Abstract: in clinical populations and is common among

Abstract:

Risky decision
making has significant harmful outcomes in clinical populations and is common
among patients with neuropsychiatric disorders and serious mental illness, for
example schizophrenia. Information transfer between brain regions facilitates
decision making about risks and rewards however, there is a paucity of research
in this area. Employing a task that models risky decision making in rats and
manipulating the transfer of information between two key brain regions, the nucleus accumbens
(NAc) and basolateral amygdala (BLA) which are known to be involved in guiding decisions, will
further our understanding of the neural basis of risky decision making. This is
an important step to further neuropsychiatric research that can inform
treatment options and optimize behaviours.

                          

Background

In schizophrenics,
irregular tissue organization and volume in the right amygdala and left nucleus
accumbens causes structural and functional abnormalities resulting in abnormal
processing of information (Tomasino et al., 2011; De Rossi et al., 2016).
Schizophrenia research suggests that protuberances within the mesocorticolimbic
system may lead to dysregulation, which inhibits dopamenergic modulation of
processing emotionally salient information (Laviolette, 2007).

 

The nucleus accumbens
(NAc) and basolateral amygdala (BLA) have been identified as key brain regions within
cortico-limbic circuitry for guiding decision making in both humans and animals;
however, reliance on
interpreting functions of different brain regions in isolation lacks external
validity. Determining the way these regions interact to guide behaviour is
essential to understanding what drives risky or safe decisions and ultimately
behaviours. Furthermore, most preclinical studies employ assays to assess
risk/reward decision making using internally generated representations of
outcome contingencies, which are used to guide advantageous  choice. This is problematic because real-life
decisions are often influenced by external stimuli that inform about the
likelihoods of obtaining favourable outcomes. I propose to use a novel assay
colloquially termed the “Blackjack” task that models situations where external
stimuli indicate probabilities (Floresco et al., 2017). Ipsilateral
disconnection of the BLA and NAc, in a rat model, will allow for the
exploration of how information transfer, between these regions, facilitates
decision making about risks and rewards in an externally cued environment.

 

Laboratory
studies designed to assess human ability to make appropriate risk/reward
decisions with functional imaging studies have provided indirect evidence showing
these regions do interact to facilitate decision making about probabilistic
rewards. When choosing high-risk, compared with low-risk gambles, the anterior
cingulate and NAc are functionally connected (Cohen et al., 2005) and
functional connectivity can be observed between the cingulate and amygdala
while anticipating reward outcomes (Marsh et al., 2007).

 

Research
studies have constructed several tasks to investigate the neural basis of
risk/reward decision making in animals. Some are designed to mirror the Iowa
gambling task (Bechara et al., 1999). Other studies have introduced probabilistic
discounting tasks, whereby rats are presented the choice between smaller,
certain rewards, and larger rewards, with the odds of obtaining a larger reward
changing systematically over a session (Stopper et al. 2012).  These tasks have been criticized as not being
representative of “real-life” risk/reward decisions. They are guided by
internally-generated value representations instead of external cues to inform
animals of the likelihood of obtaining certain rewards and therefore do not
mimic “real-life”.

 

These studies, which
used a variety of different behavioural tasks, have established that various
aspects of risk/reward decision making are mediated by neural circuits linking
different regions of the prefrontal cortex, orbitofrontal cortex, nucleus
accumbens and the basolateral amygdala (Larkin et al., 2016). Furthering these
findings about how subcortical circuits mediate risk-based decision making is
important, as it provides insight into the pathophysiology underlying abnormal
decision making.

 

Research has
identified the influence that the basolateral amygdala has in biasing choice
towards larger, uncertain rewards via interactions with the nucleus accumbens. Bilateral
inactivation of either region resulted in a reduced preference of larger
uncertain rewards (Ghods-Sharifi et al. 2009). Asymmetrical disconnection and
inactivation of the BLA and NAc (St Onge et al. 2012) had the same finding. The
roles of both the basolateral amygdala and the nucleus accumbens have been
explored in isolation from one another. The basolateral amygdala reduces the
preference for larger uncertain rewards and the nucleus accumbens supresses random
choice patterns. (Ghods-Sharifi et al., 2009; Floresco et al., 2017). It reamains unclear whether various nodes within the
cortico-amygdalar-striatal circuitry communicate and influence choice and
decisions differently under conditions with external cues versus internally
generated infrormation.

 

To explore this
issue, I propose to use a task involving choice between small/certain and
large/risky rewards. The focus will be on how the BLA-NAc circuitry contributes
to decision making in conditions involving external cues. Previous studies
suggest that the NAc shell is responsible for suppressing irrelevant or
non-rewarded behaviours while the BLA mediates judgements surrounding the
relative value associated with various courses of action (Floresco, 2015;
Ghods-Sharifi et al., 2009).

 

The use of
external cues to guide decisions is essential for adaptive behaviour. Deficits
in such behaviour are associated with a range of neuropsychiatric disorders
which may be in part due to ineffective or absent subcortical circuitry.
Results will contribute to a broader understanding of the underlying
pathophysiology present in neuropsychiatric disorders and the role of the
BLA-NAc pathway.

 

Materials
and Methods

Animals

The experiment will utilize Male Long-Evan rats.
At arrival animals will be group-housed with four animals per cage and given a
week with free food to acclimate. Five days before the intended start date for
behavioural training animals will be food restricted to 16 g per day.

 

Apparatus

Behavioural testing will occur in sound
attenuated operant chambers. The boxes will be ventilated with a fan doubling
as a mechanism to mask external noise. Each chamber will have two retractable
levers, a food receptacle and house light. The retractable levers will be
fitted on either side of the food receptacle.

 

Initial Lever
Press Training Behaviours

Prior to training on the targeted task, animals will
undergo a pre-training regimen consisting of basic lever pressing, retractable
lever training and reward magnitude discrimination training. Before
beginning basic lever pressing each rodent received sugar pellet’s in their
home cage to reduce neophobia. Training will start with lever-press training
under a fixed-ratio-1 (FR1) schedule. During the sessions the house-light will
be illuminated and one lever inserted into the chamber for 30 minutes or until
60 lever presses are made, whichever occurs first. On the following day(s),
rats will be required to press the opposite lever until achieving criterion.

 

Retractable
lever training will begin after completion of basic lever training. Sessions will
consist of 90 trials and begin with both levers retracted and the house-light
off. Every 40 seconds a trial will begin with the illumination of the house
light and the insertion of one of the two levers. If the rat responds within 10
s the lever retracts and a single pellet should be delivered via the food
receptacle with 50% probability. Rats will be trained for approximately 3-6 d
to a criterion of 80 or more successful trials (i.e. < 10 omissions).     Rats will then progress and be trained to associate one lever with a larger four-pellet reward and the other with a one-pellet reward. In phase one, training will be using a 48 trail task, blocked into four groups of two forced-choice trials followed by 10 free-choice trials (12 trials per block). Every 40 seconds, one or both levers will be inserted into the chamber. One lever delivers four pellets with 100% probability whereas the other delivers one pellet with 100% probability. The lever associated with the larger reward will remain consistent for the duration of the experiment and should be left/right counterbalanced.   Subsequently, rats will be trained for another 2-3 days on a modified version of the program with the purpose of introducing the probabilistic component of the task. The sessions will be comprised of 72 trials, divided into four blocks of eight forced-choice and 10 free-choice trials. In these sessions, selection of the small reward lever will always deliver 1 pellet, whereas choice of the large reward lever will dispense four pellets with a 50% probability.   Blackjack Task In the target task of this study, one lever will be designated the large/risky option and the other the small/certain option, and will remain consistent throughout the remainder of training   Blackjack training will be comprised of two phases. In the first phase, the first 32 trials will be forced-choice, with only one lever inserted into the chamber. Following the forced-choice trails 20 free-choice trials will commence with both levers inserted. Once stable behaviour is apparent the second phase of the experiment will be initiated. The second phase is identical to the initial phase, excepting of the fact that all trials will be free-choice.   Trials will initiate every 40 s with house light illumination and presentation of one of two distinct auditory cues (3kHz pure tone or white noise, 80 dB). Tones will be presented pseudorandomly over the session. Once the house light and tone are produced, one or both levers will insert into the chamber.  If the rat responds via selection of the small/certain lever the tone will turn off immediately and result in one pellet being delivered (100% probability) irrespective of which tone was presented. Alternatively, the large/risky lever could yield four pellets, delivered in a probabilistic manner. The probability of obtaining the larger reward is based on one of either tone presented in that trial. "Good odds" trials are associated with one consistent unique tone, (eg. 3 kHz), where a risky choice delivers a reward with 50% probability. The other unique tone (white noise, dB) signals "poor odds" trials where a risky choice is rewarded with 12.5% probability.   Both levers will retract in the event of a response on either lever. If the large/risky option is chosen and a reward is received the tone and house light will remain on until all the pellets are delivered. If a reward isn't received after the selection of the large/risky choice, the house light should extinguish immediately and the tone silenced 2 s after the choice. To facilitate the learning of associations between each unique tone and the likelihood of different outcomes (50% and 12.5%), tones will continue after a risky choice has been made. One pellet will be delivered and the tone turned off, if the small/certain option is selected. In the case of an omission, both levers will retract and the house light and tone will turn off.   The second phase of training (40 free choice trails) will then commence for another 5-6 days, or until rats display stable patterns of choice. Rats will then undergo surgery and be retrained on the task for a minimum of 5 days until stable behaviour is displayed after which they will receive their first micro-infusion test day. Stereotaxic Surgery Just prior to surgery, subanesthetic doses of ketamine and xylazine (50 mg/kg and 4 mg/kg respectively) will be administered. Rats will be stereotaxically implanted with bilateral 23-guage stainless steel guide cannula into the BLA and NAC. The coordinates being anteroposterior (AP) = -3.1 mm; medial-lateral (ML) = +5.2 mm from the bregma; dorsoventral (DV) = -6.5 mm from dura and NAc (AP = +3.4 mm; ML = + 1.4 mm; DV = -2.8 mm) (St. Onge et al., 2012). Animals will be monitored daily for a minimum of a week before being retrained until stable patterns of choice behaviour return.   To familiarize rats with the infusion equipment and procedure, animals will receive a mock infusion prior to their first micro-infusion. Injectors will be placed inside of the guide cannula for two minutes with no infusion administered. Then rats will be placed in their home cage for 10 minutes before behaviour training.   The day after mock infusions, animals will receive the first of two micro-infusion test days. Drugs or saline will be infused at a volume of 0.4 ?l. A solution containing the GABAB agonist baclofen and the GABAA agonist muscimol dissolved in 09% saline will be used to achieve inactivation. Infusions will be delivered via 30-gauge injection cannulae over 89 s.  Rats will receive counterbalanced infusions on two separate days: (1) a saline infusion into both structures contralaterally; (2) drug infusions in two regions in opposite hemispheres. Animals will receive on day of retraining after the first micro-infusion. On the following day a second, counterbalanced infusion will be administered prior to behavioural testing. Histology After completion of behavioural testing, animals will be anesthetized with isoflurane before euthanization via CO2. Brains will be frozen with CO2, sectioned at 50 ?m and mounted. Placements will then be verified with reference to a neuroanatomical atlas.   Experimental Design and Statistical Analysis The primary dependent variable of interest is the proportion of choices of the large/risky option on good and poor odds trials. This can be calculated by dividing the number of choices of the large/risky lever by the total number of those trials in which the rats made a choice, separately for good and poor odds trials and analyzed using a two-way within subjects' ANOVA with treatment and odds (good vs poor) as within subjects' factors.   Expected Results Previous studies have suggested that BLA projections modulate NAc activity and influence the direction of behaviour toward reward-related stimuli (Everitt et al., 1999; Setlow et al., 2002). Further studies have suggested that the deactivation of the BLA-NAc subcortical circuits results in a bias away from larger/uncertain reward and that amygdala connectivity with the nucleus accumbens is "responsible for mediating Pavlovian influences of action" (St. Onge et al., 2012; Seymore & Dolan. 2008). Based upon these prior studies, a functional disconnection of the BLA-NAc subcortical circuits would be expected to significantly decrease the choice of the large/risky option compared with saline, under testing situations which utilize external auditory cues to indicate event probabilities. This is a reasonable assumption as previous evidence has suggested that inactivation of the basolateral amygdala increases risky choice when odds are poor. This suggests that neural activity in the basolateral amygdala is important for making judgements about the value associated with different courses of action (Ghods-Sharifi et al., 2009). Also, inactivation of the nucleus accumbens shell increases risky choice indicating it plays a role in suppressing actions that may lead to subjectively inferior rewards (Floresco et al., 2017). The presupposition that functional disconnection of the BLA-NAc circuitry would result in an increased occurrence of risk averse choices is further supported by the knowledge that NAc activity associated with choice of larger, riskier rewards is likely driven by excitatory inputs from the BLA. If the BLA is not communicating with the NAc, we expect to see a bias towards smaller, less valuable rewards, even when the risky/large option is likely to yield a greater reward as was seen in a probabilistic discounting task (St Onge et al., 2012; Ghods-Sharifi et al., 2009). BLA projections modulate NAc activity and may influence the direction of behaviour toward reward-related stimuli, therefore the disconnection of such pathways may impair the ability of animals to make decisions which yield the highest choice-reward options (Everitt et al. 1999).   Alternatively, if we do not see that functional disconnection of BLA-NAc circuitry results in risk-averse choice patterns, it may be that this subcortical circuit is not involved in the pathophysiology responsible for poor risk assessment and decisions seen in various human conditions. Additionally, there may be top-down influences which are impacting choice.   Limitations Understanding how information is relayed via subcortical circuitry in risky decision making using rat models, would offer greater insight as to the pathophysiology underlying cognitive and behavioural abnormalities in neuropsychiatric disorders; however, there are several limitations for interpreting these findings. The most prominent limitation is the extent to which behavioural and anatomical findings from rodents can be extrapolated to humans. Even so, it is reasonable to consider such findings important, given studies which identify abnormal structural configuration and volume in both the amygdala and nucleus accumbens in schizophrenic populations (Tomasino et al., 2011; De Rossi et al., 2016).   If it is the case that functional disconnection decreases preference for the large/risky lever, the current study cannot determine whether the effect is due to an impairment in discriminating between external cues and associated reward magnitude probabilities or an impairment in the ability to discriminate reward magnitudes associated with the two levers (St. Onge et al., 2012). To remedy this, a secondary experiment would be conducted using a reward magnitude discrimination task.  Furthermore, the disconnection design does not account for cross-hemispheric communication. It should be noted that this poses no major limitation to the study as projections from the BLA to the NAc are primarily ipsilateral (McDonald, 1987). Other findings have demonstrated that the prefrontal cortex also influences decision making via communication with the BLA and NAc. A secondary set of experiments exploring how the ipsilateral and contralateral descending projections of the PFC to the amygdala and NAc influence or mediate risky decision making (St Ong et al,. 2012). Both PFC-NAc and PFC-BLA asymmetrical disconnection studies would further our understanding as well.