Abstract: schizophrenics, irregular tissue organization and volume in


Abstract:Risky decisionmaking has significant harmful outcomes in clinical populations and is commonamong patients with neuropsychiatric disorders and serious mental illness, forexample schizophrenia. Information transfer between brain regions facilitatesdecision making about risks and rewards however, there is a paucity of researchin this area. Employing a task that models risky decision making in rats andmanipulating 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, willfurther our understanding of the neural basis of risky decision making.

This isan important step to further neuropsychiatric research that can informtreatment options and optimize behaviours.                           BackgroundIn schizophrenics,irregular tissue organization and volume in the right amygdala and left nucleusaccumbens causes structural and functional abnormalities resulting in abnormalprocessing of information (Tomasino et al., 2011; De Rossi et al., 2016).Schizophrenia research suggests that protuberances within the mesocorticolimbicsystem may lead to dysregulation, which inhibits dopamenergic modulation ofprocessing emotionally salient information (Laviolette, 2007). The nucleus accumbens(NAc) and basolateral amygdala (BLA) have been identified as key brain regions withincortico-limbic circuitry for guiding decision making in both humans and animals;however, reliance oninterpreting functions of different brain regions in isolation lacks externalvalidity. Determining the way these regions interact to guide behaviour isessential to understanding what drives risky or safe decisions and ultimatelybehaviours.

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Furthermore, most preclinical studies employ assays to assessrisk/reward decision making using internally generated representations ofoutcome contingencies, which are used to guide advantageous  choice. This is problematic because real-lifedecisions are often influenced by external stimuli that inform about thelikelihoods of obtaining favourable outcomes. I propose to use a novel assaycolloquially termed the “Blackjack” task that models situations where externalstimuli indicate probabilities (Floresco et al.

, 2017). Ipsilateraldisconnection of the BLA and NAc, in a rat model, will allow for theexploration of how information transfer, between these regions, facilitatesdecision making about risks and rewards in an externally cued environment.  Laboratorystudies designed to assess human ability to make appropriate risk/rewarddecisions with functional imaging studies have provided indirect evidence showingthese regions do interact to facilitate decision making about probabilisticrewards. When choosing high-risk, compared with low-risk gambles, the anteriorcingulate and NAc are functionally connected (Cohen et al., 2005) andfunctional connectivity can be observed between the cingulate and amygdalawhile anticipating reward outcomes (Marsh et al.

, 2007).  Researchstudies have constructed several tasks to investigate the neural basis ofrisk/reward decision making in animals. Some are designed to mirror the Iowagambling task (Bechara et al., 1999).

Other studies have introduced probabilisticdiscounting tasks, whereby rats are presented the choice between smaller,certain rewards, and larger rewards, with the odds of obtaining a larger rewardchanging systematically over a session (Stopper et al. 2012).  These tasks have been criticized as not beingrepresentative of “real-life” risk/reward decisions.

They are guided byinternally-generated value representations instead of external cues to informanimals of the likelihood of obtaining certain rewards and therefore do notmimic “real-life”.  These studies, whichused a variety of different behavioural tasks, have established that variousaspects of risk/reward decision making are mediated by neural circuits linkingdifferent regions of the prefrontal cortex, orbitofrontal cortex, nucleusaccumbens and the basolateral amygdala (Larkin et al., 2016).

Furthering thesefindings about how subcortical circuits mediate risk-based decision making isimportant, as it provides insight into the pathophysiology underlying abnormaldecision making.  Research hasidentified the influence that the basolateral amygdala has in biasing choicetowards larger, uncertain rewards via interactions with the nucleus accumbens. Bilateralinactivation of either region resulted in a reduced preference of largeruncertain rewards (Ghods-Sharifi et al. 2009). Asymmetrical disconnection andinactivation of the BLA and NAc (St Onge et al. 2012) had the same finding.

Theroles of both the basolateral amygdala and the nucleus accumbens have beenexplored in isolation from one another. The basolateral amygdala reduces thepreference for larger uncertain rewards and the nucleus accumbens supresses randomchoice patterns. (Ghods-Sharifi et al., 2009; Floresco et al., 2017). It reamains unclear whether various nodes within thecortico-amygdalar-striatal circuitry communicate and influence choice anddecisions differently under conditions with external cues versus internallygenerated infrormation. To explore thisissue, I propose to use a task involving choice between small/certain andlarge/risky rewards. The focus will be on how the BLA-NAc circuitry contributesto decision making in conditions involving external cues.

Previous studiessuggest that the NAc shell is responsible for suppressing irrelevant ornon-rewarded behaviours while the BLA mediates judgements surrounding therelative value associated with various courses of action (Floresco, 2015;Ghods-Sharifi et al., 2009).  The use ofexternal cues to guide decisions is essential for adaptive behaviour.

Deficitsin such behaviour are associated with a range of neuropsychiatric disorderswhich may be in part due to ineffective or absent subcortical circuitry.Results will contribute to a broader understanding of the underlyingpathophysiology present in neuropsychiatric disorders and the role of theBLA-NAc pathway.  Materialsand Methods Animals The experiment will utilize Male Long-Evan rats.At arrival animals will be group-housed with four animals per cage and given aweek with free food to acclimate.

Five days before the intended start date forbehavioural training animals will be food restricted to 16 g per day.  ApparatusBehavioural testing will occur in soundattenuated operant chambers. The boxes will be ventilated with a fan doublingas a mechanism to mask external noise. Each chamber will have two retractablelevers, a food receptacle and house light.

The retractable levers will befitted on either side of the food receptacle.  Initial LeverPress Training Behaviours Prior to training on the targeted task, animals willundergo a pre-training regimen consisting of basic lever pressing, retractablelever training and reward magnitude discrimination training. Beforebeginning basic lever pressing each rodent received sugar pellet’s in theirhome cage to reduce neophobia. Training will start with lever-press trainingunder a fixed-ratio-1 (FR1) schedule.

During the sessions the house-light willbe illuminated and one lever inserted into the chamber for 30 minutes or until60 lever presses are made, whichever occurs first. On the following day(s),rats will be required to press the opposite lever until achieving criterion.  Retractablelever training will begin after completion of basic lever training. Sessions willconsist of 90 trials and begin with both levers retracted and the house-lightoff. Every 40 seconds a trial will begin with the illumination of the houselight and the insertion of one of the two levers. If the rat responds within 10s the lever retracts and a single pellet should be delivered via the foodreceptacle with 50% probability. Rats will be trained for approximately 3-6 dto a criterion of 80 or more successful trials (i.e.

< 10 omissions).    Rats will thenprogress and be trained to associate one lever with a larger four-pellet rewardand the other with a one-pellet reward. In phase one, training will be using a48 trail task, blocked into four groups of two forced-choice trials followed by10 free-choice trials (12 trials per block). Every 40 seconds, one or bothlevers will be inserted into the chamber. One lever delivers four pellets with100% probability whereas the other delivers one pellet with 100% probability.The lever associated with the larger reward will remain consistent for theduration 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 programwith the purpose of introducing the probabilistic component of the task.

Thesessions will be comprised of 72 trials, divided into four blocks of eightforced-choice and 10 free-choice trials. In these sessions, selection of thesmall reward lever will always deliver 1 pellet, whereas choice of the largereward lever will dispense four pellets with a 50% probability.  Blackjack TaskIn the targettask of this study, one lever will be designated the large/risky option and theother the small/certain option, and will remain consistent throughout theremainder of training  Blackjacktraining will be comprised of two phases. In the first phase, the first 32trials will be forced-choice, with only one lever inserted into the chamber.Following the forced-choice trails 20 free-choice trials will commence with bothlevers inserted.

Once stable behaviour is apparent the second phase of theexperiment will be initiated. The second phase is identical to the initialphase, excepting of the fact that all trials will be free-choice.  Trials willinitiate every 40 s with house light illumination and presentation of one oftwo distinct auditory cues (3kHz pure tone or white noise, 80 dB). Tones willbe presented pseudorandomly over the session. Once the house light and tone areproduced, one or both levers will insert into the chamber.

  If the rat responds via selection of thesmall/certain lever the tone will turn off immediately and result in one pelletbeing delivered (100% probability) irrespective of which tone was presented.Alternatively, the large/risky lever could yield four pellets, delivered in aprobabilistic manner. The probability of obtaining the larger reward is basedon one of either tone presented in that trial. “Good odds” trials areassociated with one consistent unique tone, (eg. 3 kHz), where a risky choicedelivers 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 leverswill retract in the event of a response on either lever. If the large/riskyoption is chosen and a reward is received the tone and house light will remainon until all the pellets are delivered. If a reward isn’t received after theselection of the large/risky choice, the house light should extinguishimmediately and the tone silenced 2 s after the choice. To facilitate thelearning of associations between each unique tone and the likelihood ofdifferent outcomes (50% and 12.5%), tones will continue after a risky choicehas been made. One pellet will be delivered and the tone turned off, if the small/certainoption is selected.

In the case of an omission, both levers will retract andthe house light and tone will turn off.  The second phase of training (40 free choice trails) willthen commence for another 5-6 days, or until rats display stable patterns ofchoice. Rats will then undergo surgery and be retrained on the task for aminimum of 5 days until stable behaviour is displayed after which they willreceive their first micro-infusion test day.

Stereotaxic Surgery Just prior tosurgery, subanesthetic doses of ketamine and xylazine (50 mg/kg and 4 mg/kgrespectively) will be administered. Rats will be stereotaxically implanted withbilateral 23-guage stainless steel guide cannula into the BLA and NAC. Thecoordinates being anteroposterior (AP) = -3.1 mm; medial-lateral (ML) = +5.2mm from the bregma; dorsoventral (DV) = -6.5 mm from dura and NAc (AP = +3.

4mm; ML = + 1.4 mm; DV = -2.8 mm) (St. Onge et al., 2012). Animals will bemonitored daily for a minimum of a week before being retrained until stablepatterns of choice behaviour return.  To familiarizerats with the infusion equipment and procedure, animals will receive a mockinfusion prior to their first micro-infusion.

Injectors will be placed insideof the guide cannula for two minutes with no infusion administered. Then ratswill be placed in their home cage for 10 minutes before behaviour training.  The day after mock infusions, animals will receive the first of twomicro-infusion test days. Drugs or saline will be infused at a volume of 0.

4 ?l. A solution containing the GABABagonist baclofen and the GABAA agonist muscimol dissolved in 09%saline will be used to achieve inactivation. Infusions will be delivered via 30-gauge injection cannulaeover 89 s.  Rats will receivecounterbalanced infusions on two separate days: (1) a saline infusion into bothstructures contralaterally; (2) drug infusions in two regions in oppositehemispheres. Animals will receive on day of retraining after the first micro-infusion.On the following day a second, counterbalanced infusion will be administered priorto behavioural testing. Histology Aftercompletion of behavioural testing, animals will be anesthetized with isofluranebefore euthanization via CO2.

Brains will be frozen with CO2,sectioned at 50 ?m andmounted. Placements will then be verified with reference to a neuroanatomicalatlas. Experimental Design and StatisticalAnalysis The primary dependent variable of interest is the proportion ofchoices of the large/risky option on good and poor odds trials. This can becalculated by dividing the number of choices of the large/risky lever by thetotal number of those trials in which the rats made a choice, separately forgood and poor odds trials and analyzed using a two-way within subjects’ ANOVAwith treatment and odds (good vs poor) as within subjects’ factors.  Expected Results Previous studies have suggested that BLA projections modulate NAcactivity and influence the direction of behaviour toward reward-related stimuli(Everitt et al., 1999; Setlow et al., 2002). Further studies have suggestedthat the deactivation of the BLA-NAc subcortical circuits results in a biasaway from larger/uncertain reward and that amygdala connectivity with thenucleus accumbens is “responsible for mediating Pavlovian influences of action”(St.

Onge et al., 2012; Seymore & Dolan. 2008). Based upon these priorstudies, a functional disconnection of the BLA-NAc subcortical circuits would beexpected to significantly decrease the choice of the large/risky optioncompared with saline, under testing situations which utilize external auditorycues to indicate event probabilities. This is a reasonable assumption asprevious evidence has suggested that inactivation of the basolateral amygdalaincreases risky choice when odds are poor. This suggests that neural activityin the basolateral amygdala is important for making judgements about the valueassociated with different courses of action (Ghods-Sharifi et al., 2009).

Also,inactivation of the nucleus accumbens shell increases risky choice indicatingit plays a role in suppressing actions that may lead to subjectively inferiorrewards (Floresco et al., 2017). The presupposition that functionaldisconnection of the BLA-NAc circuitry would result in an increased occurrenceof risk averse choices is further supported by the knowledge that NAc activityassociated with choice of larger, riskier rewards is likely driven byexcitatory 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 therisky/large option is likely to yield a greater reward as was seen in aprobabilistic discounting task (St Onge et al., 2012; Ghods-Sharifi et al., 2009).BLA projections modulate NAc activity and may influence the direction ofbehaviour toward reward-related stimuli, therefore the disconnection of suchpathways may impair the ability of animals to make decisions which yield thehighest choice-reward options (Everitt et al. 1999).

 Alternatively, if we do not see that functional disconnection ofBLA-NAc circuitry results in risk-averse choice patterns, it may be that thissubcortical circuit is not involved in the pathophysiology responsible for poorrisk assessment and decisions seen in various human conditions. Additionally, theremay be top-down influences which are impacting choice.  Limitations Understanding howinformation is relayed via subcortical circuitry in risky decision making usingrat models, would offer greater insight as to the pathophysiology underlying cognitiveand behavioural abnormalities in neuropsychiatric disorders; however, there areseveral limitations for interpreting these findings. The most prominentlimitation is the extent to which behavioural and anatomical findings from rodentscan be extrapolated to humans. Even so, it is reasonable to consider such findingsimportant, given studies which identify abnormal structural configuration andvolume in both the amygdala and nucleus accumbens in schizophrenic populations(Tomasino et al.

, 2011; De Rossi et al., 2016).  If it is thecase that functional disconnection decreases preference for the large/risky lever,the current study cannot determine whether the effect is due to an impairmentin discriminating between external cues and associated reward magnitudeprobabilities or an impairment in the ability to discriminate reward magnitudesassociated with the two levers (St. Onge et al., 2012). To remedy this, asecondary experiment would be conducted using a reward magnitude discriminationtask.  Furthermore, the disconnectiondesign does not account for cross-hemispheric communication.

It should be notedthat this poses no major limitation to the study as projections from the BLA tothe NAc are primarily ipsilateral (McDonald, 1987). Other findings havedemonstrated that the prefrontal cortex also influences decision making viacommunication with the BLA and NAc. A secondary set of experiments exploring howthe ipsilateral and contralateral descending projections of the PFC to theamygdala and NAc influence or mediate risky decision making (St Ong et al,.

2012). Both PFC-NAc and PFC-BLA asymmetrical disconnection studies wouldfurther our understanding as well.  

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