Effects of Bilateral Prelimbic Cortex Inactivation on Contextual Biconditional Discrimination Memory Retrieval

in Adult Long-Evans Rats

by

Sadia Riaz

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

Department of Psychology University of Toronto

© Copyright by Sadia Riaz 2016

ii

Effects of Bilateral Prelimbic Cortex Inactivation on Contextual

Biconditional Discrimination Memory Retrieval in Adult Long-

Evans Rats

Sadia Riaz

Master of Arts

Department of Psychology University of Toronto

2016

Abstract

The prelimbic (PL) subregion of the medial prefrontal cortex has been widely implicated in the

contextual control of appetitive and aversive conditioning through studies of context-induced

reinstatement of drug seeking and contextual fear conditioning. However, the role of the PL in

mediating contextual processing in appetitively motivated tasks without the involvement of

abused substances remains underexplored. Thus, the present study sought to investigate the role

of the PL in this process using a previously reported contextual biconditional discrimination

(CBD) memory task. We examined the effects of temporary post-acquisition pharmacological

inactivation of the PL on CBD memory retrieval and observed robust deficits in contextual

memory retrieval following PL inactivation. Our data provide novel insight into the role of the

PL in contextual processing in appetitively motivated tasks in the intact brain, indicating a more

general role for the PL in appetitive and aversive contextual processing in the drug-free brain.

iii

Acknowledgments

I would like to thank my supervisor, Dr. Rutsuko Ito, for her guidance, and for always being

patient and encouraging. I thank my committee members, Dr. Takehara-Nishiuchi and Dr.

Arruda-Carvalho, for their time and invaluable feedback on this thesis. I would also like to thank

Dr. Schumacher and David Nguyen for all of the skills they have taught me; without their help

and input, this study could not have been successfully conducted.

Finally, I must express my very profound gratitude to my parents and to my cat for providing me

with unfailing support, love and continuous encouragement throughout my years of study and

through the process of researching and writing this thesis. This accomplishment would not have

been possible without them. Thank you.

Author

Sadia Riaz

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Table of Contents

Acknowledgments .......................................................................................................................... iii  

Table of Contents ........................................................................................................................... iv  

List of Figures ................................................................................................................................ vi  

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

  Methods .......................................................................................................................................8  2

2.1   Subjects ................................................................................................................................8  

2.2   Apparatus .............................................................................................................................8  

2.3   Behavioural Procedures .......................................................................................................9  

2.3.1   Habituation and exposure to sucrose pellets ............................................................9  

2.3.2   Magazine training ....................................................................................................9  

2.3.3   Nose poke hold training ...........................................................................................9  

2.3.4   Contextual biconditional discrimination (CBD) training ......................................10  

2.3.5   Guide cannula implantation surgery ......................................................................11  

2.3.6   CBD (recap) training ..............................................................................................11  

2.3.7   General infusion procedure ....................................................................................12  

2.3.8   CBD test .................................................................................................................12  

2.3.9   CBD post-washout training ....................................................................................12  

2.3.10   CBD extinction text ...............................................................................................13  

2.3.11   Locomotor activity test ..........................................................................................13  

2.4   Histology ............................................................................................................................13  

2.5   Data Analysis .....................................................................................................................14  

  Results .......................................................................................................................................15  3

3.1   Histological verification .....................................................................................................15  

3.2   Magazine training and nose poke hold training .................................................................15  

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3.3   CBD training ......................................................................................................................15  

3.4   CBD memory retrieval after surgery .................................................................................16  

3.5   Stable baseline CBD memory expression ..........................................................................16  

3.6   CBD test .............................................................................................................................17  

3.7   CBD extinction test ............................................................................................................17  

3.8   Locomotor activity test ......................................................................................................17  

  Discussion .................................................................................................................................19  4

4.1   PL in contextual memory processing .................................................................................19  

4.2   PL in CBD memory expression in the presence of reward ................................................21  

4.3   Locomotor activity regulation in the PL ............................................................................22  

4.4   A within-subjects experimental design ..............................................................................22  

4.5   Future directions ................................................................................................................23  

4.6   Conclusion .........................................................................................................................23  

References ......................................................................................................................................24  

Figures............................................................................................................................................30  

vi

List of Figures

Figure 1 30

Figure 2 32

Figure 3 33

Figure 4 34

Figure 5 35

Figure 6 36

Figure 7 37

Supplementary Figure 1 38

1

Introduction 1Memory can be subdivided into many types: long- or short- term (Wescourt & Atkinson, 1973),

declarative or non-declarative (Squire, 1992), and procedural (Shadmehr & Holcomb, 2000),

emotional (Arntz, Groot, & Kindt, 2005), spatial (Olton, 1977), or contextual (Maren & Holt,

2000) (etc.). Research suggests that these different types of memory are represented within

different neural circuits of the brain (Broadbent, Squire, & Clark, 2004; Eichenbaum, Yonelinas,

& Ranganath, 2007; Manns, Hopkins, & Squire, 2003; Wescourt & Atkinson, 1973). Some of

the key regions associated with different memory systems include the medial prefrontal cortex

(mPFC; Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004; Corcoran & Quirk, 2007;

Takashima et al., 2006), hippocampus (Baddeley, Jarrold, & Vargha-Khadem, 2011; Scoville &

Milner, 2002), amygdala (Cahill, Babinsky, Markowitsch, & McGaugh, 1995; Luo, Xue, Shen,

& Lu, 2013), and striatum (McDonald & White, 1994; Pennartz, Ito, Verschure, Battaglia, &

Robbins, 2011).

Context-dependent memory in particular has drawn much interest over the last decade (Smith &

Vela, 2001), and it refers to the facilitated recollection of information when memory retrieval

occurs in the same (actual or imagined) context as that in which the information was acquired

(Stefanucci, O’Hargan, & Proffitt, 2007). Many aspects of behaviour are context-dependent. In

fact, appropriate responding to environmental contextual cues may be necessary for survival; for

example, an organism may choose to avoid threatening or fearful contexts, while approaching

appetitive contexts. Behavioural patterns (e.g., foraging and mating) may also change in response

to contextual changes (e.g. threat of predation, association with drug use, etc.). Conversely,

aberrant context processing can lead to disadvantageous outcomes such as context-induced drug

relapse (Bossert et al., 2011).

Although many different types of contextual information can contribute to the formation of

context-dependent memory, such as cognitive context or mood (Balch, Myers, & Papotto, 1999),

the present study will focus on environmental context-dependent memory (Smith & Vela, 2001).

When a complex set of environmental cues is encoded and associated with incentive or aversive

properties of specific stimuli, contextual conditioning is said to have occurred (Fustiñana, Tano,

Romano, & Pedreira, 2013). Contextual conditioning can also be regarded as a type of occasion

setting (Bueno & Holland, 2008; Maren & Holt, 2000) whereby one conditioned stimulus (CS1,

2

the context) alters the response to another conditioned stimulus (CS2) by signaling whether CS2

will be reinforced. However, in contrast to simple contextual conditioning, the occasion setter

does not signal the delivery of the unconditioned stimulus (US) directly.

Several theories have emerged to explain contextual conditioning. One view of contextual

memory representation proposes that context is represented as an additive set of independent

features, which is supported in the literature by the phenomenon of pattern completion (O'Reilly

& Rudy, 2001; Rudy, Huff, & Matus-Amat, 2004). However, an alternative (and perhaps more

dominant) view of context representation supports the idea that contextual learning necessitates

the integration of numerous complex cues into a cohesive, conjunctive representation of context

(Holland & Bouton, 1999; Nadel & Willner, 1980). According to Rudy and Sutherland’s

configural association theory (1995), elemental (or discrete cue) associations differ from

configural (an assortment of stimuli or context) association such that each cue (e.g., A and B) is

independently associated with the outcome (C) in the former, while the latter involves

association of a compound AB (as opposed to individual cues) with the outcome. Although the

compound AB is composed of discrete cues A and B, the configural representation is unique, and

dissociable from its constituents (Rudy & Sutherland, 1995; Kehoe & Gormezano, 1980;

Whitlow & Wagner, 1972).

The configural association theory of context processing is strongly supported by a class of

discrimination problems that cannot be solved by forming multiple elemental associations;

rather, they require a configural association system (Rudy & Sutherland, 1995). These tasks

include biconditional discriminations (Harris, Livesey, Gharaei, & Westbrook, 2008; Whishaw

& Tomie, 1991), negative patterning (Rescorla, 1972; Whitlow & Wagner, 1972; Deisig,

Lachnit, Giurfa, & Hellstern, 2001), feature-neutral compound discriminations (Gallagher &

Holland, 1992; Rudy & Sutherland, 1995) and transverse patterning (Leirer et al., 2010; Reed &

Squire, 1999). Of particular interest to the present study is the biconditional discrimination task.

In biconditional discrimination tasks, four elements (A, B, C, D) are combined to create two

reinforced compounds and two non-reinforced compounds such that each element is equally

associated with a reinforced and non-reinforced outcome (e.g., AB+, CD+, AC-, DB-). As a

result, linear associations of discrete elements to the outcome cannot explain the observed

increase in responding to the reinforced compounds only (Harris et al., 2008). Previous studies

3

have also examined the acquisition of context-dependent biconditional discrimination whereby

identical stimuli (or pairs thereof) have opposing outcomes based on the context in which they

are presented (Ramirez & Colwill, 2012; Gonzalez, Welch, & Colwill, 2013). In other words,

one element of the compound stimulus is the static context in which the stimulus presentation

occurs; for example, in one context (A) a tone may be reinforced (T+, e.g. with a sucrose reward)

and a light may be non-reinforced (L-) and in another context (B) the stimuli may be T- and L+.

Responding is reinforced when T is presented in context A (AT+) but not when T is presented in

context B (BT-), and responding to L is rewarded when L is presented in context B (BL+) but

not when L occurs in context A (AL-). As a result, the summation of the associative strengths of

individual elements in each compound (AT, AL, BL, BT) would be the same, and only a

configural representation of the compounds could account for the differential responding to the

rewarded and non-rewarded composites (AT+, AL-, BL+, BT-). Such contextual biconditional

discrimination (CBD) has been shown in a variety of species including rats (Wilson & Pearce,

1989), mice (Gonzalez et al., 2013), monkeys (Ramirez & Colwill, 2012), humans (Harris &

Livesey, 2008; Lober & Lachnit, 2002) and rabbits (Saavedra, 1975).

In the existing literature, the hippocampus (HPC) has been widely implicated in contextual

learning (memory acquisition) through evidence that permanent pre-training hippocampal lesions

result in impaired contextual learning and discrimination (Good & Honey, 1991; Maren & Holt,

2000; Penick & Solomon, 1991; Sutherland & McDonald, 1990). Moreover, a recent study (Riaz

et al., in preparation) has provided evidence for a role of the ventral, but not dorsal, HPC in CBD

memory retrieval. The present study aimed to extend our investigation into the neural substrates

of CBD memory retrieval to the medial prefrontal cortex (mPFC) subregions for two reasons:

Firstly, research evidence supports the role of the mPFC in contextual processing (Hyman, Ma,

Balaguer-Ballester, Durstewitz, & Seamans, 2012; Kalisch et al., 2006) but there is limited

evidence of the potentially differential roles of different subregions of the mPFC in the

contextual control over appetitively motivated behaviours for natural reward. Secondly, the

strong anatomical connections between mPFC subregions and the ventral HPC (Euston, Gruber,

& McNaughton, 2012) suggest that these regions may form a functional circuit in appetitive

context processing.

Evidence for the role of the mPFC in context processing comes primarily from studies of

context-induced reinstatement of drug seeking (Di Pietro, Black, & Kantak, 2006; Peters,

4

Kalivas, & Quirk, 2009; Bossert et al., 2011), encoding of contextual representations (Hyman et

al., 2012), the expression of contextual fear (Corcoran & Quirk, 2007), and context-dependent

discriminative cue learning (Ashwell & Ito, 2014). It has been hypothesized that the functions of

the mPFC in context processing likely depend on its strong connections with the HPC, a region

with a well-established role in spatial and contextual processing (Hyman et al., 2012; Laroche,

Jay, & Thierry, 1990; Vertes, 2006). One proposed view is that information about context

reaches the mPFC via the HPC, through the direct (and interestingly, unidirectional) inputs from

the HPC to the mPFC (Euston et al., 2012). Further evidence for a functional interaction between

the mPFC and HPC comes from electrophysiology studies that have reported HPC theta wave

phase-locking to mPFC neurons during tasks of memory acquisition and retrieval, such as spatial

memory tasks and memory-guided choice tasks (Hyman, Zilli, Paley, & Hasselmo, 2005; Siapas,

Lubenov, & Wilson, 2005; Benchenane et al., 2010; Fujisawa & Buzsa´ ki, 2011; Jones &

Wilson, 2005). Reductions in this synchronous activity are also predictive of errors, suggesting

that mPFC-hippocampal phase-locking may be necessary for correct memory retrieval (Euston et

al., 2012).

The rat mPFC is typically divided into four main subregions: (dorsal to ventral) the anterior

cingulate (AC), the prelimbic (PL, analogous to Brodmann area 32 in humans; Gass & Chandler,

2013), the infralimbic (IL, analogous to Brodmann area 25 in humans; Gass & Chandler, 2013),

and the dorsopeduncular cortex (DP) (Öngür & Price, 2000; Heidbreder & Groenewegen, 2003;

Paxinos & Watson, 1998; Vertes, 2006). The mPFC is sometimes simply divided into

dorsomedial PFC (dmPFC) and ventromedial PFC (vmPFC) due to a lack of a distinct boundary

between the different subregions and the small volume of the rat brain (Peters, Pattij, & De

Vries, 2013); the dmPFC includes the PL and portions of the AC, whereas the vmPFC includes

the IL and portions of the DP (Peters et al., 2013). Increasing evidence suggests that different

subregions of the mPFC subserve distinct functions (Vertes, 2006); while the AC has been linked

to various motor behaviours, the PL and IL have been associated with a range of cognitive,

emotional, and mnemonic processes (Heidbreder & Groenewegen, 2003; Vertes, 2004).

Anatomically, a majority of the HPC inputs to the mPFC (including IL and PL) arise from the

ventral HPC (Euston et al., 2012; Hyman et al., 2012; Jung, Qin, McNaughton, & Barnes, 1998).

While the PFC is also connected to the dorsal HPC, these projections are indirect (via the

thalamus) and appear to be significantly weaker (Thierry, Gioanni, Dégénétais, & Glowinski,

5

2000; Yoon, Okada, Jung, & Kim, 2008; Jay & Witter, 1991; Verwer, Meijer, Van Uum, &

Witter, 1997; Ishikawa & Nakamura, 2006; Chudasama, Doobay, & Liu, 2012). Further, despite

the absence of any direct projections from the mPFC to the HPC, some evidence suggests that

there are likely to be indirect inputs from the mPFC to HPC through relays in the midline

thalamus (Wouterlood, Saldana, & Witter, 1990; McKenna & Vertes, 2004; Vertes, Hoover, Do

Valle, Sherman, & Rodriguez, 2006; Chudasama et al., 2012). Given the evidence for the role of

the ventral, but not dorsal, HPC in contextual control of appetitive behaviour (Riaz et al., in

preparation), these anatomical connections are consistent with the view that mPFC’s role in this

process is dependent on its functional connectivity with the HPC.

Of the different mPFC subregions, the IL and PL regions have been well researched in their

distinct roles in context-driven control over behaviour. Most prominently, these regions have

been shown to differentially control context-dependent fear (Corcoran & Quirk, 2007) and drug

seeking (Di Pietro et al., 2006; Peters et al., 2009). While activity in the PL region has been

shown to promote the expression of conditioned fear and drug seeking behaviour, IL activation

has been associated with the extinction and inhibition of conditioned fear and drug seeking (I.

Vidal, B. Vidal, Rauch, & Quirk, 2006; Willcocks & McNally, 2013). Evidence for this

functional dichotomy comes from a variety of different types of experiments: Pharmacological

manipulation studies demonstrate that inactivation of the PL reduces freezing to a fear context

(Corcoran & Quirk, 2007) and context-induced drug seeking behaviour (Di Pietro et al., 2006),

while inactivation of the IL enhances context-induced reinstatement (Peters, LaLumiere, &

Kalivas, 2008). In contrast, pharmacological activation of the IL enhances extinction of

contextual fear conditioning (Thompson et al., 2012) and inactivation of the IL impairs this

process (Laurent & Westbrook, 2009). Further, lesions of the PL have been shown to impair

context dependent fear expression (J. Kim, N. Kim, T. Kim & Choi, 2013), while the expression

of contextual fear suppresses IL excitability (Soler-Cedeño, Cruz, Criado-Marrero & Porter,

2016).

Thus, behavioural data reveal opposing roles of the IL and PL regions in both the expression of

contextual conditioned fear and context-driven drug reinstatement, despite the fact that different

motivational values (valences) are associated with the context in each case. Indeed, while fear

conditioning is concerned with aversively motivated behaviour (Wang et al., 2013), drug seeking

is appetitively motivated (Wanat, Willuhn, Clark, & Phillips, 2009). This is important to note

6

because of evidence indicating that the underlying neural circuitry responsible for each process

differs; while context-driven drug seeking is mediated by divergent projection patterns from the

mPFC to the nucleus accumbens (with the PL preferentially targeting the core, and the IL

preferentially targeting the shell; Euston et al., 2012; Peters et al., 2009), contextual fear

conditioning is under the control of mPFC projections to the amygdala (Sierra-Mercado, Padilla-

Coreano, & Quirk, 2011; Euston et al., 2012; Arruda-Carvalho & Clem, 2015). In terms of

hippocampal inputs to the mPFC, both the dorsal and ventral HPC subregions have been

implicated in contextual fear conditioning (Hunsaker & Kesner, 2008; Wang et al., 2013). In

contrast, though limited evidence exists on the differential contributions of the dorsal versus

ventral HPC to contextual control of appetitively motivated behaviours, we have shown that only

the ventral HPC is necessary for this process in a CBD task (Riaz et al., in preparation). Thus, it

is important to consider the role of the mPFC in appetitively and aversively motivated contextual

processing independently.

The majority of what is currently known about the role of the mPFC in the contextual control of

appetitively motivated behaviour is derived from studies involving drugs of abuse. While

important, drugs of abuse are known to cause neuronal changes and alterations in baseline brain

functioning (Koob & Volkow, 2010), and therefore studies examining context-driven drug-

seeking do not speak to the causal role of brain areas in context processing under normal

conditions. As such, it is unclear whether the IL/PL functional dichotomy will be upheld in

context-dependent natural reward-seeking. The present study therefore sought to utilize a CBD

memory retrieval (as opposed to acquisition) task to investigate the role of two mPFC subregions

(IL, PL) in the contextual control of appetitively motivated behaviours using natural reward,

instead of drugs of abuse. To this end, we examined the effects of transient post-acquisition

pharmacological inactivation of the PL and IL on CBD memory retrieval. More specifically,

animals were trained to nose poke in response to the presentation of one stimulus (e.g. X+) for

the delivery of sucrose reward, and to withhold a nose poke response to the presentation of a

second stimulus (e.g. Y-) in a context-specific manner (e.g. AX+, AY-; BX-, BY+). Upon

successful acquisition, animals were subjected to inactivation of the PL/IL and an additional

CBD training session as well as a CBD extinction test. Due to unforeseen circumstances

(unexpectedly long acquisition time and high incidence of non-learning), we were only able to

collect and analyse the data from PL manipulations in this thesis.

7

As hypothesized, we observed robust deficits in CBD memory retrieval in the PL-inactivated

treatment group. Additionally, congruent with existing research evidence, we found that

locomotor activity was unaffected following inactivation of the PL cortex (Jiang et al., 2014).

Our data help to bridge the gap in the existing literature by demonstrating a role for the PL in the

retrieval of contextual memories for natural reward.

8

Methods 2

2.1 Subjects

45 experimentally naïve, adult, male Long-Evans rats (Charles-River Laboratory, Quebec,

Canada) were used in this experiment. All rats were maintained at 85-90% of their free-feeding

weights for the entire duration of the experiment (~300-400 g) and had access to water ad

libitum. The rats were pair-housed in a room held at a constant temperature of 24°C and relative

humidity of 30-60%, under a 12hr light/dark cycle (lights on at 7:00 am). All experiments were

conducted during the light phase, between 0700 and 1900 h, and in accordance with the

Canadian Council of Animal Care standards, and approved by the University Animal Care

Committee of the University of Toronto.

2.2 Apparatus

Six operant boxes (Med Associates, Georgia, VT), housed in light-resistant and sound-

attenuating chambers were used in this experiment. Each operant box consisted of a floor made

of stainless-steel rods (0.5 cm diameter rods, spaced 1.6 cm apart), and two sidewalls (right and

left) containing a recessed food magazine in the center, one of which was associated with the

delivery of 45mg sucrose pellets (i.e. the active receptacle; TestDiet, Richmond, IN). Each food

magazine was equipped with an infrared beam detector to monitor the number, timing and

duration of nose pokes made into the magazine. In addition, a 2kHz Sonalert tone generator was

mounted high on the wall opposite the wall with the active receptacle. A white noise generator

was also affixed lower down on the same wall. The chamber was illuminated by a house light

(28 V) mounted on the top left wall (center).

The 6 boxes were divided into two sets of three boxes to represent two different ‘contexts’ based

on a number of distinguishing features; the dimension and appearance of the chambers (Med

Associates chambers ENV01: Set 1: 30cm (W) x 20cm (H) x 20cm (D) vs. Med Associates

chambers ENV08: Set 2: 30cm (W) x 20cm (H) x 25cm (D)) and the odours of the chambers (Set

1: Woody sandalwood, Set 2: Bitter almond; Sunrise Botanics, Mississauga, ON). Each operant

box was cleaned with an odourless 1% Liquinox solution (Alconox, White Plains, NY) before

and after each session to remove any traces of sucrose or odours from the previous rat in the

same box.

9

All operant boxes were controlled via a computer with MED-PC software (Med Associates),

which also automatically recorded the data generated during the experiment.

2.3 Behavioural Procedures

See Figure 1 for an overview of experimental procedures. It should be noted that out of the 45

animals ran in the current paradigm, only 9 animals successfully learnt the task and were

eventually used for this study.

2.3.1 Habituation and exposure to sucrose pellets

All rats received two, 20 min sessions in which they were exposed to one of each type of operant

box (1 & 2). The same two operant boxes were used for all subsequent training and testing

sessions for each rat. For the purpose of the CBD training phase, one of the two boxes that each

rat was habituated to was assigned as context A and the other box was assigned as context B.

Context assignments were carefully counterbalanced for box type; for 5 rats the small/

sandalwood chambers (Set 1) served as context A and the large/ bitter almond chambers (Set 2)

served as context B, while the context assignment was switched around for the remaining 4 rats.

During habituation and for each subsequent training day, the order of context presentation was

changed across days (e.g. A-B, B-A, B-A, A-B) such that there were no more than two

consecutive days of the same context in the first session of the day. After the two habituation

sessions, all rats were exposed to three sucrose pellets (per rat) which were placed in their home

cage to overcome any neophobia.

2.3.2 Magazine training

Following habituation, all rats received one session of magazine training in each context to learn

to retrieve sucrose pellets from the active receptacle. Each session lasted for 20 min during

which a total of 60 sucrose pellets were delivered on a variable interval 20 s schedule (VI20).

The number of nose pokes made into each receptacle (active (right) or inactive (left)) was

recorded to assess learning.

2.3.3 Nose poke hold training

Each rat received a maximum of two days (four sessions; one session per context per day) of

nose poke hold training. During each session, successful nose pokes (held for ≥ 0.5 s) in the

10

active receptacle were rewarded on a continuous reinforcement (fixed ratio 1) schedule. An inter-

response interval (latent period) of 10 s followed each successful nose poke during which no

rewards were dispensed. Nose poke holds in the inactive receptacle had no consequence. Each

session lasted for 20 min or until a maximum of 50 sucrose pellets were dispensed. Once a

subject obtained all 50 rewards within the 20 min session, in both contexts, they were transferred

to the next phase of behavioural training.

2.3.4 Contextual biconditional discrimination (CBD) training

Rats received a maximum of 70 days of CBD training, in which they were trained to acquire

discriminative nose poke hold responses in the presence of two discriminative stimuli in a

context dependent manner. In one context (e.g., A), the tone served as the reinforced

discriminative auditory stimulus (S+) and the white noise as the non-reinforced discriminative

auditory stimulus (S-), while in the other context (e.g., B) the contingencies were reversed. Each

rat received two 25-30 min sessions of training each day (one in each context). Each session

consisted of a total of 40 trials (20 S+ and 20 S-), and began with a 90 s pre-stimulus period.

Each trial began with the presentation of the S+ or S- for a maximum of 7.5 s. A nose poke hold

(for ≥ 0.5 s) emitted in the active receptacle during stimulus presentation elicited an appropriate

consequence and terminated the auditory stimulus 1s later; a successful response to the S+

resulted in the delivery of three sucrose pellets, while a nose poke held for ≥ 0.5 s in response to

the S- resulted in a 5 s timeout period with the house light off and the session timer paused. Nose

poke holds in the inactive receptacle had no consequence. In the absence of any successful

responses, the auditory stimuli terminated after 7.5 s. The intertrial interval (ITI) was set at 30 s.

The order of S+ and S- presentation was pseudo-randomised to ensure that the same stimulus

was not presented for more than 2 consecutive trials in each session (e.g. S+, S-, S-, S+, S-, S+,

S+, S- ...). Additionally, there were no more than two consecutive sessions that started with the

same auditory stimulus (tone or white noise). The number of nose pokes made during each

stimulus presentation was recorded and a discrimination score was used to assess CBD memory

acquisition. The discrimination score was calculated for each rat, per day, by dividing the

number of successful responses during the S+ by the total number of nose poke holds emitted

during the S+ and S- in each context and averaging the ratio scores from the two contexts. All

animals underwent CBD training until they obtained a ratio score of > 0.75 in each context for 5

consecutive days of training, within a maximum of 70 days of training.

11

9 animals successfully reached the learning criterion, and proceeded to the next stages in the

experiment. In most non-learners, discriminative responding was achieved in one context, but

not the other. The number of days of CBD acquisition training ranged from 18-65 days for the

animals that successfully learned.

2.3.5 Guide cannula implantation surgery

All rats underwent bilateral guide cannula implantation after acquiring the contextual

biconditional discrimination. Each rat was anaesthetized with isoflurane gaseous anaesthetic (3-

4% isoflurane delivered in O2 at 1 L min−1; Baxter, Mississauga, ON), and body temperature

was kept constant (37°C) during the surgery with a heating blanket. The head was shaved and

placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) with the incisor bar set at 3.3mm

below the interaural line. A small scalp incision was made to implant guide cannulae (26 gauge;

Plastics One, Roanoke, VA) bilaterally into the PL (in mm from Bregma: AP +2.2, ML ±0.75,

DV -2.5; PL group n = 9), according to Paxinos and Watson (1998). The cannulae were secured

on the skull using dental cement (Lang Dental, Wheeling, IL) and three anchoring screws

(Plastics One). In order to maintain the patency of the guide cannulae, solid stainless steel

dummy cannulae (Plastics One) were inserted into the guide cannulae following surgery. All rats

were given a 7-day post-operative recovery period before continuing CBD training.

In a within-subjects experimental design, 8 (of 9) animals underwent the remaining behavioural

procedure twice- once with a saline (control) treatment and once with a drug (inactivation)

treatment. The order in which rats received the two cycles of testing was counterbalanced across

animals, such that 4 rats received the PL inactivated treatment cycle first, followed by a one day

washout period and then the PL saline treatment cycle, while this order was reversed for the

remaining 4 animals. 1 of the 9 cannulated rats, however, only received the saline treatment

cycle due to loss of headcap prior to the completion of the inactivation treatment cycle. As a

result, data from all 9 animals was included in the PL control group, while data from 8 animals

was included in the PL inactivated group.

2.3.6 CBD (recap) training

Training was resumed after the post-operative recovery period for a total of two days for CBD

recap training. All rats underwent the same training procedure as before to ensure that the

12

surgery and post-operative rest period did not affect the expression of CBD memory. In the

second cycle of testing, animals underwent recap training to ensure that the extinction test (see

below) did not affect CBD memory expression.

2.3.7 General infusion procedure

On the second day of recap training, each rat was infused with 0.3µl saline solution per side

(0.9% saline; B. Braun Medical, Bethlehem, PA), using bilateral microinjectors (33 gauge;

Plastics One) extending 1 mm below the tip of the guide cannulae, to minimize the mechanical

effects of subsequent drug infusions, as well as to habituate the animal to the infusion procedure.

All infusions were made at a rate of 0.3 µl/min using an infusion pump (Harvard Apparatus,

Holliston, MA) mounted with a 5 µl Hamilton syringe. Following each injection, the injector was

left in place for 1 min to allow for diffusion of the drug or saline away from the injector tip and

to minimize its spread along the needle tract. For all subsequent infusions, each rat was given a

15 min interval before the start of behavioural testing to allow the drug to take effect.

2.3.8 CBD test

The PL cortex was temporarily inactivated using drug MB, a gamma-aminobutyric acid A and B

(GABAA and GABAB) receptor agonist cocktail of muscimol and baclofen (Sigma-Aldrich, St.

Louis, MO; in equal parts at a concentration of 250ng/µl), respectively. In the inactivation

treatment cycle, rats were infused with 0.3µl (75ng) of MB per side (PL inactivated group, n =

8), while in the saline treatment cycle, rats were infused with 0.3µl of saline solution per side

(PL control group, n = 9). Following infusions, rats received two sessions of CBD training, one

per context, as described above.

2.3.9 CBD post-washout training

Following the CBD test day, rats received a one-day washout period before an additional

retraining session per context was administered to all rats to ensure that the infusions did not

have a lasting effect on the expression of CBD memory. Post-washout training data from the first

testing cycle was also compared to the first day of recap training of the second testing cycle to

ensure that the first extinction test (see below) did not have a lasting effect on CBD memory

expression.

13

2.3.10 CBD extinction text

The following day, rats were infused with MB or saline solution again (as above) before testing

the effect of PL manipulation on the expression of CBD memory in extinction. The operant

boxes and test stimuli used in the extinction test were identical to those used in the CBD training

sessions. The extinction test was modified from the training sessions by changing the total

number of trials administered (20; 10 S+ and 10 S-) and the duration of stimulus presentation (10

s). There was no consequence to nose poking to stimuli presented during the extinction test. Each

rat received one session of the extinction test per context. Half of the rats in each treatment group

(PL control and PL inactivated) underwent extinction in context A and then context B, and this

order was reversed for the remaining rats. Successful (held for ≥ 0.5 s) and unsuccessful nose

pokes were recorded separately although neither had any consequence during the presentation of

either auditory stimulus. As before, only responses held for ≥ 0.5 s were used to calculate the

discrimination scores.

2.3.11 Locomotor activity test

Following the second extinction session, all rats were administered a locomotor activity test in

novel opaque plastic chambers measuring 45 cm x 25 cm x 20 cm. A video camera and

EthoVision XT software (Noldus, Wageningen, The Netherlands) were used to measure the total

distance travelled by each rat (in cm) over a 60 min period. Distance traveled was recorded in

10-minute bins. Locomotor activity measurement of the PL control group (saline-infused) was

used as a baseline for comparison with the locomotor activity of the PL inactivated group (drug-

infused).

2.4 Histology

Following the completion of behavioural testing, 4 (of 9) rats were infused with 0.3µl of cresyl

violet to facilitate location of injector tip placement. All rats were then given a lethal dose of

Euthanyl (2 mL/4.5 kg; Bimeda, Cambridge, ON) and were intracardially perfused with 100 ml

saline, followed by 100 ml of 4% Paraformaldehyde solution (PFA; Sigma-Aldrich) to fix the

brain. Brains were then removed, stored in PFA, and transferred to a 30% sucrose cryoprotectant

solution before sectioning. All brains were cut coronally in 50 µm slices, and stained with cresyl

14

violet for the verification of cannula and injector tip placements via comparison with the rat

brain atlas of Paxinos and Watson (1998).

2.5 Data Analysis

SPSS statistical package version 20.0 (SPSS, Chicago, IL) was used for all statistical analyses

with the level of significance set at p < 0.05. Repeated measures analysis of variance (ANOVA)

was carried out on data collected from each phase of the experiment. The within-subjects factors

in each analysis varied across tasks and are described individually for each task in the Results

section.

15

Results 3

3.1 Histological verification

Figure 2 shows schematic diagrams (Paxinos and Watson, 1998) and representative

photomicrographs of the placement of the injector tip within the PL. All rats sacrificed for

histological verification (n = 9) showed correct injector tip placement. Thus, no animals were

excluded from statistical analyses.

3.2 Magazine training and nose poke hold training

A receptacle x context repeated measures ANOVA comparing the number of nose pokes made

by rats into each receptacle (active (right) or inactive (left)), across the two contexts (A, B)

during magazine training revealed a significant preference for nose poking in the active

receptacle over the inactive receptacle (F(1,8)=30.655, p=0.001). There was no significant main

effect of context (F(1,8)=2.140, p=0.182), and no significant receptacle x context interaction

(F(1,8)=2.610, p=0.145), indicating that all animals nose poked preferentially into the active

receptacle, across both contexts (Figure 3).

Within two days of nose poke hold training, all rats acquired the instrumental behaviour of

holding nose pokes for ≥ 0.5 s in the active receptacle to obtain a reward, as assessed by a

learning criterion of obtaining the maximum 50 rewards within the 20 min session in both

contexts.

3.3 CBD training

For CBD acquisition, learning was assessed on the basis of a criterion performance of obtaining

a ratio score of > 0.75 in each context for 5 consecutive days of training. Since the number of

days of CBD acquisition training ranged from 18 to 65 days for the 9 animals that successfully

acquired the task, only data from the first 9 and last 9 days of training were further analysed.

Figure 4 shows CBD acquisition training data from the first 9 days and last 9 days of CBD

training for all animals. A days x context repeated measures ANOVA comparing discrimination

scores from the first 9 days and last 9 days of CBD training across both contexts revealed

significant learning taking place across the 18 days (Days: F(17,136)=17.731, p< 0.001). There

was no significant effect of context (F(1,8)=0.030, p=0.867), and no significant days x context

16

interaction (F(17,136)=1.173, p=0.337), demonstrating that all animals showed similar CBD

learning in each context prior to intra-cerebral pharmacological manipulations.

3.4 CBD memory retrieval after surgery

The discrimination scores from the last day of CBD training and the first day of recap training

after the surgery across both contexts were compared and analysed using a days x context

repeated measures ANOVA. The analysis revealed no significant effect of surgery and post-

operative rest period on CBD memory retrieval (no significant effect of days; F(1,8)=1.092,

p=0.327). Moreover, there was no significant effect of context (F(1,8)=0.520, p=0.492) and no

significant days x context interaction (F(1,8)=0.234, p=0.641), indicating that CBD memory

retrieval was unaffected across both contexts by guide cannula implantation surgery.

3.5 Stable baseline CBD memory expression

A days x context repeated measures ANOVA comparing discrimination scores from the 4 days

of recap training, both post-washout trainings and training following saline infusions (for each

animal), across both contexts, revealed no significant change in discrimination scores across the

7 days (F(6,42)=0.958, p=0.455). There was no significant effect of context (F(1,7)=2.287,

p=0.174) and no significant days x context interaction (F(6,42)=1.598, p=0.225). In brief,

discrimination memory retrieval was consistent during the recap and post-washout trainings

across both cycles of testing (inactivation and saline treatments), as well as during CBD training

following saline infusions.

These data demonstrate stable baseline CBD memory expression and establish that (1) the drug

MB only temporarily affected performance after drug infusions (see below), since performance

from the recap training prior to PL inactivation did not differ significantly from the post-washout

training in the inactivation treatment cycle; (2) saline infusions had no significant effect on CBD

memory expression since discrimination scores from CBD training following saline infusions did

not deviate from baseline memory expression; (3) the extinction test in the first testing cycle did

not have a lasting effect on CBD memory expression since performance in the post-washout

training of the first cycle (prior to the first extinction test) did not differ significantly from the

recap training sessions in the second testing cycle (following the first extinction test).

17

3.6 CBD test

A treatment x context repeated measures ANOVA, comparing discrimination scores from CBD

training following saline and drug infusions, for each animal (across both contexts), was used to

assess the effect of PL inactivation on CBD memory expression. Since animals demonstrated a

stable baseline CBD memory expression before and after the inactivation and during training

following saline infusions (see above), CBD test data from the PL inactivated group were

compared to CBD test data from the PL control group in order to eliminate any effect of the

infusion procedure from the analysis.

A significant difference in performance across the two treatment groups (F(1,7)=21.429,

p=0.002) was observed. There were no significant main effect of context (F(1,7)=2.939,

p=0.130) and no significant treatment x context interaction (F(1,7)=0.118, p=0.742). Thus, on

the day of infusion and training, the PL-inactivated group performed significantly worse than the

PL control group (Figure 5). These data suggest that PL inactivation impaired performance on

the CBD training task.

3.7 CBD extinction test

A treatment x context repeated measures ANOVA was used to analyse the CBD extinction data

from both treatment cycles (PL inactivation and PL control). There was a significant effect of

treatment (F(1,7)=8.691, p=0.021) but no significant main effect of context (F(1,7)=0.050,

p=0.829) and no significant treatment x context interaction (F(1,7)=1.606, p=0.246). These data

indicate that the PL-inactivated group was significantly impaired in comparison to the PL control

group (Figure 6), further supporting the hypothesis that PL inactivation significantly impairs

biconditional discrimination memory retrieval.

3.8 Locomotor activity test

Locomotor activity data (Figure 7) from both treatment groups were subject to a treatment x time

(in 10 min bins) repeated measures ANOVA. There was a significant decrease in locomotor

activity over the 1 hr interval (Time: F(5,35)=11.359, p<0.001), but no significant main effect of

treatment (F(1,7)=0.582, p=0.470), nor a treatment x time interaction (F(5,35)=0.080, p=0.982).

In Summary, baseline locomotor activity of animals did not differ across the two treatment

18

groups (PL-inactivated, PL-control), indicating that locomotor activity was unaffected following

PL inactivation.

19

Discussion 4The present study provides evidence for a role of the PL in context-dependent biconditional

discrimination memory retrieval. Selective temporary post-acquisition pharmacological

inactivation of the PL impaired retrieval and expression of CBD memory, but had no significant

effect on locomotor activity. As such, our findings indicate that the PL is necessary for

contextual processing in appetitively motivated instrumental responding for natural reward.

4.1 PL in contextual memory processing

The present study provides strong evidence for a role of the PL in mediating contextual memory

retrieval as inactivation of the PL led to a significant impairment in CBD memory retrieval

during the extinction test. These results were not mediated by the effects of surgery or post-

operative rest period as memory retrieval during post-surgery recap training matched pre-surgery

levels. Tissue damage and/or stress caused by the infusion procedure also did not affect memory

retrieval because all rats maintained stable baseline memory expression following saline

infusions (PL control group). The effects of the pharmacological inactivation on the PL-

inactivated group were also shown to be temporary, lasting no more than two days following

infusion of MB. Thus, the current study demonstrates that the PL mediates contextual memory

retrieval in appetitively motivated tasks, in the absence of reward, in the intact brain.

These novel findings are consistent with the sparse existing literature on the role of the PL in

appetitively motivated contextual processing in the drug-free brain; in a previous study, Ashwell

and Ito (2014) demonstrated that the PL is involved in the acquisition of sucrose reward-related

spatial contextual memories. In their study, Ashwell and Ito (2014), utilised a radial maze task to

train rats to preferentially respond to a reward-associated discrete cue (S+; flashing light)

presented in three (out of six) spatially-defined contexts (3 of 6 radial maze arms), and to avoid

the same discrete cue presented in the other three spatial locations. Lesions of the PL resulted in

slower learning of the S+ approach training in the appropriate spatial contexts. However, PL-

lesioned animals did eventually acquire the task, demonstrating that other brain regions can

compensate for the loss of PL function in an acquisition task (Ashwell & Ito, 2014; Euston et al.,

2012). Together with the findings of the present study, these studies suggest that the PL is

important in mediating the use of contextual information to retrieve/disambiguate the meaning of

20

motivationally significant cues. It is unlikely that the results from the two studies are mediated

by deficits in discrete cue processing, as the acquisition of discrete cue conditioning was not

impaired as a result of excitotoxic PL lesions (Ashwell & Ito, 2014). Further evidence suggests

that PL lesions do not interfere with the ability of PL lesioned rats to acquire fear conditioning to

a tone cue (Morgan and LeDoux 1995), indicating that discrete tone cue processing remains

intact in the absence of PL function.

Interestingly, despite the absence of drugs of abuse in the current paradigm, we observed a

similar role of the PL in CBD memory retrieval as that observed in studies of context-induced

reinstatement of drug seeking (Di Pietro et al., 2006; Fuchs et al., 2005). While previous drug

reinstatement studies have demonstrated that inactivation of the PL leads to abolished context-

induced drug reinstatement (Fuchs et al., 2005), PL inactivation in the current study resulted in

impaired contextual biconditional discrimination. These analogous findings suggest that the role

of the PL in the intact, drug-free, brain in appetitively motivated contextual processing is not

qualitatively different from that in addiction states, despite evidence of alterations in baseline

brain functioning and neuronal changes with drug use (George, Mandyam, Wee & Koob, 2008;

Koob & Volkow, 2010; Porrino & Lyons, 2000).

Moreover, our findings are also congruent with studies of context-dependent fear conditioning

(Corcoran & Quirk, 2007; Kim et al., 2013) that have demonstrated a role for the PL in the

retrieval and expression of contextually conditioned fear. Although these fear conditioning

studies tap into distinct underlying aversively motivated contextual processes that are mediated

by different neural circuits than those implicated in appetitive contextual processing (with mPFC

projections to the nucleus accumbens implicated in context-driven drug seeking and mPFC

projections to the amygdala implicated in contextual fear conditioning; Euston et al., 2012;

Peters et al., 2009; Sierra-Mercado et al., 2011), it is important to note that these studies are

typically conducted without the involvement of drugs of abuse (Kim et al., 2013). Therefore, it is

important to consider the present study in light of findings from these contextual fear

conditioning studies, in order to infer a role of the PL in the intact brain.

The present novel finding that the PL is necessary for CBD memory retrieval sheds light on a

more general role for the PL in contextual processing in a drug-free state. Our data, together with

previous reports of PL function that have implicated the region in the mediation of the

21

expression of contextual fear (Corcoran & Quirk, 2007), indicate that contextual processing

within the PL may be independent of the valence of the outcome associated with the contextual

memory. Taken together, these findings suggest that the PL may subserve a more general

function in contextual processing, representing a common node in the distinct circuits involved

in appetitive and aversive contextual processing, which may diverge in the subsequent

downstream effectors responsible for the expression of each type of behaviour.

4.2 PL in CBD memory expression in the presence of reward

Intriguingly, inactivation of the PL also impaired performance on the CBD task in the presence

of reward (during the CBD test) in the present study. This was somewhat of a surprising result,

as we had expected that the presence of outcomes in the test session might mitigate the effects of

PL inactivation despite impairments in CBD memory retrieval, as successful performance of a

CBD session could have been achieved on the basis of within-session information (cue

information), even in the absence of contextual information (especially since subjects were well-

trained on the rules and format of the training sessions). One potential explanation for the poor

performance of the PL-inactivated rats is simply that the CBD task is a complex paradigm that

cannot be performed well solely on the basis of within session information (i.e., within a single

session of training in each context). The sizeable number of days of acquisition training that was

required to successfully acquire the task (18-65 days, in rats that did learn it) suggests that the

biconditional discrimination is difficult to acquire, even in animals without PL manipulations.

Moreover, it is not known whether other brain regions can compensate for loss of PL function in

CBD memory reacquisition in such a short period of time, if at all. In the afore mentioned study

by Ashwell and Ito (2014), which demonstrated compensation of lost PL function in the

acquisition of sucrose reward-related spatial contextual memories, PL lesioned rats required

more training sessions than corresponding controls (with sham lesions) to acquire the contextual

memory, indicating that reacquisition of the CBD task in the absence of PL function is unlikely

to occur within a single session (per context).

Alternatively, the CBD test data may also indicate the involvement of the PL in more than just

the retrieval of contextual memories. These findings may be a reflection of PL-inactivation

induced impulsivity; previous studies have shown that lesions of the mPFC result in increased

choice impulsivity in the presence of a rewarding outcome (Gill, Castaneda & Janak, 2010). In a

22

study by Gill et al. (2010), rats were trained to emit nose poke hold responses for increasing

durations to obtain increasing reward magnitudes (higher volume of sucrose reward) in response

to different cues; for example, a green and yellow light cue was associated with a 75 µL sucrose

reward and required a 400ms long nose poke hold, while a yellow light cue was associated with a

100 µL sucrose reward and required an 800ms long nose poke hold response. Post-training

mPFC lesions resulted in a decrease in overall nose poke hold duration and reward efficiency

(i.e., animals nose poked preferentially for cues associated with shorter nose poke hold durations,

instead of longer durations, and therefore received smaller rewards), while the total number of

nose poke holds emitted remained unchanged. Similarly, in the present study, there was no

significant difference in the total number of nose poke holds emitted (averaged across contexts)

by each treatment group during the CBD test (Supplementary Figure 1), but there was a

significant decrease in discrimination scores across the two treatment groups. These results

indicate that despite similar overall levels of nose poking in either treatment group, preference

for nose poking to the discrete cues shifted away from the S+ (hence a decrease in discrimination

scores), resulting in an overall decrease in reward efficiency. Hence, these data suggest that the

PL may play a critical role in keeping outcome-driven impulsivity in check.

However, because there are multiple possible explanations for the observed changes in behaviour

following PL inactivation in the CBD test, the role of the PL in the expression of CBD memory

in the presence of reward is inconclusive from the present study, and therefore warrants further

investigation.

4.3 Locomotor activity regulation in the PL

Locomotor activity test results for the PL-inactivated group were also in agreement with existing

literature; previous studies of PL function have reported no significant changes in locomotor

activity following inactivation of the PL (Jiang et al., 2014; Kim et al., 2013).

4.4 A within-subjects experimental design

In the current study, we demonstrated stable baseline CBD memory expression during recap and

post-washout trainings, as well as CBD training following infusions. Though CBD memory

proved difficult for animals to acquire (evidenced by the low rate of learning), animals that did

successfully acquire the contextual discrimination exhibited persistent memory expression,

23

which rendered the repeated measures experimental design of the current study particularly

powerful. Since memory expression successfully returned to baseline following the first

extinction test and after each infusion of drug MB, the observed effects of PL inactivation cannot

be attributed to carryover effects of the first testing cycle in the within subjects design.

Moreover, running the same animals in both treatment conditions also minimized variation due

to individual differences in the data from both treatment groups.

4.5 Future directions

While our results are consistent with the hypothesized functional dichotomy of the IL and PL in

contextual control over appetitively motivated behaviours in drug-free states, the role of the IL in

this process remains to be elucidated. Further enquiries could include an investigation of the

relationship between GABAergic PL manipulations and the function of other local

neurotransmitters including dopamine, glutamate, and serotonin (Steketee, 2003). It would also

be interesting to explore the functional connectivity between input and output structures in

relation to the PL and ventral HPC to facilitate mapping of the neural circuitry underlying

contextual control of appetitively motivated behaviours. For instance, both the ventral HPC and

ventral PFC (including IL and PL regions) provide direct input to the nucleus accumbens (French

& Totterdell, 2002, 2003), with the PL preferentially targeting the core, and the IL preferentially

targeting the shell (Euston et al., 2012; Peters et al., 2009). The nucleus accumbens is another

region implicated in context processing (Day & Carelli, 2007) and is therefore a suitable target

region for a follow-up study.

4.6 Conclusion

In conclusion, the current study is the first analysis of the role of the PL in contextual memory

retrieval in appetitively motivated tasks in the intact, drug-free, brain. Our findings suggest that

the PL is necessary for contextual processing in appetitively motivated tasks and may serve a

more general role in context processing regardless of the valence of the outcome associated with

the contextual memory. At present, the role of the IL in this process remains to be elucidated.

Furthering our understanding of the neural correlates of context processing in the intact brain has

important implications for understanding mental disorders, such as addiction and anxiety, which

are characterized by aberrant context induced changes in behaviour.

24

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Figures

Figure 1. Overview of experimental procedures. Animals were trained to receive reward (sucrose

pellets) by nose poking (>0.5s) into a magazine inside the operant box. During CBD training,

animals were trained to associate two distinct auditory cues with an appetitive outcome (sucrose)

or neutral outcome (house light off), in a context dependent manner. After CBD memory

acquisition, animals received bilateral cannula implantation surgery, before being subjected to

saline (PL control) and drug (PL inactivation) treatment testing cycles in a within-subjects

31

experimental design. Each testing cycle (order counterbalanced) began with 2 days of CBD recap

training. Once stable CBD memory expression was established, animals received bilateral

infusions of either saline or GABA agonists and underwent CBD training. After a one-day

washout period, animals were once again trained on the CBD task, before receiving bilateral

infusions (as before) and undergoing a CBD extinction test. Animals were also tested on a

locomotor activity task under the influence of saline or drug infusions. There was a one-day

washout period between the two treatment cycles.

32

Figure 2. Schematic diagrams and representative photomicrographs showing the position of the

injector tip in the PL. In all rats euthanized for histological verification (n = 9), the injector tip

position was within the PL. Representative photomicrographs shown are from animals infused

with cresyl violet.

33

                                               

Figure 3. Result of magazine training. Data are presented as mean number of nose pokes

averaged across the 2 contexts ± SEM. All rats (n = 9) made significantly more nose pokes into

the active (right) receptacle. **p < 0.01.

0  

50  

100  

150  

200  

250  

Inac*ve   Ac*ve  

Num

ber o

f Nos

epok

es

Receptacle

**

34

Figure 4. Acquisition of CBD memory. All animals showed significant learning from the first 9

days to the last 9 days of CBD training (p < 0.001). Mean discrimination scores averaged across

the 2 contexts ± SEM are plotted (n = 9).

0  

0.1  

0.2  

0.3  

0.4  

0.5  

0.6  

0.7  

0.8  

0.9  

1  

1   2   3   4   5   6   7   8   9   9th  last    

8th  last    

7th  last    

6th  last    

5th  last    

4th  last    

3rd  last    

2nd  last    

last    

Dis

crim

inat

ion

Scor

e

Days of CBD training

35

Figure 5. Effect of PL inactivation on CBD memory expression during training. Mean

discrimination scores averaged across the 2 contexts ± SEM, from biconditional discrimination

training following drug and saline infusions are plotted (PL inactivated and PL control groups,

respectively). PL inactivation significantly impaired biconditional discrimination memory

expression. **p < 0.01.

0  

0.1  

0.2  

0.3  

0.4  

0.5  

0.6  

0.7  

0.8  

0.9  

1  

PL  inactivated   PL  control  

Dis

crim

inat

ion

Scor

e

Treatment group

**

36

Figure 6. Effect of PL inactivation on CBD memory retrieval during extinction. PL inactivation

significantly impaired biconditional discrimination memory retrieval in extinction. Mean

discrimination scores averaged across the 2 contexts ± SEM are plotted. *p < 0.05.

0  

0.1  

0.2  

0.3  

0.4  

0.5  

0.6  

0.7  

0.8  

0.9  

1  

PL  inactivated   PL  control  

Dis

crim

inat

ion

Scor

e

Treatment group

*

37

Figure 7. Effect of PL inactivation on locomotor activity. Mean distance moved over 10 min

intervals (cm) ± SEM is plotted for each treatment group. PL inactivation had no significant

effect on locomotor activity; activity decreased in both groups over time (p < 0.001).

0  

1000  

2000  

3000  

4000  

5000  

6000  

10   20   30   40   50   60  

Dis

tanc

e m

oved

(cm

)

Time (min)

PL inactivated

PL saline

38

Supplementary Figure 1. Effect of PL inactivation on total number of nose poke holds emitted

during the CBD test. Data are presented as mean number of nose poke holds (in S+ and S-)

averaged across the 2 contexts ± SEM. A two-tailed paired t-test comparing the total number of

nose poke hold responses emitted (averaged across contexts) by each treatment group revealed

no significant difference in the total number of nose poke holds across the two treatments

(t(7)=0.5434, p= 0.6038).

0  

5  

10  

15  

20  

25  

30  

PL-­‐inac*vated   PL-­‐control  

Num

ber o

f Nos

epok

e ho

lds

Treatment Group