; Di Chiara & Imperato, 1988), and

intracranial self-administration studies have demonstrated that animals will work to receive

infusions of dopamine agonists into this structure (Hoebel et al., 1983; Ikemoto, Glazier,

Murphy, & McBride, 1997; Phillips, Robbins, & Everitt, 1994). Most recently, optogenetic

techniques, allowing precise temporal and spatial precision in the activation and inactivation of

neurons, have strengthened support for the role of the nucleus accumbens in mediating

reinforcement (Witten et al., 2011) and in eliciting reward-seeking behaviour in response to

drug-associated cues (Stefanik, Kupchik, Brown, & Kalivas, 2013; Stuber et al., 2011).

The precise role of the accumbens and its dopamine innervation in mediating reward continues to

be debated. For example, researchers have divergently emphasized roles in reinforcement and

hedonia (Wise, 2008), learning associations between stimuli or behaviours and reward (Cardinal

& Everitt, 2004; Day & Carelli, 2007), reward prediction error processing (Schultz, 1998), the

attribution of incentive salience or desirability to reward cues (Berridge & Robinson, 1998;

Berridge, 2007), and in the energization of behaviour toward a given goal (Roitman, Stuber,

Phillips, Wightman, & Carelli, 2004; Salamone, Correa, Mingote, & Weber, 2003). Some of the

debate may originate in a neglect to consider specific localization of function and the role of

neurotransmitters beyond dopamine within the accumbens (Pennartz, Groenewegen, & Lopes da

Silva, 1994). That is, the accumbens is likely to execute myriad functionally and anatomically

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distinguishable processes in the service of maximizing adaptive behaviour, given the attendant

computational complexity (Berridge, 2012; Zhang et al., 2009).

The abundance of studies focusing on the accumbens’ role in appetitive behaviour may have also

overshadowed evidence that it is additionally implicated in mediating aversive states; however,

there is an increasing emphasis on viewing this structure as a site of integration of both positively

and negatively valenced information (Carlezon & Thomas, 2009; Levita et al., 2009;

McCutcheon et al., 2012). Functional magnetic resonance imaging studies in humans have

demonstrated that the accumbens undergoes increased activation during exposure to physically

noxious stimuli and cues associated with disgust or fear, as well as in anticipation of aversive

stimuli and the prospect of both gains and losses (Becerra, Breiter, Wise, Gonzalez, & Borsook,

2001; Cooper & Knutson, 2007; Jensen et al., 2003; Klucken et al., 2012). The firing patterns of

cells within the accumbens have been found to be innately tuned to aversive as well as appetitive

stimuli, to develop predictive responses to them, and to be associated with aversive behavioural

output (Roitman, Wheeler, & Carelli, 2005).

Electrophysiological studies have also found that dopamine neurons projecting to the lateral

accumbens shell encode both rewarding and aversive stimuli during primary experience

(Lammel, Ion, Roeper, & Malenka, 2011), and some dopaminergic neurons have been found to

increase their firing rates in response to conditioned and unconditioned aversive stimuli

(Guarraci & Kapp, 1999; Horvitz, 2000). Microdialysis studies have further shown that aversive

or stressful experiences and conditioned aversive stimuli can result in dopamine release in the

accumbens (Salamone, 1994; Young, 2004). Dopamine has been posited to be necessary for

learned avoidance, fear and Pavlovian aversive conditioning (Levita, Dalley, & Robbins, 2002;

Parkinson, Robbins, & Everitt, 1999; Oleson & Cheer, 2013; Zweifel et al., 2011) as well as to

mediate defensive behaviours (Blackburn, Pfaus, & Phillips, 1992). Beyond the effects of

dopaminergic inputs, it has been hypothesized that the accumbens may represent affective state

along a continuum which can be modulated through specific neurotransmitter signatures

incorporating acetylcholine and endogenous opioids (Umberg, Pothos, & Emmanuel, 2011). For

example, acetylcholine may have an important role in mediating aversion, as suggested by its

release in response to satiation, conditioned taste aversion and aversive brain stimulation

(Hoebel, Avena, & Rada, 2007). Selective κ-opioid receptor agonists mimicking the endogenous

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ligand dynorphin also produce conditioned place aversions and elicit anhedonic and dysphoric

states in animal models of depression (Bals-Kubik, Ableitner, Herz, & Shippenberg, 1993;

Mague et al., 2003).

The capacity for traditionally reward-associated circuitry to mediate aversive states is also

suggested by the fact that exposure to drugs of abuse can cause neuroadaptations resulting in

depressive symptoms such as anhedonia and diminished motivation. While these effects of drug

usage may be viewed as homeostatic responses to supraphysiogical neurotransmitter levels, the

underlying circuitry appears to be naturally prepared to mediate aversive states. That is, aversive

states such as occur during drug withdrawal are suggested to result not only from a rebound

diminishment of reward system function, but due to the active recruitment of an “antireward”

system associated with stress and anxiety, and mediated by neurotransmitters such as

corticotropin releasing factor, norepinephrine and dynorphin, all of which can act upon the

nucleus accumbens (Koob & Le Moal, 2008).

The accumbens is densely innervated by the amygdala, a structure which has traditionally been

implicated in fear (Ledoux, 1995) but is now thought to signal the salience of both positive and

negative stimuli (Breiter et al., 1996; Hamman, Ely, & Hoffman, 2002; Morrison & Salzman,

2010). It is also innervated by other regions which have been found to process both rewarding

and aversive information, including the orbitofrontal cortex, cingulate cortex and the insula

(Hayes & Northoff, 2011; Vogt, 2005). Within subregions of the prefrontal cortex, there appears

to be regionally distinct processing of rewards and punishments (Monosov, Ilya, Hikosaka, &

Okihide, 2012). Other input and output structures of the accumbens have also been found to be

segregated in terms of response to positively or negatively valenced information. For example, a

contrast was found between dorsal and ventrally located dopamine neurons in the ventral

tegmental area, such that they responded reciprocally to noxious footshock stimulation

(Brischoux, Chakraborty, Brierly, & Ungless, 2009). Thus it appears that a reiterative

representation of both appetitive and aversive information may occur over multiple network

sites, including within the accumbens. The incorporation of bivalenced information in the

accumbens coheres with its postulated role as an arbitrator of behavioural output, whereby

multiple potentially conflicting outcomes must be considered (Humphries & Prescott, 2010). In

this light, human neuroimaging studies have reported accumbens activation to correlate with the

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amount of risk involved in decision-making (Kuhnen & Knutson, 2005; Tom, Fox, Trepel, &

Poldrack, 2007).

Given the heterogeneity of functions which have been attributed to the accumbens and the

evidence that other brain structures appear to have dedicated subregions in the processing of

contrasting valence, researchers have increasingly attempted to refine the localization of different

processes and neurochemical characteristics. The nucleus accumbens has been subdivided

morphologically, functionally and histochemically into two major subterritories: a core and a

shell which extends medially, ventrally and laterally around it (Meredith, Baldo, Andrezjewski,

& Kelley, 2008; Voorn, Gerfen, & Groenewegen, 1989). The two subregions differ significantly

in their afferent input and efferent projections. For example, different subcompartments of the

amygdala, hippocampus, and prefrontal cortex project to the shell versus the core. The output of

the core connects extensively to basal ganglia motor output structures including the ventral

pallidum, subthalamic nucleus and substantia nigra, whereas the shell projects preferentially to

subcortical limbic regions including the lateral hypothalamus, ventral tegmental area and

brainstem autonomic centers (Heimer, Zahm, Churchill, Kalivas, & Wohtlmann, 1991; Zahm &

Brog, 1992). Functional magnetic resonance imaging studies have also found distinct patterns of

connectivity with prefrontal cortical and subcortical limbic targets between the core and shell,

along with differentiable activation patterns in response to both rewarding and aversive events

and their predictive cues (Baliki et al., 2013).

The shell in particular has been posited to mediate the primary or unconditioned rewarding

effects of drugs. Supporting this idea, it has been found that drugs and natural reinforcers

preferentially increase dopamine release in the shell (Aragona, Cleaveland, Stuber, Day, &

Carelli, 2008; Ito et al., 2001; Pontieri, Tanda, & Di Chiara, 1995), and animals will self-

administer dopamine agonists and reuptake inhibitors specifically into the shell, as opposed to

the core (Carlezon, Devine, & Wise, 1995; Rodd-Henricks, McKinzie, Li, Murphy, & McBride,

2002). Primary reward functions are closely linked with learning; that is, it is thought that the

primary reinforcing experience, as can be elicited by dopamine in the accumbens shell, is an

integral agent in forming associations between conditioned and unconditioned stimuli (Pavlovian

learning), and in the acquisition of instrumental behaviour (Di Chiara et al., 2004; Gambarana et

al., 2003). The accumbens core, on the other hand, has often been implicated in mediating the

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motivation or elicitation of instrumental and approach behaviours in response to previously

associated cues (Ghitza, Fabbricatore, Prokopenko, & West, 2004; Ito, Robbins, & Everitt, 2004;

Parkinson, Willoughby, Robbins, & Everitt, 2000; Sellings & Clarke, 2003). These potentially

dissociable functions may explain the results of a study indicating that dopamine release in the

shell was necessary for the acquisition, but not the expression of, conditioned place preference

(Fenu, Spina, Rivas, Longoni, & Di Chiara, 2006).

Another dimension on which the functions of the core and shell have been contrasted is that of

processing discrete cue versus spatial or contextual information. Lesions to the shell region have

been found to impair the acquisition of spatially-sensitive place preference, whereas lesions of

the core have been found to impair the acquisition of conditioning to a discrete cue, behaviours

which may depend upon their respective connections to the hippocampus and amygdala (Ito,

Robbins, Pennartz, & Everitt, 2008). The accumbens core has also been implicated in cue-

induced reinstatement of heroin-seeking, while the shell has been implicated in context-induced

reinstatement (Bossert, Poles, Wihbey, Koya, & Shaham, 2007), and analogous results were

found in cue versus context-elicited ethanol seeking (Chaudhri, Sahuque, Schairer, & Janak,

2010).

Beyond the delineation of core and shell, there is increasing evidence of structural and functional

differentiation arranged topographically within these boundaries. A series of studies has

suggested, for example, that within the shell there is a rostrocaudal gradient in the processing of

valence; that is, it has been found that disruption of normal activity in the rostral shell leads to

increases in appetitive behavior, whereas disruption in the caudal shell leads to aversively biased

behavior. This line of inquiry originated in an attempt to replicate and extend upon findings from

the lab of Ann Kelley, where it was found that local hyperpolarizations in the accumbens shell

using either an AMPA glutamate receptor antagonist or a GABAA receptor agonist resulted in

large increases in food consumption (Basso & Kelley, 1999; Stratford & Kelley, 1998). Reynolds

and Berridge (2001) sought to confirm these findings, and further to assay regions more caudal

than had previously been probed. In fact, as noted by Richard, Castro, DiFeliceantonio, Robinson

and Berridge (2013), most localized injection studies before the year 2000 were directed

primarily to the rostral half of the accumbens, a phenomenon they term “caudal neglect”. A

reason the authors posited for this oversight is that stereotaxic atlases previously represented the

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accumbens shell in a compressed form, such that the entire rostrocaudal distance depicted has

increased by approximately 1 mm from the time of the popular atlas of the 1960s to that

commonly used today (Paxinos & Watson, 2007; Pellegrino & Cushman, 1967). In particular,

the represented distance between the anteroposterior midpoint of the medial shell to the caudal

edge has increased by approximately 0.5 mm in Paxinos and Watson between 1998 to 2007, and

a rostral migration of stereotaxic coordinates is seen in the coronal sections of the successive

atlas editions. As such, locations considered to be posterior in the 1990s are now recognized to

have been only central within the medial shell.

Reynolds and Berridge (2001) thus confirmed that microinjection of the GABAA receptor agonist

muscimol into the rostral to middle regions of the medial shell elicited intense eating, as

previously found. But as they progressively probed more caudal locations, they discovered that

the manipulation failed to generate feeding behavior, and at the most caudal regions, food intake

was in fact suppressed. Moreover, they observed an elicitation of intense aversive behaviour in

the form of anti-predator reactions such as defensive treading and burying. These behaviours are

seen in wild rodents when they eject debris or dirt toward a predator (Coss & Owings, 1978; De

Boer & Koolhaas, 2003) and in laboratory paradigms in response to shock sources or predator

odors (Treit, Pinel, & Fibiger, 1981). The same rostrocaudal pattern of feeding and fear

behaviour was found to be elicited through localized inhibition using the AMPA glutamate

antagonist DNQX (Faure et al., 2008; Reynolds & Berridge, 2003). Caudally elicited fear

behaviours were further documented to be more diverse than the specific action pattern of

defensive treading: animals were found to respond to experimenters’ approach and touch with

distress vocalizations, escape attempts and defensive biting (Reynolds & Berridge 2002, 2003).

In addition to the aforementioned motivated behaviours, rostrocaudal gradients were also found

in primary hedonic experiences modulated by GABA receptor (GABAR) inhibition in the shell;

that is, in “liking” or “disliking” taste reactions (Faure, Richard, & Berridge, 2010; Reynolds &

Berridge 2002). Animals given a bittersweet solution of sucrose and quinine displayed more

positively valenced reactions, such as lip licking, when infused with muscimol in the rostral

shell, and increasingly negative hedonic reactions, such as mouth gaping, when infusions were

more caudally administered. However, unlike in the assays of food consumption or defensive

behaviours, this same pattern was not elicited through inhibition via glutamate antagonism,

7


whereby hedonic taste reactions were unaffected (Faure et al., 2010). In addition to amino acid

manipulations, a mapping using mu opioid receptor agonism has localized a hedonic

enhancement site to the rostral half of the medial shell (Pecina & Berridge, 2005).

Further evidence of a rostrocaudal gradient in the mediation of valence was found using an

additional behavioural measure. The conditioned place preference/avoidance paradigm is a

traditional measure of rewarding or aversive properties of natural or pharmacological stimuli,

wherein an increase in time spent in an environment previously paired with the stimulus is taken

to indicate its reinforcing properties, and decreased time spent therein is thought to index

negatively reinforcing properties (Tzschentke, 2007). The choice made during exploration may

then be interpreted in terms of the expression of conditioned approach versus avoidance

motivation (Huston, de Souza Silva, Topic, & Muller, 2013). Reynolds and Berridge (2002)

found that GABAR agonism via muscimol microinjections into the accumbens shell resulted in

conditioned place preference at most rostral sites, but place avoidance at most caudal sites.

However, analogously to the contrast observed in hedonic taste reaction, glutamate antagonism

in the rostral shell via DNQX injections failed to enhance the positively valenced behaviour and

in fact established week place avoidance A rostrocaudal gradient was still however apparent, in

that more caudal injections established strong conditioned place avoidance (Reynolds &

Berridge, 2003). This diversity of behaviours demonstrating a rostrocaudal gradient in valence

suggested that the manipulations were not merely eliciting specific action patterns, but were

more generally capable of evoking a central motivational state of reward or aversion (Kelley,

Baldo, Pratt, & Will, 2005).

It should be noted that the sites of enhancement of appetitive behaviours do not all perfectly

overlap between measures; thus in more central locations along a rostrocaudal gradient, it was

possible to elicit strongly augmented feeding concurrently with place aversion and aversive

hedonic taste reactivity (Reynolds & Berridge, 2002). Moreover, the regionally mediated

valence was found not to be absolutely determined; manipulations of environmental context

could to some extent retune the bias of behaviour by altering the topographically valenced

boundaries. For example, Reynolds and Berridge (2008) found that the rostral 25% region

wherein glutamate disruptions were previously shown to generate appetitive behaviour could be

expanded caudally to fill up to 90% of the shell if testing occurred in a preferred home

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environment, whereas caudal fear generating zones could similarly be expanded if testing

occurred in a stressful environment. This serves to highlight the role of the accumbens not only

in conditioned and unconditioned aversive or appetitive behaviour, but in the integration of

information about the animal’s current situation in order to select the appropriate response at the

time of behavioural expression (Humphries & Prescott, 2010).

Approximately 90-95% of neurons in the NAc are medium-sized spiny neurons, a class of

GABAergic neurons which project inhibitory signals to downstream sites including the lateral

hypothalamus, ventral pallidum and mesencephalic dopaminergic regions (Chang & Kitai,

1985). These neurons receive extensive glutamatergic input from the prefrontal cortex,

hypothalamus, basolateral amygdala and hippocampus (Groenewegen, Wright, & Beijer, 1996),

and GABAergic input from local fast-spiking interneurons as well as subcortical structures such

as the ventral pallidum and ventral tegmental area (Taverna, Dongen, Groenewegen, & Pennartz,

2004). As such, both GABAR agonists and glutamate antagonists are expected to result in local

hyperpolarization and a reduction in action potential firing, and thereby provide a disinhibition to

efferent sites (Faure et al., 2010; Koos, Tepper, & Wilson, 2004). Where differences have been

found between the effects of GABAR agonism and glutamate antagonism (Faure et al., 2010),

the authors suggest that there are differences in mechanism which may result in a stronger

influence of GABAergic activation on the resultant output of the accumbens. For example,

muscimol acts on GABAA receptors located on somata and proximal dendrites, whereas AMPA

receptors are more likely to be found on distant spines and require interaction with endogenous

dopamine at the same site (Chen, Veenman, Knopp, Yan, & Medina, 1998). The observed

differences may also reflect the relative influence of the differentiated GABA and glutamate

input structures relevant to a given behaviour.

Thus one interpretation for the increased consummatory behaviour observed in response to

rostral accumbens manipulations is the release from inhibition of a site known to evoke feeding,

in particular within the hypothalamus. Kelley, Baldo, Pratt, and Will (2005) posit that the

inhibition of direct GABAergic projections arising exclusively within the medial shell (Heimer,

Zahm, Churchill, Kalivas, & Wohltmann, 1991) results in a release of motor feeding patterns that

are instantiated in lateral hypothalamic circuitry. Other functional contrasts observed along the

rostrocaudal axis may also be attributed to differentiated patterns of accumbens projection

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neurons, as there is increasing evidence that the accumbens should be viewed not in terms of a

monolithic function and output, but instead as a collection of distinguishable neuronal ensembles

(Pennartz, Groenewegen, & Lopes da Silva, 1994).

Although the precise nature of the relationship between connectivity and functionality remains to

be elucidated, many distinctive rostrocaudal patterns of afferent and efferent input are already

known. For example, the ventral region of the subiculum projects predominantly to the

caudomedial region of the accumbens, whereas progressively more dorsal regions of the

subiculum project to successively more lateral and rostral areas of the accumbens (Brog,

Salyapongse, Deutch, & Zahm, 1993). The mediodorsal nucleus of the thalamus may also

preferentially target the rostral half of the accumbens (Phillipson & Griffiths, 1985).

Additionally, within the accumbens, afferent projections from specific hippocampal regions

converge together with inputs from particular sub-nuclei of the basal amygdaloid complex in a

site-specific manner: In the rostral accumbens there is a convergence of inputs from the

intermediate septotemporal hippocampus and the intermediate rostrocaudal amygdala, whereas

in the caudal accumbens, there is a convergence between projections from the ventral

hippocampus and the caudal basal amygdaloid complex (Groenewegen, Wright, Beijer, &

Voorn, 1999).

Regional differences in histochemistry may also underlie the observed functional differences.

Differential regional patterns within the accumbens have been found in the expression of

calcium-binding protein, enkephalin and dynorphin in the rostral versus caudal core (Berendse,

Groenewegen, & Lohman, 1992; Voorn, Gerfen, & Groenewegen, 1989). These variations may

in turn reflect differential connectivity; for example, calbindin-poor regions receive deep layer V

cortical input and project to non-dopaminergic cells, whereas neurons in calbindin rich regions

receive superficial layer V cortical input and project to non-dopaminergic cells (Berendse et al.,

1992; Humphies & Prescott, 2010). Rostrocaudal differentiations have also been found in

substance P immunoreactivity and acetylcholinesterase activity (Jongen-Relo et al., 1994). In a

study using fast-scan cyclic voltammetry and pharmacological manipulations, it was found that

norepinephrine signaling was restricted to the caudal accumbens (Park, Aragona, Kile, Carelli, &

Whiteman, 2010). Rostrocaudal gradients in both the shell and core were found in the effects of

dopamine depletion on enkephalin, dynorphin and substance P mRNA levels (Voorn, Docter,

10


Jongen-Relo, & Jonker, 1994), suggesting to the authors that the rostral and caudal regions of the

accumbens are likely to be involved in different functions, particularly in relation to interactions

with dopamine. Moreover, a decrease in the effect of D2 receptor activation on acetylcholine

release was also found along the rostraudal axis (Henselmans & Stoof, 1991). Finally, a

rostrocaudal gradient in both dopamine D1 and D2 receptors has been measured, such that the

density of both types has been found to be greater in rostral than in caudal regions (Richfield,

Young, & Penney, 1987). The gradient appears to be more gradual for D2 receptors, and results

in a 50% lower quantity in caudal as compared with rostral regions, whereas D1 density is

largely homogenous until the most caudal regions, where they appear to be 30% lower in density

than in anterior regions (Bardo & Hammer, 1991). The gradient was found to be independent of

the core and shell dichotomy (Voorn, Jongen-Relo, & Jonker, 1994).

Since many of the aforementioned gradients have been observed in the accumbens core in

addition to the shell, analogous functional differences may also be found in the core region,. An

example of a potential functional gradient in the core derives from research in fear conditioning.

In considering possible reasons underlying discrepant results in studies measuring dopamine

release in the accumbens core during exposure to aversively conditioned stimuli, Levita, Dalley,

and Robbins (2002) noted that there was variance between research labs in the placement of

probes along the rostrocaudal axis. The authors pointed out that in a study finding evidence of

increased dopamine release, probe placements were positioned in the rostral region (Wilkinson et

al., 1998), whereas in their own study, using placements in more caudal regions, the authors

found no evidence of enhanced release. In studies having more variability in probe placement,

the resulting neurochemical data in turn tended to have considerably high variability (Murphy,

Pezze, Feldon, & Heidbreder, 2000; Pezze, Heidbreder, Feldon, & Murphy, 2001). The authors

suggest that cue conditioned dopamine release may be a regionally specific phenomenon, and

advised that in general it may be important to consider anatomical heterogeneity in the

accumbens, in particular along the rostrocaudal gradient, in interpreting research findings.

The accumbens core has been less fully characterized in terms of a potential gradient in valence

processing as compared with the shell. In one study by Reynolds & Berridge (2003), the authors

failed to find a rostrocaudal gradient in the accumbens core in fear or feeding behaviours as

elicited by glutamate antagonist microinfusions. However, the behaviours measured constituted

11


only innate unconditioned reactions, as opposed to measures of motivation in relation to

conditioned cues. The effects of localized GABA receptor manipulations within the accumbens

core have also not yet been assessed. Given the evidence for a particular role of the nucleus

accumbens core in the elicitation of motivated behaviour in response to conditioned cues (Ito,

Robbins, & Everitt, 2004; Stefanik, Kupchik, Brown, & Kalivas, 2013; Stuber et al., 2011), in

the current study we sought to determine whether inactivations in the core might yield

differential effects on cue-triggered Pavlovian approach behaviour depending on the rostrocaudal

site. In particular, an assessment was made of a rostral as compared with a historically neglected

caudal site. The study employed a modified conditioned place-preference paradigm using

discrete cues which were associated with the presence of appetitive and aversive outcomes.

GABA receptor (GABAR) agonism via microinfusion in the accumbens was used to assess the

potential biasing effects of localized inactivation on an approach/avoidance exploratory decision

in the face of signals indicating both appetitive and aversive outcomes. It was hypothesized that

animals receiving rostral infusions of the GABAR agonist would display a bias toward approach

in the expression of conditioned approach/avoidance, as evidenced by an increased amount of

time spent in the conflicting cue arm as compared with saline control animals, and that animals

receiving infusions in caudal regions would display a bias toward aversion in the face of the

conflicting cues, as evidenced by decreased time spent in the conflicting cue arm.

2 Methods

2.1 Subjects

Experimental procedures were performed using male Long-Evans rats (Charles River

Laboratories) weighing between 350 and 400g at the time of surgery. They were housed in pairs

under a 12 hour light/dark cycle, with lights turning off at 7:00 pm. Experiments occurred during

the light phase of the cycle. Water was available ad libitum, but beginning 2 days prior to

training, food was restricted sufficiently to reduce and then maintain their body weight at 85% of

their baseline value. All procedures were performed in accordance with the guidelines of the

Canadian Council of Animal Care.

12


2.2 Surgery

Animals were anaesthetized using 3-4% isofluorane and placed in a frame for stereotaxic

surgery. A 26-gauge stainless steel bilateral guide cannula (Plastics One) was implanted into one

of the following coordinates (in mm from bregma): rostral accumbens core (AP = + 1.7, ML = ±

1.5, DV = -5.6) or caudal accumbens core (AP = +0.7, ML = ± 1.5, DV = -5.7). At the time of

infusions, injector tips extended by 1 mm below the guide cannulae, and thus the final targeted

location was at 1mm ventral to the above coordinates. The guide cannulae were affixed to the

skull using dental cement and jeweler’s screws. Stainless steel obdurators were inserted into the

guide cannulae in order to maintain patency. Rats received injections of 5mg/kg Anafen (Merial

Canada Inc.) 20 minutes prior to awakening in order to minimize pain. Animals underwent a

minimum recovery period of 7 days in their homes cages before beginning experimental training.

2.3 Conditioned Cue Preference Task

Radial Maze Apparatus. Behavioral training and testing was conducted using a six-arm radial

maze apparatus (Med Associates). The six arms converged at a hexagonal hub where automated

steel guillotine doors controlled access into each arm (45.7 cm length, 9 cm width, 16.5 cm

height). The arms were enclosed with Plexiglas walls, a removable lid and a steel grid floor

which was connected to a footshock-generating device (Med Associates). A receding well was

located at the end of each arm, which was connected to input tubing and a syringe for the

delivery of sucrose solution. The apparatus was covered with red cellophane in order to obscure

extra-maze stimuli. Med PC IV software (Med Associates) was used to control the timing of

maze door opening. A ceiling-mounted camera was positioned above the apparatus to allow for

monitoring and recording of test sessions. Sessions occurred under illuminated conditions in

order to ensure cue visibility.

Discrete Cues. During training and test trials, wooden rectangular inserts measuring 45 x 2.5 cm

and covered with either gray duct-tape, blue denim cloth material or exposed wooden finish were

placed along both bottom lengths of maze arms and affixed using Velcro. The inserts were thus

discriminable in terms of texture, colour and reflective properties. The inserts become cues

predictive of either sucrose availability, footshock administration, or neutral conditions (no

13


scheduled events) in a given arm during the training sessions. The valence of cues for a given rat

was determined following the unvalenced cue habituation session (described below), whereby

any innate preference for a cue was counterbalanced by assigning it to the opposite valence

during conditioning.

2.4 Training Procedures

Habituation (day 1). Two habituation sessions were conducted on the first day of the

experiment. No unconditioned aversive or appetitive stimuli were presented in either session.

During the first habituation session, no cues were inserted into the arms. Rats were placed

individually in the central hub to begin the session with all doors closed. After one minute, three

doors were raised in order to allow free exploration of the identical arms and the hub for a period

of 5 minutes. After this time, all doors were lowered and the animal was removed from the

apparatus. During the second habituation session, the cues were inserted into 3 separate arms

and the same sequence of events occurred as above. The amount of time spent exploring each

arm was measured and used to determine the valence of each cue for a given rat, as explained

above. Following the apparatus habituation session, rats were given access to sucrose solution in

their home cages for 5 minutes in order to habituate to its consumption.

Training (days 2-10). Training sessions were conducted once daily over a period of 9

consecutive days. The appetitive, aversive, and neutral cues were placed in randomized arms

prior to each trial, varied between subjects within a session, and within subjects between

sessions, in order to minimize any conditioning to extraneous intra-maze cues or odors. A

syringe for sucrose administration was connected via polyethylene tubing to the well in the arm

containing the appetitively valenced cue. The flooring in the arm containing the aversively

valenced cue was connected to the footshock generator. At the start of each session, a rat was

confined in the central hub. After 30 seconds, an initial door was elevated to allow access to one

arm. Upon entry of the animal, the door was lowered to restrict the rat to that arm for a period of

120 seconds. During this time, the animal was administered either the unconditioned appetitive,

aversive, or no stimulus, depending on the intended valence of the cue contained within that arm.

The appetitive stimulus was administered as an infusion of 2.0 ml of sucrose delivered 4 times at

an interval of approximately 30 seconds. The aversive stimulus was a footshock lasting 0.5

14


seconds of approximately 0.25 mA, as calibrated to the level at which the animal demonstrated a

startle response. The footshocks were administered at approximately 30 second intervals. At the

end of the period in each arm, the door was opened to allow reentry of the animal into the central

hub, whereupon it was confined for 30 seconds until a second arm was opened. The same

procedure then followed for the arms of the other 2 valences, and the animal was subsequently

removed from the apparatus to terminate the session. The order of entry into arms of each

valence was varied across trials. The arms were cleaned with 70% ethanol in water between

animals and sessions. The maze was rotated by varying degrees at the end of each testing day in

order to prevent any conditioning to extra-maze spatial or contextual cues. (See Diagram 1 for a

visual depiction of the apparatus).

2.5 Drugs and Infusions

For three days prior to infusion sessions, animals were habituated to gentle hand restraint in the

manner and environment in which infusions were to be administered. On the day before the first

drug session, all animals received an infusion of the saline vehicle, in order to minimize the

effects of subsequent infusions and to further habituate the animals to the procedure. On

infusion days, animals received 0.3µl bilateral intracerebral microinjections of a solution

containing a mixture of the GABAA receptor agonist muscimol and the GABAB receptor agonist

baclofen, (75 ng of each drug per infusion) (Sigma-Aldrich) dissolved in physiological saline, or

the saline vehicle only. The drug was infused via 33 gauge microinjectors projecting 1 mm

below the indwelling guide cannulae using an infusion pump (Harvard Apparatus) mounted with

5µl Hamilton syringes. The infusion occurred at a rate of 0.3µl/46 sec, and the injector was left

in place for an additional 1 min in order to ensure complete diffusion of the drug from the

injector tip. Approximately 10-15 minutes following the end of each infusion, behavioural

testing occurred in the conflict task (described below).

2.6 Testing Procedures

Acquisition of Conditioned Cue Preference. In order to assess the acquisition of conditioned

cue preference, testing sessions were conducted under drug-free conditions after training day 8.

Testing sessions followed the protocol used during the second habituation session. The rat was

15


permitted free access to three arms which each contained one of the three sets of valenced cues,

but no unconditioned stimuli will be presented. The amount of time spent exploring each arm

was measured and recorded via video monitoring. A rat was determined to have entered a given

arm when both its front and hind paws crossed the door threshold, and likewise to have exited

the arm when all of its paws had entered the central hub. Conditioned cue preference tests were

followed by an additional session of training on day 9.

Conflict Test for the Expression of Approach vs. Avoidance. A final test session was

conducted on the 10th

day. The aversively conditioned cue was superimposed with the appetitive

cue within a single arm. A second arm contained the neutral cue. The animal was placed inside

the central hub at the start of the session with all doors closed. After a 1-minute period, two

doors were raised, allowing access to the conflicting-cue and neutral arms. The rat was allowed

to explore both arms in addition to the central hub for a period of 5 minutes. No unconditioned

stimuli were administered. The amount of time spent exploring each arm was measured and

recorded via video monitoring. (See Diagram 2 for a visual depiction of the apparatus).

Novelty Preference Test. Rats performed a novelty preference task in order to determine

whether the drug manipulation might result in an altered preference for novel visual cues which

could explain any difference observed in exploration of the superimposed cues during the

conflict test, given that it was a relatively novel configuration. At the beginning of the exposure

phase, animals were introduced to the central hub of the radial maze for a thirty second

habituation period. Doors to two arms were subsequently opened, and for 10 min the rats were

free to explore the arms, which were characterized by distinct visual patterns on their walls

(black and white circles, diagonal, or horizontal lines) (See Diagram 3). The animals were then

removed from the apparatus for an interval period of 10 minutes. During the testing phase,

animals were returned to the apparatus for 5 min, whereupon three arms will be open, two of

which contained the previously observed cues and one of which contained a novel pattern. The

average time spent exploring the familiar arms was measured and compared with that spent

exploring the novel arm (See Diagram 4).

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2.7 Data Analysis

In order to assess motivational bias during the conflict test, a within-subjects ANOVA was

performed with the factors of drug condition (saline vs. baclofen/muscimol) and location of the

microinfusion (rostral accumbens core or caudal accumbens core). The dependent measure was

the amount of time spent in the conflict arm versus the time spent in the neutral arm. In order to

assess animals’ learning of the valenced cue associations during the training period, a within

subjects ANOVA was conducted for the dependent measure of time spent in each arm

(appetitive, neutral, aversive). The factors of future drug condition (saline vs.

baclofen/muscimol) and cannula location (rostral core vs. caudal core) were evaluated to

determine whether there were any preexisting group differences in learning.

2.8 Histology

After completion of the behavioural testing, animals were sacrificed using 1200mg/kg chloral

hydrate (Sigma-Aldrich) and perfused intracardially with 100 ml saline, followed by 100 ml of

4% paraformaldehyde (PFA) in phosphate buffered saline. Brains were then removed and stored

in PFA before being transferred to a sucrose cryoprotectant. Coronal slices of 50µm diameter

were cut with a freezing microtome, and then stained with cresyl violet for viewing under a

microscope in order to verify the placement of cannulae. Only animals having the correct

targeted cannula placements were included in the data analysis.

3 Results

3.1 Histology

Brain slices were verified for the placement of injector tips in the nucleus accumbens core, with

reference to the stereotaxic atlas of the rat brain of Paxinos and Watson (1997). Data from 11

animals was excluded from statistical analyses due to incorrect cannula placement (n = 6), loss of

the cannula during training (n = 5) and infection (n = 1). The final group numbers were as

follows: caudal core inactivation, n = 7; caudal core saline, n = 9; rostral core inactivation, n =

8; rostral core saline, n = 11. (See Diagram 5 for cannula placements).

17


3.2 Training

A three-way within subjects ANOVA was conducted on exploration time in the three arms

during a final preference test at the end of training in order to determine whether the animals had

learned to associate the valenced cues with the aversive and appetitive outcomes. Degrees of

freedom were adjusted using a Huynh-Feldt correction as Mauchly’s Test indicated a violation of

sphericity. A significant main effect of arm was found, F(1.72, 51.51) = 27.35, p < .001. Paired

samples t-tests revealed that animals spent a greater amount of time in the appetitive vs. the

neutral cued arm, t(33) = 2.46 , p = .019, and less time in the aversive as compared with the

neutral arm, t(33) = 6.39, p = < .001 (Figure 1). There were no significant main effects found for

cannula location, F(1, 30) = .72 , p = .40 or drug condition, F(1, 30 ) = .257, p = .62, nor were

there any significant interactions, indicating that the groups did not exhibit any pre-existing

differences in learning after training (Figure 2).

3.3 Conflict test

A three-way within subjects ANOVA was conducted to compare the amount of time spent in the

neutral vs. conflict arm for the two different drug conditions (GABAR agonist inactivation and

saline) and two different cannula placement locations (rostral vs. caudal accumbens core). The

results indicated a main effect of arm valence on exploration time, with animals overall spending

a greater amount of time in the neutral as compared with the conflict arm, F(1, 31) = 6.91, p =

.013. Significant main effects were also found for drug condition, F (1, 31) = 13.49, p = .00 and

cannula location, F(1, 31) = 15.23, p = .00. Significant two-way interactions were found between

arm and drug condition, F(1, 31) = 17.22, p = .00 and drug condition and cannula placement,

F(1, 31) = 9.20, p = .005, and a three-way interaction was observed between arm, drug condition

and cannula placement, F(1, 31) = 8.87, p = .006.

Tests of simple effects were conducted in order to specify the differences between conditions as

observed in the significant interactions. Among animals having cannula placements in the caudal

core it was found that saline animals did not differ significantly in the time spent exploring the

conflict versus neutral arms, F(1, 14) = 1.91, p = .19, whereas the inactivated animals spent

significantly less time in the conflict as opposed to the neutral arm, F(1, 14) = 15.82, p = .001

18


(Figure 3). Among animals having cannula placements in the rostral core, the saline group again

did not differ in time spent exploring the conflict versus neutral arms, F(1, 17) = .053, p = .82.

The inactivation group spent more time in the neutral as opposed to the conflict arm, however

the difference was not statistically significant, F(1, 17) = 2.99 , p = .10 (Figure 4). Thus the

saline animals in both cannula placement groups displayed ambivalence in their approach

behaviour toward the arm containing both appetitive and aversive cues, whereas animals

receiving GABAR agonists in the caudal region of the core displayed an increased aversion

toward the arm containing the mixed valence cues. Animals who underwent GABAR agonist

inactivation in the rostral region of the core did not demonstrate the same increased aversion to

the conflict arm as the caudal group, and instead displayed an ambivalence between the arms.

A separate ANOVA was conducted to assess whether total exploration time outside of the central

hub differed based on cannula location and drug condition. Significant main effects were found

for both cannula location, F(1, 31) = 15.23, p = .00 and drug condition, F(1,31) = 13.49, p = .001

and a significant interaction was found between drug condition and cannula location, F(1, 31) =

9.20, p = .005. Tests of simple effects revealed that among saline animals, total exploration time

did not differ between animals in the rostral and caudal location groups, F(1, 31) = .441, p = .51,

whereas among the inactivation animals, those having rostral cannula placements displayed

significantly lower total exploration times than those having caudal cannula placements, F(1,31)

= 21.09, p = .00. Among animals having caudal cannula placements, total exploration time did

not differ significantly between saline and inactivation animals, F(1,31) = .190, p = .67, whereas

among animals having rostral cannula placements, the animals undergoing inactivation displayed

significantly reduced total exploration times, F(1, 31) = 24.46, p = .00.

An observation of the behaviour of the animals during the conflict test suggested a qualitative

difference contributing to the decrement in total exploration time in the rostral core inactivated

animals. It was observed that these animals spent a significant amount of time performing

unusual chewing behaviour. Many of the animals did not exit the central hub, and instead spent

the duration of the testing period performing chewing motions with either no substrate in their

mouths or directed toward substrates not seen in the control animals, such the edge of the

apparatus or their own body parts. Among animals who departed the central hub, most of the

animals made only one arm entry in total, and spent the majority of time in one place chewing on

19


a cue, edge of apparatus, or body part. This chewing time was subtracted from the arm

exploration times used in the analysis. Chewing time in the central hub was not quantified due to

visibility limitations.

3.4 Novelty Preference Test

A test of novelty preference was conducted in animals with caudal core cannula placements in

order to determine whether inactivation resulted in an altered exploratory bias for novel visual

cues. Paired sample t-tests were conducted to compare the average time spent in the familiar vs.

the novel arms. Saline animals spent a greater amount of time in the novel arm, t(7) = 2.72 , p =

.030, indicating a preference for the novel spatial environment. While animals undergoing

inactivation in the caudal core spent a greater amount of time in the novel as opposed to familiar

arm, the difference was not statistically significant due to a high level of variability, t(5) = .36, p

= .73 (Figure 5). The data collected for animals with rostral core cannula placements was

insufficient for analysis, as the majority of the animals did not complete all required phases of

the experiment, such as by failing to explore all arms during the exposure phase, or failing to exit

the central hub during testing.

4 Discussion

The current results provide evidence for a topographical differentiation in function along the

rostrocaudal axis of the nucleus accumbens core for the processing of valenced cues in the

motivation of approach and avoidance. Inactivation of the caudal accumbens core via GABAR

agonism resulted in a bias toward aversion as compared with normal controls, whereas

inactivation of the rostral core resulted in an ambivalence similar to that observed in control

animals. An additional behavioural difference was observed in rostrally inactivated animals in

that they displayed a reduction in exploratory activity and a significant augmentation of chewing

behaviour.

Previous experiments involving pharmacological manipulations of the nucleus accumbens shell

found opposite effects of inactivation in the caudal vs. rostral regions in the elicitation of

negatively vs. positively valenced consummatory and defensive behaviours (Faure, Reynolds,

20


Richards & Berridge, 2008; Reynolds & Berridge, 2001). A similar assessment in the nucleus

accumbens core did not demonstrate any difference in these behaviours along a rostrocaudal

gradient, suggesting to the authors that the core may not observe the same regional bivalent

gradation in valence processing (Reynolds & Berridge (2003). However, these inactivations were

performed using a glutamate antagonist, as opposed to via a GABAR agonist as employed in the

current study. Additionally, the behaviours previously measured constituted only innate

reactions to unconditioned stimuli. Given the evidence for a particular role of the nucleus

accumbens core in the elicitation of motivated behaviour in response to conditioned cues

(Stefanik, Kupchik, Brown, & Kalivas, 2013; Stuber et al., 2011), we sought to determine

whether inactivations in the core might yield differential effects on cue-triggered Pavlovian

approach behaviour depending on the rostrocaudal site. The finding that caudal inactivation

caused greater avoidance than that observed under rostral inactivation may be analogous to the

results of investigations in the accumbens shell. That is, in both cases there is evidence for a

rostrocaudal gradient in the processing of valence, with disruption in activity in more caudal

regions resulting in greater aversive behaviour.

The augmentation in avoidance behaviour observed during the conflict test in animals

undergoing GABAR agonsim in the caudal core suggests that this region may have a particular

role in motivating avoidance behaviour in response to aversively predictive cues. While

GABAR agonism is considered to result in localized neural inactivation, this may in effect

activate a functional output pathway. That is, given that the output neurons of the accumbens are

GABAergic in nature (Chang & Kitai, 1985), a reduction in activity of these inhibitory output

neurons may in turn result in greater activation of a downstream structure effecting the avoidance

behaviour. In light of the postulated function of the nucleus accumbens as a site of integration of

information and a selector of appropriate behaviour (Humphries & Prescott, 2010), it may be the

case that the caudal core has the capacity to either elicit or inhibit aversive behaviour, according

to current circumstances as informed by environmental cues. The function of the GABA

neurons in the accumbens is moderated by dopaminergic input from the VTA, whose modulatory

effect is state dependent and may result in either increased or decreased activation of striatal

neurons depending in part on current glutamatergic and other neuromodulator input (Onn, West

& Grace, 2000). The GABAergic output neurons are also modulated by acetylcholine

interneurons and noradrenergic input (Hoebel, Avena, & Rada, 2007; Park, Aragona, Kile,

21


Carelli, & Whiteman, 2010). These mechanisms may result in the capacity for the caudal core to

inflect the level of inhibitory output in either the positive or negative direction through the

integration of environmental information.

Unlike in previous studies assaying a topographical gradient in the accumbens shell, in the

current study there was no evidence of an inversion in the direction of the elicited of

approach/avoidance behaviour in the rostral core region such as that previously seen after

inactivation of rostral shell sites (Faure, Reynolds, Richards & Berridge, 2008; Reynolds &

Berridge, 2001). That is, the rostrally inactivated animals in the current study did not increase

their motivation to approach the reward cued environment above control levels.

A limitation in the interpretation of the current behavioural results with reference to a

rostrocaudal gradient in approach and avoidance was the qualitatively different nature of the

behaviour of the rostrally vs. caudally inactivated animals. As noted, the former animals

displayed lower total exploration times, which appeared to be largely mediated by a compulsive

chewing behaviour. Given that a significant percentage of time was spent chewing on an object

or surface, which was immediately in front of the rat, the animals may not have had a sufficient

opportunity to encounter the previously learned cues, explore the apparatus and display any

potential evaluative or motivational bias. However, chewing behaviour itself may be considered

to be more coherent which approach than avoidance behaviour, given that the animal is engaging

with, as opposed to moving away from the substrate. As such, the augmentation of this

behavioural pattern after rostral inactivation may in fact be supportive of a key role for rostral

core regions in eliciting approach behaviours. It is possible that the specific targeted rostral

coordinates constitute a site with connectivity to a structure representing motor output sequences

for chewing behaviour. The inactivation may have caused an inhibition of the GABAergic output

of the local medium spiny neurons, resulting in a release from inhibition of a particular

behavioural pathway. It is possible that other rostral core coordinates, perhaps also varying

mediolaterally and dorsoventrally, might mediate other appetitively valenced behaviours,

including Pavlovian approach to conditioned cues. This serves to highlight the necessity of

targeting and carefully specifying multiple coordinate sites in the determination of the functions

of the nucleus accumbens, particularly considering the historical neglect in the research literature

of more caudal accumbens sites. The varying behaviours elicited by manipulation of separate

22


subregions is also coherent with the concept that this structure should be considered as a

composite of distinguishable neuronal ensembles, as opposed to in terms of a monolithic

function and output (Pennartz, Groenewegen, & Lopes da Silva, 1994).

The current study presented the animals with a juxtaposition of positively and negatively

valenced cues, which resulted in a requirement for the animal to make an approach/avoidance

decision in the face of opposing motivations. The question may arise as to whether differences

in the bias of the animals’ exploratory allotment were due either to an altered level of approach

or avoidance motivation. Previous studies which have assessed these poles of valence

independently have found evidence for both appetitive motivational (Berridge & Robinson,

1998; Roitman, Stuber, Phillips, Wightman, & Carelli, 2004) and aversive motivational

processing in the nucleus accumbens (Carlezon & Thomas, 2009; Levita et al., 2009;

McCutcheon et al., 2012). Moreover, in studies which have differentiated rostrocaudal locations

with respect to function, manipulations within a subregion have been found to simultaneously

affect discrete measures of appetitive and aversive motivation. (Faure, Reynolds, Richards &

Berridge, 2008; Reynolds & Berridge, 2001, Reynolds & Berridge, 2003). That is, it seems

possible that the bias toward aversion observed in the caudally inactivated animals is a composite

of a decreased motivational signal as well as an increased avoidance signal. In fact it may prove

difficult to dissociate the contribution to the two proposed poles of processing at the regional

pharmacological level. This caveat to interpretation is informed by several lines of evidence

from previous research. First, there is evidence from electrophysiological assessments that

separate neuronal populations of medium spiny neurons within the same region of the nucleus

accumbens code for values of cues associated with reward vs. punishment (Roitman, Wheeler &

Carelli, 2005). This suggests that the GABAergic manipulation at regional level could

theoretically have affected both neuron population types. An additional consideration when

interpreting electrophysiological studies is the specific targeted coordinate site of the accumbens

from which the recordings are taken. In the study by Roitman, Wheeler and Carelli (2005) for

example, the anteroposterior coordinate was at +1.7mm relative to bregma, rendering it close to

the rostral coordinate in the current study. (The mediolateral and dorsoventral differed however

at 0.8 and -6.5 respectively). In this study, the authors found that a greater proportion of reward

cue-sensitive neurons exhibited phasic excitations than inhibitions, and that aversive cue-

sensitive neurons exhibited a similar proportions of excitation relative to inhibition subgroups. It

23


would be interesting to note whether the populations of neurons displaying increasing vs

decreasing activity in response to cues would inverse in the majority in more caudal regions of

the accumbens.

Additionally, there is evidence supporting the existence of some neuronal populations whose

activity patterns demonstrate that they have integrated information about both cost and benefit in

decision-making paradigms (Roesch et al., 2009). This further serves to highlight the fact that

nucleus accumbens subregions should not be considered as dichotomously aversive or appetitive

information processing locations, but that complex integrations and reiterations of valence are

likely to occur. Similarly supporting the notion of value integration at the level of the nucleus

accumbens, a functional magnetic resonance imaging study in humans has demonstrated that

cue-associated activity in the ventral striatum was modulated by the net value (calculated as the

expected reward value minus effort-based cost) of the upcoming decision (Croxson et al., 2009).

The concept of cost has most frequently been quantified in terms of the effort required to obtain

a reward, or through measuring delay discounting, wherein a reward diminishes in value

hyperbolically as the delay to obtain it increases. The accumbens may receive input that already

reflects the integrated value of certain costs and benefits. For example, individual dopaminergic

neurons in the rodent’s ventral tegmental area (VTA) as recorded by Roesch and Bryden (2011)

appeared to respond in a manner which reflected the integration of reward magnitude and delay.

A population of neurons in the ventral striatum, which receives dopaminergic input from the

VTA, also encoded both of these variables. Similarly, Day, Jones and Carelli (2011) found that a

subgroup of neurons in the nucleus accumbens displayed phasic firing rates which reflected the

cost-discounted value of the upcoming response in an effort-based, but not in a delay-based

decision-making task. The activation of other subgroups did not did not correlate with the cost-

discounted value, but instead appeared to be associated with response initiation, reward delivery,

the sustainment of high effort requirements or during waiting for delayed rewards. This serves to

illustrate the fact that the nucleus accumbens performs many distinguishable functions in the

activation of reward directed behaviour. In the foregoing study, the coordinate location in the

core from which measurements were taken was at an AP location of 1.3mm rostral to bregma,

rendering it relatively central with respect to the two anteroposterior coordinates assessed in the

current study. It remains to be determined whether the characterization of firing patterns would

be differentiable at other targeted sites in the accumbens.

24


The current study may also be considered in relation to research investigating the neural

substrates of decision making under conditions of risk or uncertainty. While our study does not

systematically vary the probability of reward or punishment, and thus the emphasis is not on the

dimension of uncertainty, there is nonetheless likely a component of risk that is processed by the

animals in this task. The arrangement of the cues in the conflict tests is a novel configuration for

which the rats have not previously experienced any valenced outcome. During initial habituation

the animals are exposed to these juxtaposed cues without any programmed contingencies, and for

the duration of their training they encounter the cues and their associated outcomes in isolation

with respect to valence. When the animal encounters the conflicting cues during testing, there is

therefore a degree of uncertainty in the potential outcome. Neuroimaging studies in humans have

suggested a role for the nucleus accumbens in mediating risk-associated processing, such as

measured during gambling tasks. For example, several studies have found that higher levels of

activation in the accumbens during deliberation was associated with the choice of a riskier

opportunity for reward (Kuhnen & Knutson, 2005; Matthews, Simmons, Lane, & Paulus, 2004).

Some animal studies have also supported a role for the accumbens in biasing behaviour toward

riskier decisions. An experiment by Cardinal and Howes (2005) found that lesions of the nucleus

accumbens core resulted in an increased aversion to risk in a decision-making task, rendering the

animals more likely to prefer a smaller, certain reward versus a larger reward which was

dispensed with varying probability. In that study, the lesions to the accumbens extended

throughout the majority of the rostrocaudal axis of the core (from AP 0.7 to 2.2 mm relative to

bregma) in all subjects, with significant damage also extending to medial regions of the shell.

Thus it is unclear which subregion may have contributed to the behavioural effect. After finding

similar results when temporarily inactivating the entire nucleus accumbens via the infusion of a

GABA agonist, Stopper and Floresco (2011) attempted to further refine the localization of the

effect by using smaller volumes of the GABA agonist, thereby limiting its spatial spread to the

subregions of core vs. shell. It was found that inactivation of the shell recapitulated the risk

aversion observed when inactivating the broader structure, whereas inactivation of the core did

not result in a risk aversion deviating from control animals. It should be noted that the

anteroposterior coordinates of 1.5mm targeted in their study was closest to rostral, as opposed to

the caudal, coordinates targeted in the current study. Given that the rostrally inactivated animals

in the current study did not demonstrate a significantly deviated bias in their approach-

25


avoidance behaviour as compared with controls, the results of the two studies may be

correspondent, to the extent that the current behavioural task may be appropriating the same

circuitry used in cost-benefit decision making under uncertainty. It is possible that risk-taking

behaviour as measured by Stopper and Floresco (2011) may be differentially altered if

inactivations were to be performed in more caudal regions of the core.

The elicitation of a compulsive chewing behaviour in animals undergoing inactivation of the

rostral core in the current study may be of interest beyond the possible interpretation of this

effect as a type of augmented appetitive response. The chewing observed in the rats was unusual

in that it was directed toward substrates that control animals did not normally chew upon,

including apparatus edges, body parts and the fur of cage-mates. Additionally, many animals

exhibited chewing motions even in the absence of any substrate in their mouths. Incidentally, this

type of behaviour has previously been characterized and is in fact used as a primary measure in

animal models of tardive dyskinesia. In humans, tardive dyskinesia is a disorder of involuntary

repetitive movements that can develop in patients undergoing long-term treatment with

dopamine antagonists most commonly used as antipsychotics medications. The symptoms most

frequently involve the orobuccal and lingual facial muscles (Sachdev, 2000). The syndrome has

been modelled in rats exposed to antipsychotic medications who demonstrate increased vacuous

chewing movements; that is, compulsive and repetitive chewing motions in the absence of

normal substrates (Kulkarni & Naidu, 2001). While some research has focused on potential

dopamine receptor aberrations in light of the primary target of the medications, there is some

evidence to suggest that deviations in the GABAergic system may be associated with this

condition (Gunne, Häggström, & Sjöquist, 1984; Inada et al., 2008). Thus it is possible that the

current site targeted in the rostral accumbens core is a key location involved in eliciting the

symptoms of tardive dyskinesia, possibly resultant from an aberration of GABA signalling

therein.

The differential effects of inactivation in the rostral vs. caudal accumbens core may result from

their distinctive rostrocaudal patterns of afferent and efferent input, given that these regions have

been found to have differential connectivity with multiple structures. For example, the ventral

region of the subiculum has been found to project predominantly to the caudomedial region of

the accumbens, whereas progressively more dorsal regions of the subiculum project to

26


successively more lateral and rostral areas of the accumbens (Brog, Salyapongse, Deutch, &

Zahm, 1993). In addition, afferent projections from specific hippocampal regions have been

found to converge together with inputs from particular sub-nuclei of the basal amygdaloid

complex in a site-specific manner: In the rostral accumbens there is a convergence of inputs

from the intermediate septotemporal hippocampus and the intermediate rostrocaudal amygdala,

whereas in the caudal accumbens, there is a convergence between projections from the ventral

hippocampus and the caudal basal amygdaloid complex (Groenewegen, Wright, Beijer, &

Voorn, 1999). Tract tracing experiments have also revealed a topographical organization of the

efferent connections originating in the prefrontal cortex and terminating in NAc subregions along

rostral-caudal and mediolateral axes. For example, the medial orbitofrontal cortex (OFC) has

been found to project preferentially to the rostral accumbens whereas the lateral OFC has been

found to project to the more caudal accumbens regions. (Berendse, Graaf & Groenewegen,

1992). Given that the prefrontal cortex, amygdala and hippocampus have been implicated in

valuation and decision-making (Bechara, Damasio & Damasio, 1999; Johnson, van der Meer &

Redish, 2007) future experiments should examine the functional connectivity between

subregions of the nucleus accumbens and the specific target sites of their projections in order to

determine whether localized circuits preferentially mediate approach vs. avoidance behaviours.

The observed functional differences in the rostral and caudal core may also be mediated by

differences in neurotransmitter receptor distributions. One example is that norepinephrine

signalling has been found to be restricted to the caudal accumbens in rodents (Park, Aragona,

Kile, Carelli, & Whiteman, 2010). In an interesting study of the human brain, it was found that

the caudal subdivision of the accumbens selectively contains strikingly high levels of

noradrenaline, and the authors state that this site represents the only area in the human brain

expressing equally high levels of both noradrenaline and dopamine (Tong, Hornykiewicz, &

Kish, 2006). A rostrocaudal gradient has been found in dopamine D1 as well as D2 receptors, in

that the density of both subtypes has been found to be greater in the rostral as opposed to the

caudal regions (Richfield, Young, & Penney, 1987). The effects of agonizing or antagonizing

dopamine D1 or D2 and noradrenergic receptors in rostral vs. caudal accumbens regions might

therefore be investigated, especially considering the evidence of a role for dopamine in

mediating both reward-directed and aversive behaviours (Lammel, Lim & Malenka, 2014) and

27


for norepinephrine in mediating the processing of motivational salience and anxiety (Bremner,

Krystal, Southwick & Charney, 1996; Ventura et al., 2008).

The results of the current study emphasize the importance of taking measurements or performing

manipulations in multiple subregions of the nucleus accumbens in order to fully characterize the

topography of its heterogeneous functions. One future direction would include a probing of

additional coordinate sites along the anteroposterior, mediolateral and/or dorsoventral axes using

the current paradigm in both the nucleus accumbens shell and core. Additionally, it would be

informative to test the effects of the current pharmacological manipulation on the acquisition of

conditioned cue preference, given previous evidence suggesting that the acquisition and

expression of motivated behaviours may be differentially represented within the accumbens. It

may be the case that GABAR manipulations in the accumbens shell would have a greater impact

on the acquisition of cue preference using the current paradigm, given the evidence supporting a

role for the shell in primary reward and reward-cue learning (Ito et al., 2001). Studies of finely

localized functional connectivity with other brain regions as outlined above will further elucidate

the representation and processing of valence in the brain.

28


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Figure 1. A test of training for cue valence demonstrated that animals spent a greater amount of time in the appetitive vs. the neutral cued arm, t(33) = 2.46 , p = .019, and less time in the aversive as compared with the neutral arm, t(33) = 6.39, p = < .001

Figure 2. In a test for cue valence training, there were no significant main effects found for cannula location, F(1, 30) = .72 , p = .40 or drug condition, F(1, 30 ) = .257, p = .62, nor were there any significant interactions, indicating that the groups did not exhibit any pre-existing differences in learning after training.

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Figure 3. Results of the conflict test in animals undergoing caudal core inactivation revealed that saline animals did not differ significantly in the time spent exploring the conflict versus neutral arms, F(1, 14) = 1.91, p = .19, whereas the inactivated animals spent significantly less time in the conflict as opposed to the neutral arm, F(1, 14) = 15.82, p = .001

Figure 4. Results of the conflict test in animals undergoing rostral core inactivation indicated that the saline group did not differ in time spent exploring the conflict versus neutral arms, F(1, 17) = .053, p = .82. There was no statistically significant difference in the inactivation group in time spent in the neutral as opposed to the conflict arm, F(1, 17) = 2.99 , p = .10.

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Figure 5. A test of novelty preference revealed that saline animals spent a greater amount of time in the novel arm, t(7) = 2.72 , p = .030, indicating a preference for novelty. While animals undergoing inactivation in the caudal core spent a greater amount of time in the novel as opposed to familiar arm, the difference was not statistically significant, t(5) = .36, p = .73.

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Diagram 1. Radial arm maze apparatus. For all training sessions, the walls of each arm were lined with a panel cue predictive of sucrose reward, footshock, or no outcome. During the final preference test, rats were allowed access to all three arms for a period of 5min. Neither sucrose nor footshock were administered.

Diagram 2. Radial arm maze apparatus. During the final conflict test, rats were allowed access to two arms for a period of 5min. One arm contained a superposition of cues predictive of sucrose reward and footshock, whereas the other arm contained the neutral cue. Neither sucrose nor footshock were administered.

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Diagram 3. Novelty preference apparatus during exposure phase. . At the beginning of the exposure phase, animals were introduced to the central hub of the radial maze for a thirty second habituation period. Doors to two arms were subsequently opened, and for 10 min the rats were free to explore the arms, which were characterized by distinct visual patterns on their walls (black and white circles, diagonal, or horizontal lines).

Diagram 4. Novelty preference apparatus during testing phase. During the testing phase, animals were returned to the apparatus for 5 min, whereupon three arms will be open, two of which contained the previously observed cues and one of which contained a novel pattern. The average time spent exploring the familiar arms was measured and compared with that spent exploring the novel arm.

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Diagram 5. Schematic representation of the locations of injector tips in the NAc rostral (left) and caudal (right) core based on the stereotaxic atlas of the rat brain of Paxinos and Watson (1997).

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