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Roles of medial prefrontal cortex subregions in modulation of active and inhibitory action selection… Capuzzo, Giulia 2019

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Roles of medial prefrontal cortex subregions in modulation of active and inhibitory action selection during aversively-motivated behaviours by   Giulia Capuzzo  B.Sc., University of Aberdeen, 2017  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIRMENTS FOR THE DEGREE OF  MASTER OF ARTS  in  The Faculty of Graduate and Postdoctoral Studies   (PSYCHOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  August 2019  © Giulia Capuzzo, 2019    ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  Roles of medial prefrontal cortex subregions in modulation of active and inhibitory action selection during aversively-motivated behaviours  submitted by Giulia Capuzzo  in partial fulfillment of the requirements for the degree of Master of Arts in Psychology  Examining Committee: Dr. Stan Floresco Supervisor   Dr. Catharine Rankin Supervisory Committee Member      Dr. Rebecca Todd Supervisory Committee Member       iii Abstract Decision making in stressful and potentially aversive situations is an evolutionary trait functionally vital to avoid dangerous situations. It can either require action performance to actively avoid negative outcomes, or behavioural suppression to stay safe from danger. Failure to coordinate behaviour discrimination in real-life conflicting threatening situations can lead to aversive consequences because of improper inhibition of motor output when action is needed or, vice versa, when defensive actions are performed instead of withheld. These disruptions of appropriate functioning in avoidance behaviours can lead to improper action selection and increase negative outcomes as seen in disorders such as substance abuse, anxiety and depression.  It has already been shown that striatal regions (namely the core and shell of the nucleus accumbens) are involved in regulation of avoidance behaviours with distinct roles in suppression and promotion of behaviour. Following the cortico-striatal connections with the prefrontal cortex (PFC), we investigated how active and inhibitory avoidance are controlled by the prelimbic cortex (PL) and infralimbic cortex (IL), which have been differentially implicated in instrumental response acquisition and expression. We also probed the extent to which the contribution of these regions is restricted to responding that is aversive and flexible. Separate groups of animals were trained to criteria on three distinct tasks and learned to avoid foot-shock delivery or to obtain sucrose by performing or suppressing lever-press behaviour. Pharmacological inactivation of these prefrontal regions revealed a role for PL in facilitating promotion and inhibition of goal-directed actions to oppose prepotent responding only when response allocation is under flexible conflicting conditions. IL inactivation, instead, was found to be    iv necessary to refine action selection by inhibiting inappropriate responses while promoting instrumental active behaviour through suppression of fearful reactions. These results add a link in the neural network of avoidance processing and help further our understanding of how conditioned instrumental behaviours in threatening situations are processed by cortical regions and how pathological avoidance can arise in neuropsychiatric disorders.                   v Lay Summary Responding to environmental threats is crucial to survival and animals can learn which course of action is most suited for to avoid aversive consequences. The prelimbic and infralimbic cortices are two subregions of the medial prefrontal cortex recruited during decision-making. How these regions are involved in behavioural control when two opposite responses compete for allocation is unknown. Here we examine their contribution by disrupting neuronal activity in well-trained rats prior to performance on an active/inhibitory avoidance task and on two variations of this designed to investigate whether their involvement is exclusive to flexible and aversive environments. We found that the prelimbic cortex regulates behaviours that are in opposition to prepotent conflicting responses, while the infralimbic cortex contributes to inhibition of actions and reactions elicited by aversive and appetitive stimuli. These results will help identify neural pathways involved in threat-responding and response-inhibition as targets for intervention in aberrant avoidance disorders.             vi Preface All experiments were designed by Giulia Capuzzo and Dr. Stan Floresco based on work from the same laboratory by Patrick T. Piantadosi and were conducted at the University of British Columbia, within the Department of Psychology. All experiments were planned and carried out by Giulia Capuzzo. In addition, Giulia Capuzzo completed all surgeries, behavioural training, drug testing procedures and histological analysis with assistance from undergraduate students under her direction. Giulia Capuzzo analysed the data with assistance from Dr. Stan Floresco and Giulia Capuzzo wrote this document with the editing help of Dr. Stan Floresco, supervisory author on this project. Research for this thesis was approved by the UBC Animal Care Committee, protocol number A14-0210 and A18-0242.               vii Table of Contents   Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................. v  Preface .......................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ ix List of Figures ................................................................................................................................ x Acknowledgements ....................................................................................................................... xi Introduction .................................................................................................................................. 1  Aversive Learning and Defensive Behaviours .......................................................................... 1  Neural Systems Mediating Avoidance ..................................................................................... 2  Prefrontal Systems Mediating Avoidance ................................................................................ 4            A Novel Active/Inhibitory Avoidance Task for Prefrontal Function ........................................ 6  Materials and Methods ................................................................................................................ 7  Animals .................................................................................................................................. 7  Apparatus …........................................................................................................................... 7  Surgery ................................................................................................................................... 8  Behavioural Training .............................................................................................................. 9  Familiarization with Lever-Pressing ........................................................................................ 9  Active Avoidance Training .................................................................................................... 10  Active/Inhibitory Avoidance Training: Blocked Design .........................................................12  Active/Inhibitory Avoidance Training: Intermixed Trials ...................................................... 13  Active/Inhibitory Reward-Seeking Training .......................................................................... 13  Microinfusions ...................................................................................................................... 15  Histology .............................................................................................................................. 16  Data Analysis ........................................................................................................................ 17  Results ......................................................................................................................................... 19 Active/Inhibitory Avoidance ................................................................................................. 19     viii Active Avoidance .................................................................................................................. 24  Active/Inhibitory Reward-Seeking ........................................................................................ 28 Active vs. Inhibitory Learning................................................................................................ 33 Discussion ................................................................................................................................... 37  Regulation of Behaviour by PL .............................................................................................. 37 Regulation of Behaviour by IL ............................................................................................... 42 Clinical Implications............................................................................................................... 46 Summary and Conclusions .................................................................................................... 47  References.................................................................................................................................... 48                  ix List of Tables  Table 1 Performance Measures During Active/Inhibitory Avoidance, Active Avoidance and Active/Inhibitory Reward-Seeking Tasks ............................................................ 36                     x List of Figures  Figure 1 Task Structure for Active/Inhibitory Avoidance and Reward-Seeking .............................. 15 Figure 2 Histology Diagram .......................................................................................................... 17 Figure 3 PL Inactivation Impaired Active, but not Inhibitory, Avoidance Performance ................. 23 Figure 4 IL Inactivation Impaired Active and Inhibitory Avoidance Performance ......................... 24 Figure 5 IL, but not PL, Inactivation Impaired Active Avoidance Performance ............................. 27 Figure 6 PL Inactivation Impaired Inhibitory, but not Active, Reward-Seeking Performance. ....... 33 Figure 7 IL Inactivation Impaired Inhibitory, but not Active, Reward-Seeking Performance ......... 34 Figure 8 Length of Training to Criteria is Different for Active and Inhibitory Trials .................... 36                xi Acknowledgements I am extremely grateful and excited to have been able to conduct my research in such a beautiful place and with the support of an exceptional supervisor, Dr. Stan Floresco who has been above and beyond in mentoring me. Thanks to his patience and guidance in these years I have learned invaluable lessons that shaped my growth as researcher and academic. Special thank you to the other members of my committee, Dr. Rebecca Todd and Dr. Catherine Rankin, for taking time to be part of my education and learn about my research as well as providing valuable feedback. I would like to thank all the wonderful members of the Floresco lab that have been incredibly supportive and helpful both in and out of the lab. I am thankful for the team of animal technicians, including Alice Chan and Alice Cheng, they were a precious support to the animal work conducted for this thesis and made long days all the better with their cheerful chats. I shall also extend my gratitude to all the amazing and beautiful people in the behavioural neuroscience office for your friendship and support.   Thanks to Emma and Huw for being my most perfect match and to Sai and Ryan for discussing life while going for groceries, all of you make my days brighter. Finally, thanks to my parents for always supporting me and for raising me to be the person I am today, for that I will always be proud of my roots.        Introduction Aversive learning and defensive behaviours Decision-making under threat of punishment can require flexible action selection in order to avoid negative outcomes and is vital to survival. When more than one defensive response is available, but outcomes are conflicting, learned instrumental behaviours guide action selection and are key to survival and safety. Successful regulation of these relatively complex behaviours involves either suppression or promotion of action and must correctly encode pairing of environmental stimuli to response-outcome contingencies. In response to cues predictive of aversive outcomes, animals can direct behaviour to reduce or prevent exposure to noxious stimuli and, if successful, be safe from danger. These responses can be innate, such as freezing and fleeing, or learned and can be associated to environmental cues. Aversive learning has been extensively studied in paradigms that use Pavlovian fear conditioning, during which a noxious unconditioned stimulus (US) is paired to a neutral stimulus that precedes it. Following consolidation of this association through repeated exposure, the neutral stimulus becomes a conditioned stimulus (CS) that can elicit the fear response originally caused by the US even when the latter is not presented. Any reaction to the CS does not have consequences on US delivery in Pavlovian conditioning, and animals here learn to perform innate reactions (freezing etc.) in response to conditioned stimuli.  In naturalistic settings animals can, instead, often escape and protect themselves from harm by performing learned goal-directed behaviours. Aversive instrumental conditioning allows control over aversive outcomes as it requires performing or inhibiting actions that are specific to the contingency to avoid US delivery. Animals can therefore control the probability of harm by learning    2 to execute the appropriate behaviour. This often involves suppressing instinctual prepotent reactions, like freezing, and focusing on the instrumental action required, which in experimental context can include lever-pressing or shuttling. In avoidance conditioning we initially see Pavlovian mechanisms that lead to a fear response (typically behavioural arrest) to the CS when this announces imminent threat. Later in training, once animals learn the relevant contingency, the innate fearful response is replaced by the active instrumental response that terminates or prevents US delivery. Active avoidance paradigms, thus, introduce instrumental components that allow reliable control over outcomes and establish a response-outcome association. Learning of this association, where promotion of behaviour leads to safety, demands that animals overcome the instinctual response (freezing in rodents) and initiate action, as we know that freezing evoked by Pavlovian conditioning impedes avoidance. In other instances, suppression of behaviour aids survival by avoiding detection by predators. These inhibitory avoidance behaviours, similarly to active avoidance, are learned through instrumental conditioning but require withholding of actions. In such cases, inhibition of inappropriate responses is necessary to avoid harm and learning these instances is equally as important to survival.  Neural systems mediating avoidance Lesion and inactivation studies have identified numerous brain structures mediating expression of different types of avoidance behaviours, although these have been primarily focused on active avoidance and required behavioural output to remain safe from punishment. Of these regions, the striatum is crucial for instrumental conditioning and appears to be key in facilitating aversive responses. Inactivation of its ventral portion, the nucleus accumbens (NAc), more specifically impairs expression of active avoidance (Salamone, 1994; Schoenbaum & Setlow, 2003; Boschen et    3 al., 2011). Conversely, stimulation of inhibitory prefrontal inputs to the NAc modulates motivational valence in a real-time place preference task, eliciting acute avoidance behaviour (Lee et al., 2014). Further evidence points to separate roles of the core (NAcC) and shell (NAcS) subregions of the accumbens in instrumental action selection and encoding of valence in aversive contexts (Parkinson et al., 1999; Corbit et al., 2001; Bassareo et al., 2002). Inhibition of punished reward-seeking is driven by NAcS activity, while NAcC promotes instrumental seeking behaviour irrelevant of punishment during motivational conflict in a conditioned punished reward-seeking task (Piantadosi et al., 2017).  A recent study explored impairments in expression of instrumental aversion following striatal inactivation using a platform-mediated avoidance task in which animals could avoid foot-shock signalled by an auditory cue by stepping onto a platform. During conditioning sessions, animals could access sucrose by pressing a lever some distance away from the platform, thus introducing a novel appetitive conflicting component in response allocation. With training, rats learned to suppress lever pressing for sucrose during tone presentation and step on the platform instead of freezing. Large inactivations encompassing both NAcC and NAcS reduced avoidance and decreased time spent on the platform during the aversive tone, while at the same time this manipulation increased freezing (Bravo-Rivera et al., 2014). These results suggest that the NAc is necessary for expression of platform-mediated avoidance in situations that may entail forms of response conflict (e.g. avoid shock vs. seek food), even though the fearful association to the tone remained intact.  Ventral striatal activity also controls expression of active avoidance responses in a shuttle avoidance task. Here, animals have to move between chambers to avoid foot-shock during a conditioned auditory stimulus and freezing to the tone more readily competes with avoidance    4 responses. Instrumental defensive reactions in this paradigm recruit the NAcS, as activity in this region, not in the NAcC, is critical for expression of signalled avoidance behaviour (Ramirez et al., 2015).   Thus, striatal subregions are differentially involved in expression of aversively-motivated instrumental behaviours depending on task contingencies and size of lesion and work in concert to direct reactions to aversive cues. The roles of NAcC and NAcS have been further explored in a context requiring discrimination of active and inhibitory avoidance contingencies. When instrumental avoidance is associated to an auditory cue and lever-press response has to be either performed or withheld to avoid foot-shock delivery, both NAcS and NAcC are necessary to drive behavioural output and inactivation of either disrupts active avoidance. Inhibitory avoidance, was not affected by NAcC inactivation, while NAcS activity facilitated the inhibition of inappropriate responses, as inactivation lead to increased lever-pressing resulting in foot-shock during these inhibitory trials (Piantadosi et al., 2018). These results show that flexible allocation of behaviour is directed by NAc subregions that play cooperative but distinct roles in action selection to avoid aversive outcomes: the NAcC selectively drives active avoidance, while NAcS modulates both active and inhibitory behavioural responses. Prefrontal systems mediating avoidance Accumbal modulation of aversively-motivated behaviours is likely mediated via interactions with inputs from frontal cortical regions. The NAc receives dense incoming projections from the medial prefrontal cortex (mPFC), and stimulation of prefrontal inputs to the NAc elicits avoidance behaviour in absence of previous contextual association (Berendse et al., 1992; Lee et al., 2014). Projections to the NAc core and shell from the two main dorso-ventral subdivisions of the mPFC,    5 infralimbic (IL) and prelimbic (PL) cortices are topographically arranged: dorsal PL neurons project more prominently to NAcC and ventral PL neurons project to NAcS, while the IL sends projections exclusively to the NAcS (Hurley et al., 1991; Brog et al., 1993; Groenewegen et al., 1999).  Recent studies investigated the contribution of mPFC regions to active avoidance and have established distinct roles for IL and PL in the regulation these behaviours (Gass & Chandler, 2013). Pharmacological and optogenetic manipulations yielded distinct patterns of functional impairment for the two regions, but the results showed inconsistency between behavioural tasks. Studies focusing on active avoidance expression found that, when using the platform-mediated avoidance task described previously, PL inactivation reduced avoidance without affecting freezing behaviour. IL inactivation had opposite effects of reducing freezing without affecting avoidance responses (Bravo-Rivera et al., 2014). In contrast to these results, shuttle avoidance behaviour requires IL, but not PL, as pharmacological manipulation of the former reduced avoidance and disinhibited freezing behaviour, while PL lesion had no effect on either measures (Moscarello & LeDoux, 2013).  The mPFC is also involved in cognitive control of impulsive actions that might have negative consequences and inhibition of innate and learned behaviours (Chudasama & Robbins, 2003; Bari & Robbins, 2013). This aspect of behavioural inhibition is beneficial when learned contingencies are not relevant anymore, as in extinction of conditioned aversive or appetitive responses. Studies focusing on the role of mPFC in regulating extinction of conditioned fear indicate that, while PL is necessary for fear expression, IL regulates recall of extinction and stimulation of this region reduced freezing response and strengthened extinction learning (Quirk et al., 2000; Milad et al., 2004; Sangha et al., 2014; Shiba et al., 2016). In contexts where it is necessary to either promote or suppress responses, behavioural flexibility required to direct instrumental actions necessitates mPFC    6 control and both IL and PL are involved in regulating conflicting goal-directed approach and avoidance behaviours (Burgos-Robles et al., 2017; Schwartz et al., 2017; Verharen et al., 2019). Despite the evidence implicating the mPFC in aversion and behavioural flexibility, the role of these regions in situations that require shifts between promotion and suppression of action to avoid punishment remains to be clarified. A novel active/inhibitory avoidance task for prefrontal function To further clarify prefrontal contributions to avoidance expression, we assessed how inactivation of IL and PL affected performance of rats trained on a task requiring discrimination between signalled active and inhibitory avoidance trials. By using this aversive go/no-go task we could identify how instigation and suppression of actions are controlled in an aversive situation that requires flexible allocation of responses. Animals had to either perform or withhold an instrumental lever-press response upon presentation of an auditory cue to avoid foot shock punishment. This behaviour has been shown to depend on different NAc subregions that receive inputs from the mPFC (Piantadosi et al., 2018), and human research identified corticolimbic activation in similar tasks that combine active and inhibitory behaviour control under threat (Schlund et al., 2011, 2016; Levita et al., 2012). In order to parse out the discriminative component of this task, we compared performance following IL and PL inactivation during a simpler version of the avoidance task that only included active trials and did not require flexible responding. Additionally, we compared the contribution of these regions in promoting or inhibiting instrumental responses in the context of reward-seeking to ascertain how the valence of the outcome (avoiding punishment or obtaining reward) may differentially influence how these regions contribute to promoting or inhibiting behaviours.     7 Materials and Methods Animals All experimental procedures were approved by the Animal Care Committee at the University of British Columbia and conducted in compliance with guidelines provided by the Canadian Council on Animal Care.  All reasonable efforts were made to minimize the number of animals used and their suffering. Male Long-Evans rats arrived at the vivarium weighing 250-275g (Charles River) and were housed in groups of 4 per cage for about a week with ad libitum food and water to allow acclimation and were handled daily. They were then split into pairs of similar weight and food restricted to about 90% of their free feeding weight starting 2-3 days prior to behavioural training and allowed to gain weight throughout the course of the experiment on a delayed-growth curve (about 5-10g/week) to promote exploration and exploitation of the operant environment. Food consisted of standard lab rat chow delivered in the home cage. Colony temperature (21° C) and light cycle (12-h light/dark) were kept constant. Training and handling occurred during the light hours. Handling, surgery and testing was done all by the same female investigator. Apparatus Behavioral testing was conducted in operant chambers (30.5 cm x 24 cm x 21 cm, Med-Associates). Each chamber was equipped with a fan to provide ventilation and attenuation of external noise. As viewed from the opening door, two levers were situated on the right wall of each chamber and were separated by a food receptacle from which sucrose pellet reinforcement was delivered (45 mg pellet, BioServ, Frenchtown, NJ). For all experimental procedures, only the left lever was extended into the chamber. All chambers had three 100 mA cue lights: one over each retractable lever and a central one over the food receptacle. An additional 100 mA house light was situated on the wall opposite the    8 food receptacle. The floor of the chambers consisted in a grid of 19 parallel stainless-steel rods spaced 1.5 cm apart. These were wired to a shock generator and solid-state grid scrambler for the delivery of foot-shock. Four infrared photobeams located just above the grid floors on the front and back walls (those that had no lights or levers) were used to keep count of locomotor activity assessing the number of beam breaks.  All data were recorded on a computer connected to the operant conditioning chambers and task codes were written in Med-PC. Each animal was randomly assigned a box on the first day of operant conditioning that remained constant until the end of the experiment. Surgery Animals were single housed and allowed to gain weight with ad libitum food without training in the operant chambers for 3 days prior to surgery. On surgical day, animals were injected a subanesthetic dose of ketamine (50 mg/kg) and xylazine (5 mg/kg) intraperitoneally and maintained on inhalant isoflurane (1-3%) throughout surgery. Surgery was carried on in a stereotaxic frame, anaesthetized rats were secured with earbars (flat skull) and analgesia was administered subcutaneously (Anafen, 10 mg/kg). They were then stereotaxically implanted with a set of twenty-three-gauge bilateral stainless-steel guide cannula that were lowered into the PL or IL according to the following stereotaxic coordinates (in mm): PL – from bregma: Anterior/Posterior +3.2; Medial/Lateral: ±0.7; from dura: Dorsal/Ventral: -2.8 IL – from bregma: Anterior/Posterior: +2.8; Medial/Lateral: ±0.7; from dura: Dorsal/Ventral: -4.1  Cannulae were held in place by dental acrylic cement adhering to four stainless-steel skull screws at the edges of the incision. Stainless-steel thirty-gauge obturators flush with the end of the guide cannula were inserted into the guide cannula and remained in place until infusions. The animals were given a daily dose of analgesia delivered subcutaneously for two days following surgery    9 (Anafen, 10 mg/kg). Rats were given 5-7 days to recover from surgery before resuming or beginning behavioural training. Behavioural training Active/Inhibitory Avoidance training was similar to that reported by Piantadosi et al. (2018) and based on a paradigm used in humans, as described by Levita et al. (2012). Animals training on the full version of the task (Active/Inhibitory Avoidance) and on the Active Avoidance variation began training shortly after arrival at the colony, while those allocated to the Active/Inhibitory Reward Seeking received surgery prior to training and were exposed to the operant boxes after recovery. All animals received 5-7 operant sessions per week at a consistent hour (9am-1pm across groups) and were fed immediately after each session. Familiarization with Lever-Pressing  On the first day of training animals were placed in the chambers with the house light on and no lever extension or sound was produced for the entire session (1hr). This allowed animals to freely locomote and familiarize themselves with the chamber to reduce neophobia associated with this novel environment.  Subsequent sessions consisted of lever retraction training. In these 60 min sessions, the house light remained illuminated. At the start of the session, the lever on the left of the food port (the one further from the entry door) was extended. If a rat pressed the lever, this caused it to retract for 1s, after which it was reinserted. There was no limit to the number of presses animals could make during a session, this procedure allowed rats to learn about the nature of the manipulandum. Rats received daily sessions until they reached criteria of >40 presses in a session or after 4 days of training.  Rats that did not reach criteria after 2 sessions were presented with sucrose powder placed    10 on the lever to entice lever pressing.  Rats performed a mean of 87 ±11.36 lever-presses during their final lever-retraction training session (range 40-673 presses). Six of rats whose data were included in the final analysis of the active only cohort did not reach criteria after 4 days of training but were still advanced next phase of training (range 0-30 presses). Active Avoidance training After reaching criteria for lever pressing, animals started training on the first phase of avoidance instrumental learning. In this phase daily sessions consisted of 20 active trials with an inter-trial interval of 105s (± 30s). At the beginning of each trial the left lever was extended that coincided with the simultaneous presentation of an auditory cue. For these experiments, the cue initially associated with active avoidance was white noise cue (80 dB), as pilot studies in our laboratory revealed that rats acquire lever press avoidance more rapidly when using this cue versus a high pitch pure tone (9 kHz, 80 dB). Archival data from male rats trained on the same Active/Inhibitory Avoidance task showed those that had the high tone (n=23) associated with active trials took significantly longer to reach criterion performance than those that learned to lever-press on presentation of white noise cues (n=25) during both this first phase of only active trials (F(1,46)=8.90, p<0.01; high tone: 16.65 ±1.47 days; white noise: 11.12 ±1.6 days) and, for those that achieved criteria, the subsequent blocked trial design (F(1,43)=13, p<0.001; high tone (n=20): 21 ±2.61 days; white noise (n=25): 10.64 ±1.34 days). Crucially, those animals that did reach criteria for the final intermixed trial design did not differ significantly in the number of days to criteria (F(1,28)=0.63, p=0.43; high tone (n=8): 12 ±2.02; white noise (n=22): 10.23±1.13) and their performance over the last four days of training was comparable for both active (F(1,28)=0.16, p=0.69; high tone: 82.27 ±2.63; white noise:    11 84.16 ±2.66) and inhibitory trials (F(1,28)=0.17, p=0.68; high tone: 81.83 ±4.33; white noise: 84.37 ±3.37).  If a rat pressed the lever within 15 s of lever insertion, this terminated the sound and retracted the lever and the trial was recorded as a “Successful Active Avoidance”. In addition, these responses also extinguished the house light and illuminated a central stimulus light on the same wall as the lever for 30 s, that served as a signal safety to reinforce the operant response. Presentation of a safety signal reinforces avoidance learning by explicitly signaling successful avoidance, and thus, safety (Berger & Brush, 1975; Dinsmoor, 2001; Fernando et al., 2014, 2015). These 30 s periods were added onto the intertrial interval, so that, depending on the number of successful avoidance responses, sessions could last between 40-50 min. If animals failed to press lever within the first 15 s, this was followed by a 10 s escape period.  Here, the lever remained inserted, but auditory cue terminated and the first of up to 3, 0.5 s foot-shock were delivered. During the escape period, rats could lever-press and that would retract the lever, cease shocks and result in presentation of the safety signal. If no lever press was performed after the first shock, two additional foot-shocks were delivered after 5 s and 10 s (i.e. 25 s and 30 s from the start of trial). Pressing of the lever during the escape period (between delivery of the first and third shock) was recorded as an “Escape”. Complete omission of lever pressing resulted in delivery of a total of 3 shocks and retraction of the lever without presentation of safety signal; these trials were recorded as “Active Avoidance Failure”. During this initial stage of avoidance training, rats were given daily sessions until they made ~70% active avoidance (i.e. < 30% failures) for at least 3 consecutive days before progressed to the next phase of training.     12 Over the course of training, the shock current was initially set to 0.2 mA for all animals and individually titrated by increments of 0.05 mA over the course of training to reinforce avoidance behaviour if avoidance behavior did not improve or performance declined for over 3 consecutive days. Shock intensities (range 0.2-0.4mA) were kept constant after surgery. Individual shock intensity for each session was the same for active and inhibitory avoidance trials. Active/Inhibitory Avoidance training: Blocked design Rats were then introduced to the active/inhibitory avoidance version of the task. Initial training used a blocked version of the task. These daily sessions consisted of 12 active and 12 inhibitory trials (Fig. 1A). Active trials were identical to those described for the initial phase of training. In contrast, inhibitory trials were associated with a 9 kHz, 80 dB tone. Each inhibitory trial started in a manner similar to active trials, in that the tone would play and the lever was inserted. However, during these trials, rats were required to withhold pressing of the lever for 15 s to avoid foot-shock, and pressing of the lever during this period while to tone was on immediately delivered a foot-shock. During these trials, animals could emit more than one lever press, each resulting in foot-shock, as the lever remained extended and tone stayed on for the entire 15 s period.  Trials where the animal pressed the lever at least once were recorded as “Inhibitory Avoidance Failures” and number of lever presses was recorded for each trial. At the end of the 15 s period, the tone was silenced and the lever retracted. If no lever press was performed by the end of the trial, the same safety signal used in active trials was presented (i.e. 30 s light cue). Trials were no lever presses occurred were recorded as “Successful Inhibitory Avoidance”. The latency to press the lever on Successful Active Avoidance, Escape and Inhibitory Avoidance failure trials were recorded.     13 The blocks of trials were initially presented with 12 active trials at the start of the session followed by 12 inhibitory trials.  Over subsequent training sessions, the order of active/inhibitory blocks alternated each day. Rats were trained on this blocked version of the task until they achieved performance criteria of ~70% successful trials in both active and inhibitory avoidance for at least 3 consecutive days.  This design allowed animals to learn active and inhibitory trial presentation was not constant and strengthen the tone-trial type association rather than rely on temporal cues.  During this phase of training, a small proportion of rats that initially displayed criterion performance on active trials began to show a decline in performance over additional training (even though performance on inhibitory trials remained at criterion levels).  In these situations, rats were given remedial sessions and trained on the previous phase of training (with only active avoidance trials) until criterion performance was reached again.  Active/Inhibitory Avoidance training: Intermixed Trials The final version of the task consisted of 12 active and 12 inhibitory trials as in earlier phases (Fig. 1A). However, active and inhibitory trials were now presented pseudorandomly such as that for every block of 4 consecutive trials, 2 would be active and 2 would be inhibitory. All other parameters remained identical to the previous stage of training and animals were trained on this intermixed task until they reached criteria of >70% successful trials in both active and inhibitory trials for 3 consecutive days. During this phase of training, a small proportion of rats began performing poorly on the intermixed task design, and was given remedial sessions of the blocked design to rescue performance. After reaching criteria in this final portion of training, animals were subjected to surgical implantation of cannulas in PL or IL and allowed to recover before being retrained to criterion for pharmacological testing.     14 Active/Inhibitory Reward-Seeking Training This task was designed to resemble the basic structure of the active/inhibitory avoidance task as closely as possible, except here animals had to either emit or withhold a lever press response to obtain food reward (Fig. 1B). The task used the same auditory and visual cues and ITI as in the avoidance task, and a high tone or white noise auditory cue were associated with either active or inhibitory trials. Each trial started with lever insertion and initiation of one of the two auditory cues that instructed the animals to either press the lever within 15 s of its insertion or withhold pressing for 15 s to obtain a two-sucrose pellet reward. On active trials, a lever press terminated the tone, retracted the lever, illuminated the stimulus light/extinguished the house light for 30s and delivered 2 pellets in the food port 3 s after the press marking the trial as “Successful Active Reward-Seeking”. Failure to press the lever during active trials resulted in lever retraction and the trial ended with no reward delivered, these trials were recorded as “Active Reward-Seeking Failure”. On inhibitory trials, if a rat withheld pressing for the 15 s period, the tone terminated, the lever was retracted, the house light was illuminated for 30 s, 2 pellets were delivered 3 s later and the trial was recorded as “Successful Inhibitory Reward-Seeking”. If animals pressed the lever during the 15 s the tone was played on inhibitory trials, the lever retracted, no reward was delivered but the tone continued for the entire 15 s period and the trial was recorded as “Inhibitory Reward-Seeking Failure”.  After receiving initial lever exposure, animals were first presented 2-5 daily session with 20 active trials and were then introduced to inhibitory trials in a blocked design equivalent to that used in avoidance training with 12 active and 12 inhibitory trials. During the initial stages of training, we observed that performance in active trials was substantially higher than in inhibitory trials. As such, on some training days, rats received sessions of 20 exclusively inhibitory trials. After at least 21 days    15 of training, animals were introduced to the intermixed presentation of active and inhibitory trials for 7 days or until criteria of >50% successful active and inhibitory trials for 4 consecutive days. In practice, rats pressed the lever on active trials close to 100%, whereas performance on the inhibitory trials was considerably poorer. Animals that received at least 7 intermixed days and performed >25% successful inhibitory trials before mock infusion but had at least 41% successful inhibitory trials on saline infusion days were included in our analysis. This permissive criterion was rationalized because of the difficulty encountered to train some animals to withhold lever pressing.    Figure 1. Task Structure for Active/Inhibitory Avoidance and Reward-Seeking. Potential scenarios and outcomes of each trial during (A) active/inhibitory avoidance and (B) active/inhibitory reward-seeking. Grey outlined lightning bolt = foot-shock delivery; pair of grey circles = sucrose delivery. Time in seconds (s) describes duration of cues, escape period and safety signal.  Microinfusions Rats received their first of two microinfusion test days after displaying criterion performance for at least three consecutive days. On the day before the first infusion, rats received a mock infusion 10 					 				   	 	 				!"#$		!"#$!"!"#$!"	!"#$  	 	 	   16 min prior to the start of their daily training session, during which they were brought to the same room used for the following infusion days, obturators were removed, and a stainless-steel mock injector flush with the end of the guide cannula was inserted for approximately 2 min.  If performance remained stable on mock infusion day, the next day animals received their first test days, whereas if performance dropped, animals were given 2-3 additional days of training and mock procedure was repeated. On infusion days, animals received 0.4 µl of either 0.9% saline (SAL) or a solution containing 100 ng each of the GABA-B agonist baclofen and the GABA-A agonist muscimol in saline (BM) through injectors inserted in the cannula guides. Each infusion lasted 60 s and injectors were left in place for an additional 60 s to ensure proper drug/vehicle diffusion. Obturators were put back in place and animals returned to their home cage before starting their daily avoidance session 10 min after the end of the infusion. Rats were retrained for at least 2 days until they again displayed criterion performance, after which they received their second counterbalanced infusion. The order of the first infusion (drug or vehicle) was counterbalanced based on their performance relative to the rest of the group in order to maintain comparable pre-infusion performance and animal numbers in both scenarios. Histology Following two infusion days, animals were anaesthetized with 4% isofluorane and euthanized with CO2. Brains were removed and fixed in a 4% formalin buffered saline solution and stored until sectioning. Using a cryostat, 50µm sections were collected and mounted on glass slides. All brains were Nissl stained with Cresyl Violet and acceptable cannula placements are reported in Figure 2. Data from animals whose placement was not correct was removed from analysis.     17  Figure 2. Histology Diagram Schematic of coronal sections, the ventral extent of acceptable microinfusions is labeled by a filled grey triangle for PL and by a black square for IL. Placements are shown for, A, Active/Inhibitory Avoidance, B, Active Avoidance and, C, Active/Inhibitory Reward-Seeking.  Data analysis For all tasks and trial types, successful trials, failures and escapes were converted to a percentage of total trials of each trial type (i.e. active or inhibitory). Percentage of successful active and inhibitory trials were analyzed separately for each brain region using two-way repeated measures ANOVAs with Treatment (SAL vs. BM) and Trial Type (active or inhibitory) as two within-subject factors. The percentage of escapes, the number of shocks received, and response latencies were each analyzed with separate one-way repeated measures ANOVAs with Treatment as the within-subject factor. In situations where rats did not make any response on active or inhibitory trials during a test session, latency values were set to 15 s (the maximum time allotted). Locomotor data were converted into  							   ! 							   ! 							   !"#$%&'	&(# 	&(# "#$%&'	)*(+# 	,-./		,-/		-./	    18 beam-breaks/min, and these values were analyzed separately for each region and task group using one-way repeated measures ANOVAs. Shock intensities used during testing measured in mA for each animal were analyzed separately for the active/inhibitory and the active avoidance tasks using one-way ANOVAs to compare groups assigned to either brain region. Supplementary analyses of how treatments affected performance over the course of a test session were conducted by grouping the percentage of successful trials were into blocks of two trials and analyzed via two-way repeated measures ANOVA with Treatment and Trial Block as within-subject factors.  Lastly, learning rates for different types of trials (active vs. inhibitory) of each task were calculated by counting the number of training days each rat required to display criterion performance for two consecutive days. For each type of response, the data were analyzed separately with one-way ANOVAs. Prescribed alpha level for all analyses was 0.05. Data are reported as mean ±SEM.            19 Results Active/Inhibitory Avoidance Initial learning All rats (n=26) successfully learned the initial active avoidance contingency, taking between 6-27 sessions to reach criterion for an average of 11.2 ±1.1 sessions. On the final day of their active avoidance training, rats performed 78.1% ±3 active avoidance responses, 5.4% ±1.2 escapes, and 16.5% ±2.8 failures. Rats then progressed to the blocked design, during which they received active avoidance followed by inhibitory avoidance trials and vice versa on alternate days. Out of the initial 26 animals, n=23 reached criterion on this blocked design after 18 ±1.8 sessions, over a range of 7-35 session. On the final day of their active avoidance training, rats performed 84.8% ±3.1 active avoidance responses, 4.5% ±1.8 escapes, 10.6% ±2.3 active avoidance failures, and 82.4% ±2.4 successful inhibitory avoidance trials.  Of the 23 animals that progressed on to the last stage of intermixed active/inhibitory avoidance training, 21 rats reached criterion taking on average 11.9 ±1.1 sessions with a range of 3-27 sessions. On their last day of intermixed avoidance training, animals that successfully reached criterion performed well on both active and inhibitory trials with averages of 84.7% ±3.8 successful active avoidance responses, 5.1% ±1.8 escapes, 10.2% ±2.5 active avoidance failures and 88.4% ±2.1 successful inhibitory avoidance trials. Data from the 5 animals that did not reach criterion were not included in the final analysis. Of the 21 rats that achieved the final task criteria and were implanted with guide cannula into either the PL or IL, 6 were excluded from the final analysis due to either cannula placements outside of the region of interest (n=4), unexpected mortality following surgery (n=1) , or poor    20 performance following surgery (n=1). This resulted in final sample sizes of 7 and 8 for the PL and IL groups, respectively. The mean shock intensity for PL group was 0.32 mA ±0.03 (range: 0.30- 0.35 mA), while for the IL it was 0.30 mA ±0.04 (range: 0.25-0.35 mA). These intensities were not significantly different (F(1,13)=1.56 ,p=0.23). PL inactivation Analysis of successful active and inhibitory trials with two-way repeated measure ANOVA exposed a significant main effect of Treatment (F(1,6)=24.39, p<0.01) and, in particular, a Treatment x Trial Type interaction (F(1,6)=36.37, p<0.001), suggesting that PL inactivation differentially affected performance on active vs inhibitory trials (Fig. 3A). Interestingly, this analysis revealed a significant main effect of Trial Type (F(1,6)=61.71, p<0.001), indicating that animals performed significantly better on inhibitory (91.07% ±2.69) vs. active avoidance trials (41.07% ±8.97) across treatment conditions. Simple main effects analyses further confirmed that PL inactivation markedly impaired performance during active trials, significantly decreasing the percentage of successful avoidance responses (F(1,6)=59.44, p<0.001; Fig. 3A, left). Escapes on active trials were comparatively low, and did not differ across treatments (F(1,6)=0, p=1 ; Fig. 3B).  In contrast, PL inactivation did not alter performance on inhibitory trials (F(1,6)=0.67, p=0.44; Fig. 3A, right) nor did it alter the number of lever-presses on these trials (F(1,6)=0.22, p=0.654; Fig. 3C). Reflecting the impairment on active trials, rats received more foot-shocks on PL inactivation days (F(1,6)=48.59, p<0.001; Table 1).  PL inactivation tended to increase response latencies on active trials but this effect did not achieve statistical significance (F(1,6)=4.10, p=0.089; Table 1). Latency to press the lever in    21 inhibitory trials was not affected by PL inactivation (F(1,6)=0.14, p=0.73; Table 1). Thus, rats showed disruption, accompanied by a slight delay, of lever-pressing behaviour to avoid foot-shock upon presentation of active avoidance cue and did not show changes in escape behaviour. Locomotion (beam breaks/min) across sessions did not differ across treatment conditions (F(1,6)=0.22, p=0.66; Table 1). Supplementary analyses examined how PL inactivation altered performance over the course of the test session. This analysis revealed that inactivation of this region reduced active avoidance responses from the beginning of the session that persisted for duration of the training session. This observation was confirmed by analysis of active avoidance performance over blocks of two trials during the course of a session (Fig. 3D) that yielded a statistically-significant effect of Treatment (F(1,6)=35.43, p=0.001), but no effect of Block (F(5,30)=0.87, p=0.51) and no significant Treatment x Block interaction (F(5,30)=0.86, p=0.52). Similar analysis conducted on data from inhibitory trials established the lack of effect was consistent across blocks of trials (Interaction: F(5,30)=1, p=0.44; Block: F(5,30)=0.70, p=0.63; Treatment: F(1,6)=2.27, p=0.18; Fig. 3E). Collectively, PL inactivation selectively impaired the ability to initiate instrumental responses to avoid foot-shock when signaled by discriminative cues, without affecting inhibitory avoidance. Thus, activity in this region seems to be necessary to selectively guide active defensive behaviours in response to learned cues in aversive contexts. IL inactivation A separate group of well-trained rats received IL inactivation and saline treatment prior to tests sessions on the active/inhibitory avoidance task.  In contrast to the more selective effects of PL inactivation, similar treatments in the IL impaired performance on both active and inhibitory trials    22 (Fig. 4A). This was reflected in the analysis of the proportion of successful avoidance trials that yielded a significant main effect of Treatment (F(1,7)=23.25, p=0.002) but no main effect of Trial Type (F(1,7)=1.33, p=0.29) nor Treatment x Trial Type interaction (F(1,7)=0.72, p=0.45).  The impairment on active avoidance trials (F(1,7)=22.97, p<0.01, Fig. 4A left) was not accompanied by a change in the proportion of escapes (F(1,7)=0.88, p=0.38; Fig. 4B).  On the other hand, the impairment on inhibitory trials induced by IL inactivation (F(1,7)=12.92, p<0.01, Fig. 4A right) was associated with an increased number of lever presses over the course of a session (F(1,7)=7.22, p=0.03; Fig. 4C). IL inactivation tended to slow latencies to press during active trials and this effect approached statistical significance (F(1,7)=4.57, p=0.07; Table 1), but did not alter latencies to press on inhibitory trial failures (F(1,7)=0.08, p=0.79; Table 1). In accordance with the broad disruption in performance, rats received correspondingly more shocks following IL inactivation (F(1,7)=46.49, p<0.001; Table 1). Rats, on average, made more beam breaks/min following IL inactivation but this measure of locomotion did not differ significantly across treatments (F(1,7)=3.90, p=0.09; Table 1). Analysis of active and inhibitory avoidance performance over blocks of two trials confirmed effect of Treatment remained significant for both active (F(1,7)=29.87, p<0.001) and inhibitory trials (F(1,7)=7.41, p=0.03). This two-way ANOVA also demonstrated the effect of IL inactivation remained consistent over the session across trial blocks, as analysis of these data failed to yield significant Treatment x Block interaction and effect of Block on either active (both Fs(5,35)<1.14, both ps>0.36; Fig. 4D) or inhibitory trials (both Fs(5,35)<0.94, both ps>0.47; Fig. 4E). These data implicate IL activity in the control of goal-directed behaviour motivated by aversive consequences in a task requiring flexible responding. IL is necessary to successfully allocate    23 responses in both active and inhibitory aspects of avoidance by promoting action and inhibiting inappropriate responses, respectively.  Figure 3. PL Inactivation Impaired Active, but not Inhibitory, Avoidance Performance. A, PL inactivation decreased the percentage of successful active (left) avoidance trials and had no effect on inhibitory trials (right). B, inactivation did not affect percentage of escape responses. C, number of lever presses throughout a session was un altered by inactivation. Percentage of successful, D, active but not, E, inhibitory avoidance responses partitioned over blocks of two trials, was significantly different following saline and PL inactivation treatments. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (n=7) //0/1/2///	345/,167		#$%&'	3 # #&#8&#"&9	&(##$%&'	&(#//0/1/2///:		# #&## #&#;//0/1/2///;:	4 4//0/1/2///# #&#&+	&		 &+	&		:		:		#$%&'		   24  Figure 4. IL Inactivation Impaired Active and Inhibitory Avoidance Performance. A, IL inactivation decreased the percentage of successful active (left) and inhibitory (right) avoidance trials. B, inactivation did not affect percentage of escape responses. C, number of lever presses throughout a session was increased following IL inactivation. Percentage of successful, D, active and, E, inhibitory avoidance responses partitioned over blocks of two trials, was significantly different following saline and PL inactivation treatments. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (n=8)  Active Avoidance Pharmacological inactivation of both IL and PL impaired active avoidance performance and reduced instrumental responding when animals had to discriminate between cues signaling active and inhibitory trials. Following from these results, we investigated the involvement of these prefrontal :		//0/1/# #&## #&#:		#$%&'	2///	 4//0/1/2/////0/1/2///:		# #&#8&#"&9	&(##$%&'	&(#//0/1/2///	34:	45/,167		#$%&'	3# #&#&+	&		 &+	&		;;;;;   25 regions in absence of a discriminatory component using a simpler task during which a single cue informed the rat it had to make a response to avoid foot-shock delivery. Initial learning The group of rats assigned to the active avoidance task initially comprised 31 animals, but only 29 of these achieved criterion performance. Rats received on average 14 ±1.2 session before reaching criterion and undergoing surgery (range 4-26 sessions). On their last day of active avoidance training, average performance was 86% ±3.1 active avoidance responses, 2.8% ±0.9 escapes and 11.2% ±2.8 failures.  Of the 29 rats that achieved the final task criteria for active avoidance and were implanted with guide cannula, 9 were excluded from the final analysis due to either cannula placements outside of the region of interest (n=6) or unexpected mortality following surgery (n=3). This resulted in a final n=10 for each PL and IL group. The mean shock intensity for PL group was 0.29mA ±0.05 (range: 0.20- 0.35 mA), while for the IL group it was 0.30mA ±0.06 (range: 0.20-0.35 mA). These mean intensities were not significantly different (F(1,18)=0.16 ,p=0.70). PL inactivation In stark contrast to what observed following PL inactivation in an active/inhibitory avoidance task, similar treatment did not alter performance of the same region when the task only comprised active trials. The ANOVA of these data revealed these treatments did not significantly alter the proportion of successful active avoidance responses (F(1,9)=0.21, p=0.66; Fig. 5A, left), escapes (F(1,9)=0.05, p=0.83; Fig. 5B, left), number of foot-shocks (F(1,9)=0.18, p=0.68; Table 1) or response latencies (F(1,9)=0.63, p=0.45; Table 1). Locomotion was also unchanged across conditions (F(1,9)=1.80, p=0.21; Table 1). Surprisingly, analysis of active avoidance trials across blocks of two trials (Fig. 5C)    26 revealed a significant main effect of Block (F(9,81)=2.70, p<0.01) although no significant Treatment x Block interaction (F(9,81)=1.05, p=0.41) or main effect of Treatment (F(1,9)=0.21, p=0.66) were found. Animals were less likely to avoid later in sessions, but performance did not vary across treatment conditions. These data indicate that PL activity does not appear to be essential to facilitate instrumental avoidance of punishment in response to a singular warning stimulus and when allocation of behaviour is unambiguous.  IL inactivation Unlike the lack of effect of PL inactivation on active avoidance, IL inactivation impaired performance on this task in a manner similar to that observed during active/inhibitory avoidance.  Inspection of Figure 5A (right) suggests a drop in performance following drug infusion, and ANOVA of successful active avoidance trials confirms a statistically-significant difference between treatment conditions (F(1,9)=19.40, p<0.01), but no effect on escape behaviour  (F(1,9)=2.50, p=0.15; Fig. 5B, right). Accordingly, rats received significantly more foot-shocks on days when IL activity was inhibited (F(1,9)=13.12, p<0.01; Table 1). IL inactivation did not affect latency to perform lever-press (F(1,9)=0.001, p=0.97; Table 1), and locomotion was higher but the effect did not reach statistical significance (F(1,9)=3.89, p=0.08; Table 1). When analyzed across two-trial blocks (Fig. 5D), performance did not yield significant main effect of Block (F(9,81)=1.05, p=0.41) and no Treatment x Block interaction was found (F(9,81)=0.75, p=0.67), while Treatment remained significant (F(1,9)=19.40, p<0.01). Thus, active avoidance behaviour was reduced by IL inactivation consistently throughout the session.     27 These data suggest that IL is required to perform active instrumental behaviours under threat of punishment, even when the circumstances don’t require cue discrimination and flexible responding.  Figure 5. IL, but not PL, Inactivation Impaired Active Avoidance Performance. A, PL inactivation (left) had no effect on active avoidance and IL inactivation (right) decreased the percentage of successful active avoidance trials. B, inactivation of either region did not affect percentage of escape responses. C, percentage of successful active avoidance responses, partitioned over blocks of two trials, was significantly different along the course of a training session across PL treatments. D, percentage of successful active avoidance responses, partitioned over blocks of two trials, was significantly different following saline and IL inactivation treatments. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (PL n=10; IL n=10)   	 	//0/1/2///	3	4%3		 #%3		//0/1/2///:		# #&#8&#"&9% #%:		//0/1/:		2/// //0/1/2///# #&#&+	&		 &+	&		;;;:	4   28 Active/Inhibitory Reward-Seeking Since our manipulations established PL and IL are recruited differently during aversive flexible responding, we decided to further investigate aspects of behavioural control in an appetitive setting that requires similar promotion or inhibition of lever-pressing action. Here we report how inactivation of these regions affected active and inhibitory goal-directed behaviours when appropriate cued responding would result in delivery of sucrose pellets. Initial learning 30 animals received training on the rewarded active/inhibitory contingency. They all received 2-5 initial sessions solely comprising of active trials for an average of 3.5 ±0.3 sessions. On the final day of their active training, rats performed 84.5% ±5.0 active responses. In all later training session animals were presented with inhibitory trials, either with or without active trials. Animals received on average 15.1 ±1.0 sessions of only inhibitory trials (range 25-8) and 17.7 ±0.8 sessions (range 25-11) of active trials followed by inhibitory trials or vice versa, during training with the blocked design protocol. On their last session of inhibitory training before testing, animals scored on average 57.3.5% ±4.8 successful inhibitory trials, while during the last session of active/inhibitory blocked trials rats performed 98.1% ±2.0 active responses and 52.8% ±5.6 successful inhibitory trials. The final stage of intermixed presentation of active and inhibitory trials lasted on average 9.9 ±1.1 sessions, with 21 sessions before pharmacological testing for the slowest animal and 2 sessions for the quickest to learn. On their last day of intermixed training, 5 rats failed to reach performance criterion. The remaining 25 rats all performed 100% successful active responses and an average of 63.7% ±3.4 successful inhibitory trials.    29 Of the 25 rats that achieved criterion performance and received microinfusion tests, 5 were excluded from the final analysis due to cannula placements outside of the region of interest. This resulted in final sample sizes of 11 for PL and 9 for the IL groups.  PL inactivation Inactivation of PL during this appetitive task had opposite effects on active and inhibitory trials than what we observed in the avoidance contingency. Two-way ANOVA of active and inhibitory trials following saline infusion and PL inactivation (Fig. 6A) revealed significant effects for both Treatment (F(1,10)=62.87, p<0.0001) and Treatment x Trial Type interaction (F(1,10)=18.15, p<0.01), indicating that our manipulation indeed affected performance differently between trial types.  Two-way ANOVA carried on this data also yielded a significant main effect of Trial Type (F(1,10)=92.95, p<0.0001). Examination of % Successful trials plotted in Figure 6A suggested trial type affected performance on saline days, as animals seemed to have higher rates of success during active vs. inhibitory trials. To investigate this effect, we proceeded to compare the mean scores for active and inhibitory trials on drug-free days. Our analysis exposed a significant difference between baseline performance for active and inhibitory avoidance (F(1,10)=39.53, p<0.0001) for which rats were much better at active trials (100% ±0) than inhibitory trials (60.61% ±6.27). This suggests that rats found it considerably more difficult to appropriately inhibit lever-pressing rather than make responses when goal-directed behaviour was associated with sucrose delivery. These results are directly opposite to what observed for aversively-motivated active and inhibitory trials, for which inhibition of behaviour was easier than promotion of lever-pressing.     30 Subsequent partitioning of the two-way interaction with one-way ANOVAs revealed no significant change in performance during active trials following PL inactivation vs. control treatments (F(1,10)=3.65, p=0.09; Fig. 6A, left). On the other hand, and in stark contrast to the effect of PL inactivation during active/inhibitory avoidance, inhibitory trials were markedly affected by PL inactivation and analysis of these data confirmed a strong impairment in performance (F(1,10)=62.98, p<0.0001; Fig. 6A, right).  Even though we found no effect on active performance, rats were slower at performing lever-presses during drug-infusion days (F(1,10)=6.81, p=0.03; Table 1). On the other hand, the reduction in successful inhibitory trials was not driven by differences in latency to press (F(1,10)=2.55, p=0.14; Table 1). Thus, active reward-seeking was not impaired during active trials, even though animals showed slower responding, while appropriate inhibition of behaviour was significantly reduced. No significant change in locomotion was observed (F(1,10)=0.19, p=0.67; Table 1). Animals also received significantly fewer reward pellets during infusion days compared to saline (F(1,10)=47.96, p<0.0001; Table 1). Two-way ANOVA conducted on data from active performance over blocks of two trials yielded no Treatment x Block interaction (F(5,50)=1.42, p=0.23) and no effect of Block (F(5,50)=1.42, p=0.23), but exposed a significant Treatment effect (F(1,10)=7.10, p=0.02) that was driven by a slight reduction in performance early in sessions, as apparent from inspection of Figure 6B. Similar analysis of inhibitory trials (Fig. 6C) revealed no main effect of Block (F(5,50)=1.06, p=0.39) or Treatment x Block interaction (F(5,50)=1.68, p=0.16), but a significant main effect of Treatment (F(1,10)=50.10, p<0.0001) showing PL inactivation is affecting all trial blocks consistently during training sessions. Collectively, the data gathered from this PL inactivation    31 experiment implicate this region in the regulation of reward-seeking behaviour through inhibition of inappropriate instrumental actions. IL inactivation Inactivation of IL had different effects on active and inhibitory performance (Fig. 7A), and two-way ANOVA with Treatment and Trial Type as within-subject factors reported significant main effects of Treatment (F(1,8)=169.81, p<0.0001) and a significant interaction of Treatment with Trial Type (F(1,8)=140.42, p<0.0001). Furthermore, analysis of performance using two-way ANOVA carried on these data revealed a significant main effect of Trial Type (F(1,8)=209.16, p<0.0001), suggesting a consistent difference in baseline performance for active and inhibitory trials. Analogous to what reported previously in this task, analysis of mean scores for active and inhibitory trials on saline days confirmed a significant difference in performance (F(1,8)=21.05 , p<0.01) that was driven by fewer successful inhibitory trials (70.37% ±5.40) compared to active trials (98.15% ±1.22). Animals were once again less successful at inhibiting the response than they were at performing lever-press to obtain sucrose under control conditions.  Performance during active trials was not affected by our IL manipulation and rats performed comparable amounts of successful active responses during saline and inactivation test days (F(1,8)=0.08, p=0.79; Fig. 7A left). Inhibition of behaviour during inhibitory trials was, instead, impaired and rats were not able to withhold response when necessary. This resulted in fewer successful inhibitory trials following IL inactivation (F(1,8)=271.70, p<0.0001; Fig. 7A right).  No significant difference in latency was observed during either active (F(1,8)=0.031, p=0.86; Table 1) or inhibitory trials (F(1,8)=0.75, p=0.41; Table 1). Animals displayed an increase in locomotor activity after IL inactivation but this measure only approached statistical significance    32 (F(1,8)=5.03, p=0.055; Table 1). When comparing amount of pellets received, ANOVA revealed rats received fewer reward after IL inactivation test days vs control treatments (F(1,8)=142.55, p<0.0001; Table 1). The lack of effect on active performance was confirmed to be consistent for the duration of the whole session once data analysis of performance by blocks of two trials (Fig. 7B) yielded no significant Treatment effect (F(1,8)=1, p=0.35), no significant interaction (F(5,40)=1, p=0.43) and no main effect of Block (F(5,40)=0.67, p=0.65). Analysis of inhibitory trials across two-trial blocks (Fig. 7C) confirmed Treatment effect reached significance (F(1,8)=160, p<0.0001) and established no significant Treatment x Block interaction (F(5,40)=0.87, p=0.51) and no effect of Block (F(5,40)=1.18, p=0.34), meaning performance was impaired throughout the duration of a session on drug test days.  IL, therefore, is not necessary to drive behavioural output required to gain a sucrose reward. Its activity is, instead, required for successful inhibition of inappropriate responses that are involved in the same goal-directed behaviour.    33  Figure 6. PL Inactivation Impaired Inhibitory, but not Active, Reward-Seeking Performance. A, PL inactivation had no effect on active trials (left) but decreased the percentage of successful inhibitory trials (right). Percentage of successful, B, active and, C, inhibitory reward-seeking responses partitioned over blocks of two trials, was significantly different following saline and PL inactivation treatments. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (n=11) :		//0/1/:		#$%&'	2///	 //0/1/2/////0/1/2///:		# #&#8&#"&9	 #$%&'	# #&#&+	&		 &+	&		;;;   34  Figure 7. IL Inactivation Impaired Inhibitory, but not Active, Reward-Seeking Performance. A, IL inactivation had no effect on active trials (left) but decreased the percentage of successful inhibitory trials (right).  Percentage of successful, C, inhibitory but not, B, active reward-seeking responses partitioned over blocks of two trials, was significantly different following saline and IL inactivation treatments. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (n=9)  Active vs. Inhibitory Learning When comparing the data from the active/inhibitory avoidance vs reward seeking tasks, it became apparent that it was easier for animals to learn to either make an instrumental lever press or withhold it on active or inhibitory trials depending on the task. To confirm these impressions statistically, we computed the number of days it took to reach criterion performance (70% success rate) and # #&#&+	&		#$%&'	&+	&		:		//0/1/2///	 :		//0/1/2/////0/1/2///:		#	 #$%&'	#&#8&#"&9;;   35 maintain it for at least two consecutive days for each trial type for all animals that were included in our final analysis.  Analysis of Active/Inhibitory Avoidance data showed that the length of training required for active and inhibitory trials differed significantly during avoidance learning (F(1,14)=14.61, p<0.01; Fig. 8, left). Thus, during the initial training on active avoidance, rats required 12.1 ±2 days to achieve criterion performance. However, upon introduction of the inhibitory trials in the task, rats learned to suppress lever pressing during these trials much more rapidly, requiring only 4.3 ±1 days to achieve criterion.   When an analogous analysis was carried on training data for our rewarded version of the active/inhibitory task, length of training between the two trial types was again found to be significantly different (F(1,19)=44.66, p<0.0001; Fig. 8, right). However, contrary to what was observed for Active/Inhibitory Avoidance learning, animals achieved criterion performance on active trials (much more quickly 3.15 days ±0.21) that when inhibitory trials were introduced (21.95 days ±2.77). From these data, we conclude that rats found it more difficult to learn to lever press rather than withhold lever pressing to avoid aversive outcomes. On the other hand, when lever-press action was associated with reward-delivery, inhibition of inappropriate response was more difficult and took longer to learn compared to promotion of behaviour.    36  Figure 8. Length of Training to Criteria is Different for Active and Inhibitory Trials. Number of days required to reach stable criterion performance on each trial type. Inhibitory trials were significantly quicker to acquire during avoidance training (left) and significantly slower to acquire during reward-seeking training (right). White dots represent number of days to criterion for individual animals. Error bars are SEM. Asterisk denotes p<0.05 between the SAL and B/M conditions. (Avoidance n=15; Reward-Seeking n=20)     Table 1. Performance Measures During Active/Inhibitory Avoidance, Active Avoidance and Active/Inhibitory Reward-Seeking Tasks. Latencies are measured in seconds, and locomotion is indexed by photobeam breaks. Values displayed are mean ± SEM. * = p<0.01; **= p<0.001; ***= p<0.0001   PL(SAL) PL(BM) IL(SAL) IL(BM) Active/Inhibitory Avoidance          Active Avoidance Latency (s) 5.75 ±0.79 9.49 ±1.93 6.11 ±1.27 7.6 ±1.14      Inhibitory Avoidance Failure Latency (s) 9.21 ±1.87 8.34 ±2.42 7.6 ±2.35 6.78 ±1.12      Shocks received 8.57 ±1.91 29.29 ±2.09** 5.5 ±4.05 16.88 ±2.89**      Locomotion (beam breaks/min) 15.75 ±1.66 16.76 ±3.33 19.58 ±1.96 29.52 ±5.11  Active Avoidance          Avoidance Latency (s) 5.04 ±0.44 4.56 ±0.45 5.57 ±0.41 5.59 ±0.54      Shocks received 7.3 ±3.27 9.1 ±2.65 6 ±1.33 22.9 ±4.33*      Locomotion (beam breaks/min) 18.19 ±2.55 25.52 ±3.95 12.67 ±1.69 20.05 ±2.92  Active/Inhibitory Reward Seeking          Active Response Latency (s) 1.01 ±0.13 2.34 ±0.58* 1.41 ±0.31 1.34 ±0.17      Inhibitory Failure Latency (s) 4.64 ±1.35 2.89 ±0.55 3.9 ±1.19 3.21 ±0.55      Rewards received 36.72 ±1.43 25.09 ±1.22 *** 38.44 ±1.19 26 ±0.82***      Locomotion (beam breaks/min) 18.93 ±3.26 21.33 ±4.15 21.06 ±2.83 32.67 ±5.35    37  Discussion This study investigated the roles of two mPFC subregions, namely IL and PL cortex, in regulating response suppression and promotion during expression of avoidance in well-trained animals. These regions send efferent projections to NAc nuclei that have been shown to differentially affect active and inhibitory avoidance (Piantadosi et al., 2018). In addition, we aimed to investigate how valence and response competition affect prefrontal control of these goal-directed behaviours. By using three different behavioural assays and pharmacological inactivation we were able to describe separate functions for our two regions of interest. Both IL and PL were found to be necessary to promote instrumental action to avoid aversive consequences. PL had a more prominent role in regulating behaviour under flexible conditions, while IL inactivation disrupted active responding even when no inhibitory trials were presented. Suppression of inappropriate responses to maintain safety, though, is dependent on IL, but not PL activity. Further investigation of behavioural flexibility revealed that both IL and PL are necessary for behavioural inhibition when seeking reward in a similar instrumental environment. The results we obtained are indicative of a prefrontal network controlling expression of learned instrumental behaviours in aversive or appetitive contingencies with different roles for PL and IL during response promotion or suppression.  Regulation of Behaviour by PL Active avoidance: The present results highlight that PL activity during active avoidance responses in situations that require some form of response flexibility. PL Inactivation decreased successful avoidance responses during the active/inhibitory avoidance task, where well-trained male rats had to discriminate between cues presented pseudorandomly signalling whether they had to emit or    38 withhold a lever press response to avoid shocks. Escape behaviour only aided initial learning during both tasks and was not a common strategy used in active trials at the time of pharmacological testing, as shown by low escape rates from test day data on the active/inhibitory avoidance task. To examine how mPFC inactivation affects lever-press active avoidance in absence of cue discrimination we trained a separate cohort of animals to only perform active instrumental responses as training inhibitory avoidance would have required to first learn the response to be inhibited. In contrast with results obtained during active/inhibitory avoidance, no effect of PL inactivation on active responding was encountered in this active avoidance only version of the task when a single cue signalled the need to press the lever to avoid shock and no discrimination or cycling between emitting and withholding responses was required. Previous studies investigating active avoidance showed PL is necessary for expression of conditioned place avoidance (Jiang et al., 2014), but not for acquisition and expression of shuttle avoidance during which animals have to run to a different chamber to avoid shock upon a single cue presentation (Moscarello & LeDoux, 2013). This lack of PL involvement is true for when trials are signalled by cues (Tran-Tu-Yen et al., 2009), as well as during Sidman unsignalled shuttle avoidance (Martinez et al., 2013). On the other hand, pharmacological inactivation of the PL impaired platform-mediated avoidance without affecting measures of freezing when rats had to disengage from lever pressing for food to avoid a shock (Bravo-Rivera et al., 2014). Furthermore, inhibitory activity from PL has been shown to drive avoidance responses during this task (Diehl et al., 2018) with aberrant persistent avoidance displaying increased PL activity (Bravo-Rivera et al., 2015). Thus, learning and expression of different forms of avoidance recruit PL differently across different tasks.    39 To further explore these differences in PL involvement and draw a more detailed picture of its role in avoidance we must take into account how response competition plays a role in recruiting this subregion. Avoidance tasks such as platform-mediated avoidance that involve conflicting drives of reward-seeking and punishment aversion or, in the case of the active/inhibitory avoidance, promotion and suppression of behaviour, recruit PL activity to guide active responding. PL circuitry has been recently showed to be recruited for action selection when competing approach and avoidance cues are presented simultaneously (Burgos-Robles et al., 2017) and it is required in other domains of flexibility to inhibit expression of previously learned instrumental contingencies during extinction of avoidance responses (Fragale et al., 2016; Rosas-Vidal et al., 2018; Martínez-Rivera et al., 2019). On the other hand, when no response competition is present, as in shuttle avoidance and our active avoidance task, PL is not necessary for expression of avoidance responses (ARs). Based on these observations, it appears that the involvement of PL activity is linked to flexibility requirements of behavioural tasks, activity within this region is crucial when expression of active avoidance is accompanied by response competition. Its involvement in the expression of lever-press active avoidance is, therefore, critical when animals have to overcome competing drives and flexibly allocate behaviour between active and inhibitory responses. Inhibitory avoidance:  PL was not necessary during inhibitory trials and animals were able to successfully inhibit lever-pressing in these trials during the active/inhibitory avoidance task. PL, though, has been shown to be involved in conditioned suppression of reward seeking under threat of punishment when failure to suppress appetitive behaviour leads to aversive outcomes (Chen et al., 2013; Verharen et al., 2019). Thus, its role in withholding action is dependent on the valence of the    40 conflicting stimuli, and it is seemingly not required when both competing behaviours are aversively-motivated. To summarize, PL is not needed for withholding of inappropriate lever-pressing action to avoid punishment. It instead regulates active avoidance to produce learned actions and avoid threat and its function is restricted to discriminative conditions that require flexible response allocation. Notably, the effects of PL on active and inhibitory avoidance mirror the outcomes of NAc core inactivation during the same task (Piantadosi et al., 2018) and strengthen the assumption for an underlying avoidance circuitry involving cortico-striatal connections. Appetitive behaviour: To assess whether contributions of PL to instigation and/or inhibition of instrumental responding are specific to aversively motivated situations, we designed a complementary task closely resembling our active/inhibitory avoidance paradigm. Rats had to discriminate between the same two auditory cues presented in avoidance trials, but successful trials lead to sucrose delivery instead of foot-shock. Here, PL inactivation showed a pattern of impairment opposite to that of avoidance where performance was disrupted on inhibitory, but not active, trials. These contrasting effects once again suggest that PL involvement in instrumental actions is dependent on valence of conditioned stimuli and in presence of appetitive cues it aids suppression of action to obtain reward while it promotes instrumental responding in an aversive setting. In the appetitive domain, though, PL has also been shown to be necessary to distinguish response-reward contingencies in goal-directed instrumental actions (Balleine & Dickinson, 1998) and facilitating cue-evoked reward-seeking (Ishikawa, Ambroggi, S. M. Nicola, et al., 2008).   Another explanation for the opposing results in aversive and appetitive domains takes into account innate responses and how easily active vs. inhibitory responses are acquired in each task. Our tasks involved a degree of complexity that increased the cognitive demand as animals had to continuously update their    41 action selection between opposed active and inhibitory cued responding. Over the course of training, we observed that acquisition of active responses in an appetitive context was much more rapid compared to inhibitory ones, whereas inhibitory avoidance responses were acquired more rapidly than active avoidance response for rats trained to avoid foot-shock. These observations lead to further rationalisation of our seemingly contrasting findings in active/inhibitory avoidance and reward-seeking based on which response is easier to perform and thus prepotent in that setting. When, in appetitive situations, the prepotent and easier response is to promote behavioural output (i.e. press the lever), PL activity ensures that actions that oppose such drive (i.e. withhold pressing) can be performed when appropriate. In avoiding shock delivery, however, animals found it hardest to press the lever and this active responding contrasting the prepotent response (i.e. inhibition of pressing) in the aversive context required PL activity. We therefore propose that PL is necessary to facilitate learned behaviours that oppose prepotent responses, it provides the cognitive control needed to overcome competing innate drives in both aversive and appetitive situations that involve conflicting options for action initiation or inhibition.   The human homologous of dorsal PL, the dorsal anterior cingulate cortex (dACC), has been associated with cognitive control over instrumental behaviours along with the ability to direct behaviour away from automatic prepotent responses to allow for goal-directed action when evaluating alternative choices of action (Kolling et al., 2016; Shenhav et al., 2016). Results from human research further strengthen this theorization of PL function as neural response within prefrontal regions during approach-avoidance behaviours indicate these are recruited when overriding automatic emotional responses is necessary to guide goal-directed behaviour (Roelofs et al., 2009). Disruption of human anterior prefrontal cortex during conflicting events effectively reduces such behavioural control with a concomitant increase in prepotent emotionally-driven responding (Volman et al., 2011).    42   Regulation of Behaviour by IL  Active avoidance:  In contrast to the effects of PL inactivation on active/inhibitory avoidance, similar treatment administered within the IL to well-trained rats revealed that activity in this region of the mPFC supports active avoidance while at the same time suppresses inappropriate punished responding during inhibitory trials. For animals in the active avoidance cohort, inactivation also led to impaired active avoidance when unequivocal instrumental contingencies only necessitated active responding. The correspondence of results obtained for active avoidance under both flexible and certain response allocation, contrary to what observed for PL, suggests IL is a key substrate for the expression of lever-press avoidance in both presence and absence of response competition.  The role of this region in promoting avoidance responses might rely on its ability to modulate aversive associations and downregulating expression when they are no longer relevant. This behavioural flexibility is exploited, for example, during extinction of aversive conditioning when fearful reactions need to be suppressed to adapt to contingency alterations. IL lesion and inactivation studies found it is indeed necessary for inhibition of fear responses once these have been extinguished (Quirk et al., 2000; Sierra-Mercado et al., 2011; Milad & Quirk, 2012; Gass & Chandler, 2013; Sangha et al., 2014) and IL stimulation alone reduced the fear response by simulating the extinction memory (Milad & Quirk, 2002). In conflicting situations involving opposite drives such as approach-avoidance tasks, IL modulates behavioural flexibility by suppressing avoidance in trained animals during reward consumption and inactivation reinstates fear response to a pain-predicting cue (Schwartz et al., 2017). Similarly, to allow expression of avoidance, animals have to suppress    43 innate fearful reactions and perform an instrumental response. In this regard, IL stimulation has anxiolytic effects that would aid action promotion in fearful circumstances (Shimizu et al., 2018). During shuttle avoidance, fearful reactions interfere with avoidance and running to safety directly competes with freezing behaviour. Learning and expression of instrumental responses in this instance is facilitated by IL activity suppressing freezing and inactivation impairs previously acquired avoidance expression while increasing CS-evoked freezing (Martinez et al., 2013; Moscarello & LeDoux, 2013).  In contrast with these reports, IL inactivation does not affect responding during tasks, like conditioned place avoidance, for which Pavlovian reactions have less interference with the AR (Jiang et al., 2014). Platform-mediated avoidance provides an additional example of how IL controls suppression of fearful reactions without necessarily affecting avoidance under conditions that do not involve behavioural competition. Here, reversible IL inactivation increased freezing to the conditioned tone while reducing reward-seeking but had no effect on avoidance responding and animals could still perform the AR even though the fear to the stimulus was reinstated (Bravo-Rivera et al., 2014). The role of IL in active avoidance depends on the degree to which the response is in competition with Pavlovian reactions that lead to behavioural arrest. IL contributes to control of active behaviour motivated by impending punishment that requires suppression of fearful reactions to mediate avoidance responses. Even though significant, the magnitude of impairment in active avoidance performance observed following IL inactivation was more modest than that caused by PL inactivation during active/inhibitory avoidance suggesting the latter has a more prominent role in    44 this behaviour. IL functioning might therefore be supported by PL activity as animals must use acquired instrumental responses to shape goal-directed actions.  It has been shown that such fearful reactions are present in the form of freezing that interferes with active avoidance responding during tasks such as shuttle avoidance. If exposure to the tone caused excessive freezing during IL inactivation, we would expect to see reduced locomotion and slower latencies to press the lever during those trials. Data from latencies and beam breaks, though, does not support this hypothesis as we observed no significant change in latency during active trials and a tendency towards higher locomotor values indicating freezing is likely not an interference to active responding in our avoidance contingency. Inhibitory avoidance:  IL inactivation impaired inhibition of lever pressing during inhibitory avoidance trials and rats received more shocks as they increased punished responding. This is in line with IL lesion studies that reported an increase in punished responding in Vogel conflict test during which a shock is delivered every 20 licks for water and suppression of licking behaviour is required for safety from punishment (Resstel et al., 2008). In a step-down paradigm, latency to step down a grid floor that had delivered foot-shock the previous day was used to measure passive avoidance and electrolytic lesioning lead to faster stepping down showing IL mediated inhibition of previously punished behaviour (Jinks & McGregor, 1997).  IL therefore refines action selection under threat of punishment and is necessary for inhibition of inappropriate actions and/or reactions during conflict to support both active and inhibitory avoidance in well-trained male rats. Inhibition of fearful reactions that interfere with the AR allows expression of active avoidance, while inhibition of punished actions prevents aversive outcomes and is necessary for inhibitory forms of avoidance. Our findings following IL inactivation    45 closely resemble NAc shell inactivation profile (Piantadosi et al., 2018) and add important insights into the neural network of avoidance as we know this striatal region receives dense projections from IL.  Appetitive behaviour: As we did for the PL, we assessed whether IL activity is involved when promotion and inhibition of behaviour are motivated by reward instead of aversion using a variation of the original active/inhibitory avoidance task. IL was necessary to inhibit inappropriate reward-seeking lever-press responses during inhibitory trials, but inactivation had no effect on active trials. In agreement with our findings in the appetitive domain and the inhibitory nature of IL functioning, inhibition impaired recall of extinction and produced reinstatement of lever-press cocaine-seeking in rats that previously extinguished the behaviour while pharmacological activation reduced cocaine-induced reinstatement (Peters et al., 2008). When animals had continuous access to a lever paired with cued operant sucrose delivery, IL inactivation increased unrewarded responding showing impaired cue discrimination and poor allocation of active behaviour (Ishikawa, Ambroggi, S.M. Nicola, et al., 2008). IL activity also suppresses impulsive actions in a 5-choice serial reaction time task where animals have to direct approach to the appropriate target aperture. Under these test conditions IL lesions increased premature responding that delayed obtainment of reward (Chudasama et al., 2003).  It is interesting to highlight that even though inactivation of this region did not affect expression of active reward-seeking, it impaired active responding when motivated by aversive consequences during our avoidance tasks. Following from our discussion of how IL facilitates active and inhibitory avoidance likely by inhibiting fear reactions and punished actions, respectively, the lack of effect during active trials in this rewarded task can be explained by the absence of conflicting    46 drives to be suppressed. During reward-seeking inhibitory trials lever-pressing action must be withheld and this was controlled by IL for our well-trained animals, but promotion of behaviour in active trials didn’t require behavioural inhibition as no fearful reaction interfered with it and was not mediated by IL activity. IL is therefore necessary for behavioural suppression of inappropriate goal-directed actions and Pavlovian reactions. More specifically, it promotes expression of active behaviour in those instances when interfering fearful reactions need to be suppressed and it facilitates learned inhibitory responding motivated by aversive and appetitive cues. Clinical Implications Avoidance behaviours are beneficial when properly directed to avoid harm, but excessive avoidance of external stimuli reinforced by reduction in anxiety prevents healthy functioning in non-threatening situations. Maladaptive responding to innocuous stimuli by means of exaggerated defensive reactions and avoidance is associated with neuropsychiatric conditions including PTSD (Pineles et al., 2011), autism (Richer, 1976), major depression (Ottenbreit et al., 2014) and anxiety disorders (Heuer et al., 2007). Identifying crucial neural networks underlying such impairments will help in reaching a more comprehensive understanding of the disorders and further research on how to help those suffering from them.  Human studies of approach-avoidance conflict highlight mPFC regions as key targets for anxiolytic treatments aimed at reducing disproportionate avoidance (Aupperle et al., 2015; Chrysikou et al., 2017). Activation in human mPFC also correlates with anticipatory anxiety (Simpson et al., 2001) and incoming threats increase mPFC activity as different goal-directed behaviours are evaluated to avoid the aversive predicted outcome (Mobbs et al., 2007). Even though active avoidance behaviours have been the main focus of anxiety research and coping mechanisms,    47 behavioural inhibition is a key component of these disorders (Chamberlain et al., 2005; Murray et al., 2009). Thus, animal research aimed at developing behavioural assays and uncovering neural correlates of behavioural control in aversive situations is critical to support research into human dysfunction of avoidance regulation. Summary and Conclusion The findings reported in this study provide novel insights into prefrontal modulation of competing active and inhibitory behaviours in the face of aversive outcomes, with separate roles for PL and IL cortices. By employing variations of our active/inhibitory avoidance behavioural contingency we were also able to gather additional information on how these regions are recruited at times when only active behaviour is necessary and during appetitively-motivated responding. Combined analysis of results from these sets of studies offered a profile for PL in regulating promotion or inhibition of behaviour during conflicting response allocation that is dependent on valence of stimuli and prepotent responding. This region is necessary to facilitate behavioural goal-directed actions that are in competition with those easiest to perform. More specifically, it is required for lever-pressing motivated by impending shock and to inhibit inappropriate responding during reward-seeking. Results from IL inactivation studies instead revealed it has a more prominent role in refining action selection in appetitive and aversive contingencies through inhibitory control. It is necessary to suppress fearful reactions that interfere with active avoidance as well as to withhold action when this disrupts inhibitory reward-seeking and avoidance. These data improve our understanding of aversively-motivated behaviours controlled by PL and IL to form a hypothesis of avoidance control that complements previous literature and recent findings regarding NAc function. Exposing precise    48 neural networks underlying these behaviours is crucial for our understanding of avoidance under optimal and aberrant states.      49 References Aupperle, R. L., Melrose, A. J., Francisco, A., Paulus, M. P. and Stein, M. B. 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