The Role of the Ventral Dentate Gyrus in Learned Approach-

Avoidance Conflict Resolution

by

Dylan CM Yeates

A thesis submitted in conformity with the requirements

for the degree of Master of Arts

Graduate Department of Psychology

University of Toronto

© Copyright by Dylan Yeates 2017

ii

The Role of the Ventral Dentate Gyrus in Approach-Avoidance Conflict Resolution

Dylan Yeates

Master of Arts

Graduate Department of Psychology

University of Toronto

2017

Abstract

Approach-avoidance conflicts occur when organisms face stimuli linked to opposing affective

outcomes, and must choose to either engage or disengage. It is increasingly acknowledged that

the hippocampus, particularly its ventral aspect, is part of a critical network involved in

recognizing and resolving approach-avoidance conflicts. Less is known about how the ventral

hippocampus’ subdivisions along its transverse axis mediate affective conflicts. Using transient

pharmacological lesions in Long-Evans rats, the present study observed that temporary

inactivation of the ventral dentate gyrus resulted in an increased bias towards approaching

affectively conflicting information, despite also being associated with aversive outcomes. Further

testing showed that this could not be explained by an increased bias towards either positive or

negative information alone, and was only observed when the conflicting stimuli were presented

together. These findings suggest that the ventral dentate gyrus is a critical actor in resolving

learned approach-avoidance conflicts.

iii

Acknowledgments

This thesis was the result of an entire community, not just one person. I would first and

foremost like to thank Dr. Rutsuko Ito and Dr. Andy Lee for their guidance, encouragement and

genuine support throughout this whole bumpy process; I feel genuinely lucky to have found my

way to them. I would also like to thank Dr. Junchul Kim for his participation on my master’s

thesis committee and insightful discussion.

I must thank Dr. Anett Schumacher for her extensive help throughout every step of this

experiment, David Nguyen for at least attempting to teach me his magic touch when it comes to

animal training, Laurie Hamel and Christina Gizzo for teaching me the quirks of our radial maze

room, Bilgehan Cavdaroglu for always being available to talk statistics, and Sadia Riaz for being

there when the ear bars just would not go in properly during surgery. Special thanks to Dean

Carcone for letting me use his thesis as a formatting cheat sheet, and Alicia Ussling, who

swooped in halfway through the experiment to assist with data collection; I could not have

finished this in time or sane without her help. I also would like to thank all my animal

participants for making the ultimate sacrifice in the name of science.

I would not be where I am today without the extensive and tireless support of my

wonderful parents. Finally, I would like to thank my tireless and loving fiancé Harveer Athwal

for sticking by me throughout this whole experience, and always believing I could do it.

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Table of Contents

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

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

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

Chapter 1 The Role of the Ventral Dentate Gyrus in Approach-Avoidance Conflict

Resolution ...................................................................................................................................1

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

1.1 Approach-Avoidance Conflict .............................................................................................1

1.2 The Function of the Hippocampus along the Dorso-Ventral Axis ......................................2

1.3 Functions of the Hippocampal Subregions ..........................................................................7

1.4 The Ventral Dentate Gyrus and Learned Approach-Avoidance Conflicts ..........................9

Methods .....................................................................................................................................10

2.1 Subjects ..............................................................................................................................10

2.2 Surgery ...............................................................................................................................10

2.3 Radial Arm Maze Apparatus .............................................................................................11

2.4 Habituation .........................................................................................................................11

2.5 BAA Y-Maze Cue Conditioning .......................................................................................12

2.6 Conditioned Cue Acquisition Test and Group Allocation .................................................13

2.7 Drug Microinfusion ...........................................................................................................13

2.8 Mixed Valence Approach-Avoidance Conflict Test..........................................................14

2.9 Novelty Detection ..............................................................................................................14

2.10 Retraining and Conditioned Cue Preference Test ..............................................................15

2.11 Elevated Plus Maze ............................................................................................................15

2.12 Metric Pattern Discrimination............................................................................................15

2.13 Locomotor test ...................................................................................................................16

2.14 Histological Procedure .......................................................................................................16

v

2.15 Data Analysis .....................................................................................................................16

Results .......................................................................................................................................17

3.1 Histological Verification ....................................................................................................17

3.2 Habituation .........................................................................................................................17

3.3 Conditioned Cue Acquisition .............................................................................................17

3.4 Mixed Valence Approach-Avoidance Conflict Test..........................................................18

3.5 Novelty Detection ..............................................................................................................18

3.6 Conditioned Preference ......................................................................................................19

3.7 Elevated Plus Maze ............................................................................................................19

3.8 Metric Pattern Detection ....................................................................................................19

3.9 Locomotion ........................................................................................................................20

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

4.1 The Ventral Dentate Gyrus is Involved in Approach-Avoidance Conflicts with

Conditioned Cues ...............................................................................................................20

4.2 The Results cannot be explained by Novelty or Pattern Separation Deficits ....................21

4.3 Inconsistent EPM Results ..................................................................................................22

4.4 Locomotion results Unexpected ........................................................................................22

Limitations and Future Directions ............................................................................................23

Figures .......................................................................................................................................26

Bibliography ..................................................................................................................................34

vi

List of Figures

Figure 1. Placements of injector tips. Red stars are BM infusion sites, while black crosses are

saline infusion sites. ...................................................................................................................... 26

Figure 2. Time spent in the conflict and neutral arms in the 4th habituation session. ................... 27

Figure 3. Time spent in each arm during the final cue conditioning acquisition test. .................. 27

Figure 4. Time spent in each arm during the mixed valence approach-avoidance conflict test. .. 28

Figure 5. Number of entries during mixed valence approach-avoidance conflict test ................. 28

Figure 6. Number of retreats during mixed valence approach-avoidance conflict test. ............... 29

Figure 7. Time spent in start and familiar arms during novelty habituation................................. 29

Figure 8. Time spent in familiar arms and novel arms during novelty test. ................................. 30

Figure 9. Time spent in appetitive and neural arms during final preference test. ......................... 30

Figure 10. Time spent in aversive and neural arms during final preference test. ......................... 31

Figure 11. Time spent in the open arm of elevated plus maze. .................................................... 31

Figure 12. Number of entries into the open arm of elevated plus maze. ...................................... 32

Figure 13. Combined time exploring both objects during metric pattern discrimination. ............ 32

Figure 14. Difference in time spent exploring objects in the MPD test between final test and last

training session.............................................................................................................................. 33

Figure 15. Distance travelled with each 5-min time block during locomotor test. ....................... 33

1

Chapter 1 The Role of the Ventral Dentate Gyrus in Approach-Avoidance Conflict

Resolution

Introduction

1.1 Approach-Avoidance Conflict

Animals, both human and non-human, must navigate a complex world that requires knowledge

about which objects and situations are adaptive and which are non-adaptive. By understanding

and learning about what is going to help or harm them, animals can tailor their behaviour to

approach stimuli that predict good outcomes (such as pleasurable food and safe places), and

avoid stimuli linked to bad outcomes (such as bodily harm). However, oftentimes the adaptive

value of stimuli is not simply good or bad. For a small animal like a rat, a stash of food in a

wide-open space is desirable, but approaching said food could expose the animal to predation.

This is an example of an approach-avoidance conflict, which arises when an organism is

confronted with a goal or situation that predicts both appetitive and aversive outcomes, making it

drawn towards and repelled away from the stimulus simultaneously (McNaughton & Corr,

2014). It is thought that abnormal approach-avoidance conflict resolution is involved in many

clinical disorders, particularly anxiety.

Initially, behaviours associated with approach-avoidance conflicts were thought to simply result

from the combination and mixing of approach and avoidance behaviours (McNaughton, 2011).

However, further investigations found that under conflict conditions, laboratory animals adopt

specific behaviours that cannot be explained by the linear mixing of approach and avoidance

systems, such as an increase in vigilance (Hinde, 1966). These behaviours were later found to be

attenuated when extensive lesions to the hippocampus (HPC) were made, as well as when

clinically effective anti-anxiety medications were given, leading to a hippocampal theory of

approach-avoidance conflict resolution and anxiety (Gray & Mcnaughton, 2000).

While much of the work on the hippocampus has focused on its role in memory, recently more

attention has been paid to its role in affective processes. Damage to the ventral hippocampus

(vHPC) in animals has been found to blunt the expression of anxiety in situations that naturally

2

evoke it, while leaving spatial memory relatively untouched (Moser & Moser, 1998) indicating

the presence of dissociable functions along the longitudinal axis (Strange, Witter, Lein, & Moser,

2014). These tests, which typically include the elevated plus maze (EPM) and open field (OF),

can be considered forms of approach-avoidance conflicts, which require the animal to balance an

innate drive leading to approach (the desire to explore novel environments) with an innate drive

leading to avoidance (caution of places where harm is a possibility). Further studies found that

approach-avoidance conflicts brought about by contrasting conditioned cues also recruit the

vHPC, as indicated by abnormally high approaches towards conflict stimuli when the brain

region is damaged (Schumacher, Vlassov, & Ito, 2016).

While the increased interest in the vHPC and its role in affective processes has lead to significant

progress in our understanding of the region, much is still unknown, such as if the subfields of the

region play specialized roles in approach-avoidance conflict resolution, how the inputs to the

vHPC are transformed along the transverse axis, and how these processes are related to what is

known about the dorsal hippocampus (dHPC). This study examined the role that a prominent

region of the vHPC, the ventral dentate gyrus (vDG), has in resolving approach-avoidance

conflicts. This thesis will begin by reviewing the literature surrounding the HPC, its anatomical

subregions, and its functional properties regarding spatial and mnemonic processing, before

examining the specific roles that the vHPC plays in affective processes. It will then look at the

specific role the vDG plays in approach-avoidance conflict resolution and other paradigms the

region is suspected to be involved in, based on existing literature. This study found that in the

absence of vDG activity animals display abnormal behaviour in an approach-avoidance conflict

with conditioned stimuli. Furthermore, the results cannot be explained by abnormal novelty

processing, spatial pattern separation, or processing of learned valences alone. Given the

anatomical connections and important functional role the dentate gyrus has in general

hippocampal function, the outcome of this study suggests the vDG is a key player in approach-

avoidance conflict processing.

1.2 The Function of the Hippocampus along the Dorso-Ventral Axis

Early behavioural testing of animals with hippocampal lesions indicated that damage to the

region had striking effects in approach-avoidance conflicts (Crespi, 1942; Jarrard, Isaacson, &

3

Wickelgren, 1964; Niki, 1965). Under conditions of affective conflict, animals with hippocampal

lesions would seemingly ignore the aversive consequences of their actions when faced with the

possibility of reward, overexert themselves for small rewards, or resist extinction when reward

was withheld. These results were also seen with the application of clinically effective anti-

anxiety medication, and these medications also have the effect of reducing the influence of

hippocampal theta rhythms generated by interactions with the septal nuclei (Gray &

Mcnaughton, 2000). These primary observations led to a septo-hippocampal theory of anxiety,

represented in a mature form as the idea that the regions act together as a coherent behavioural

inhibition system (BIS). The function of the system in normal animals is to enhance the salience

of negative outcome predicting stimuli and decrease approach behaviours under situations where

motivational conflicts are detected. Conversely it has also been suggested that most of the same

effects could be seen if the system normally blunts the salience of positive outcome predictions

(Ito & Lee, 2016).

The HPC’s role in behavioural inhibition has historically been overshadowed by the prominent

effects it has on mnemonic processes, as indicated by the dramatic deficits in declarative and

spatial memory caused by HPC lesions (Black, Nadel, & O’Keefe, 1977; Scoville & Milner,

1957). Although the BIS theory does attempt to explain how hippocampal damage could result in

mnemonic deficits, other theories in turn could plausibly explain away the results of at least

some of the behavioural inhibition studies. Many of the earlier studies could be explained as a

failure to use place strategies to associate certain locations with particular outcomes, with cue

learning intact (Black et al., 1977). Since much of the early evidence for behavioural inhibition

functions had a spatial component, this appears to be a plausible explanation for the results.

Regardless of the merit behavioural inhibition theories had to explain certain evidence other

theories struggled to explain, the notion that the HPC primarily mediates some form of memory

or spatial reasoning became and now remains the dominant view (Burgess, Maguire, & O’Keefe,

2002).

However, the view that the HPC is a unitary structure with one function has been challenged by

work demonstrating anatomically, genetically, and functionally specialized domains along its

dorsoventral axis (Moser & Moser, 1998; Strange et al., 2014). While the dorsal part of the HPC

in rodents (dHPC) appears to be important for mediating spatial/mnemonic functions, as

4

deficiencies in spatial processing that appear when this portion is selectively damaged closely

mirror those observed with complete hippocampal lesions (Moser, Moser, Forrest, Andersen, &

Morris, 1995), the vHPC is more closely associated with various affective functions, most

typically anxiety (Fanselow & Dong, 2010). Many studies have linked damage of the vHPC with

a decrease in anxiety in innately fearful situations (Pentkowski, Blanchard, Lever, Litvin, &

Blanchard, 2006) though it has also shown involvement in reactions to conditioned stimuli as

well (Maren & Holt, 2004; Schumacher et al., 2016). Anatomically, the vHPC is notable for

showing extensive anatomical and functional connections with structures associated with

processing affective information, including the amygdala (Felix-Ortiz et al., 2013; Ishikawa &

Nakamura, 2006), nucleus accumbens shell (Friedman, Aggleton, & Saunders, 2002; Okuyama,

Kitamura, Roy, Itohara, & Tonegawa, 2016), medial prefrontal cortex (mPFC) (Ciocchi,

Passecker, Malagon-Vina, Mikus, & Klausberger, 2015; Padilla-Coreano et al., 2016; Parfitt et

al., 2017) and hypothalamus via the lateral septum (Canteras & Swanson, 1992).

In addition to the dorsal-ventral dichotomy, some have suggested that the middle third of the

hippocampus should be considered an intermediate region, a distinction based mainly on genetic

studies (Dong, Swanson, Chen, Fanselow, & Toga, 2009; Thompson et al., 2008). While this is

certainly an interesting distinction that merits further investigation, it has not been as widely

validated anatomically as the dorsal-ventral dichotomy, and it is not clear what functional

attributes this intermediate region would have. For the purposes of this study and in line with

most current literature, the dHPC will be considered the part of the structure that lies above the

diencephalon and lies horizontally along the brain, while the ventral part lies alongside and

below the diencephalon and stands vertically.

It should also be noted there is evidence the vHPC does not exclusively mediate affective

behaviours and does participate in spatial processing, at least in some limited capacity.

Pyramidal cells in the vHPC appear to acquire place fields during exploration, though these are

much larger and much less numerous than the place fields of dHPC cells (Jung, Wiener, &

McNaughton, 1994). Though the authors of the original paper speculate that these less defined

fields could reflect non-spatial information processing, another group found that the ventral place

cells in the CA1 showed little overlap with anxiety responsive cells (Ciocchi et al., 2015),

suggesting that the ventral place cells may reflect spatial processing independent of affective

5

functions. Supporting this line of reasoning, one recent study implicated vCA3 and vCA1

immediate early gene expression in spatial novelty detection, though the same regions were

involved in a non-spatial version of the same task to a lesser extent (Beer, Chwiesko, &

Sauvage, 2014). Although the studies suggesting the vHPC is involved in spatial and mnemonic

processes are less numerous than those that do not, it appears that the effects reviewed above are

real and require further examination. A proper theory of vHPC function therefore needs to

account for these non-affective processes as well as the emotional behaviours reviewed above.

A small but growing number of human studies have shown the anterior HPC (homologous to the

rodent vHPC (Strange et al., 2014)) is involved in approach-avoidance conflicts and anxiety, in

line with the evidence from animal studies. It has been found that the anterior HPC is more

active when predators threaten to impede subjects on a stimulated exploration task (Bach et al.,

2014), and when confronted with a decision to engage or not engage with stimuli associated with

opposing valenced outcomes (O’Neil et al., 2015). A recent study found that signals in the

anterior HPC not only correlated with avoidance behaviour during a gambling task, but also with

trait anxiety, in line with some of the predictions of the BIS theory (Loh et al., 2016). In all cases

above, no associations were found between behaviours/activity and posterior HPC (the human

homolog of the dHPC). In short, both animal and human studies now support the idea that the

ventral/anterior HPC are associated with affective processes.

It is possible that the results of the vHPC studies discussed above can be explained as conflict

resolution during tasks that involve both the desire to approach and avoid simultaneously (Ito &

Lee, 2016). Such a conflict is created in two common tests of anxiety in rodents, the EPM and

the OF, which require the animal to balance an innate desire to explore new environments (which

could yield positive results) with a desire to avoid situations that could be dangerous (such as

wide, bright areas that leave the animal open to predation). Inactivation of the vHPC increases

the amount of time rodents spend exploring the exposed areas of the EPM and OF compared to

their more conservative controls, indicating an increased approach tendency relative to

avoidance. Another example of conflicting affective processes is how much an animal is willing

to work towards a reward, which can be considered a conflict between the desire for known

reward and the wish to conserve time and effort. Reward omissions during operant tasks will

eventually extinguish reward seeking behaviours at a predictable rate depending on the schedule

6

of reinforcement. With vHPC disruptions however, animals are willing to work much harder for

a reward in extinction tests than controls (Schmelzeis & Mittleman, 1996). Conflict resolution

has also been discussed in relation to theories of the HPC’s role in spatial and mnemonic

functions. It has been proposed that the results of some spatial studies can also be explained

through the dHPC working to resolve conflicts during decision making that utilizes spatial and

sensory information, rather than motivational information (Bannerman et al., 2014). It has been

found that rats with deficient NMDA-dependent long-term potentiation (LTP) in the dentate

gyrus (DG) and CA1 subregions of the HPC had difficulty approaching a hidden platform in a

water maze that was signaled by a beacon that was identical to a distracter beacon when they

were placed closer to the distracter (Bannerman et al., 2012). The results bear some similarity to

the effects of studies examining affective processes, suggesting that the whole HPC implements

a common function or algorithm to resolve conflicts. It is possible that the pattern separation,

pattern completion, and mismatch detection functions commonly described in the

spatial/memory literature ultimately serves to distinguish between close alternative options,

compare representations, and resolve conflicts. What differs between the dHPC and vHPC would

be their inputs and outputs, meaning that the dHPC would work through conflicts between

sensory data and spatial locations, while the vHPC works on conflicts between valences and

responses (Ito & Lee, 2016).

While the evidence points to the involvement of the vHPC in affective behaviours, specifically

those that involve approach-avoidance conflicts, and its distinct anatomical connections could

communicate with other brain regions associated with affective processes, the details about how

the subregions of the vHPC transform the information it receives along its transverse axis

remains relatively underexplored. Much of the work on hippocampal subregions has focused on

the dHPC, and has suggested that each of the major subregions of the HPC serve distinct

functions during the encoding and retrieval of memories. Given that the dHPC and vHPC regions

mainly differ in terms of connections and the small details of their physiology (Agster &

Burwell, 2013; Jung et al., 1994), and not in terms of basic anatomical structure, it is worth

reviewing the consensus functions of the hippocampal subfields, mainly through the work on the

dHPC subregion functions on memory. We will then review the current findings on the effects of

vHPC subregion function.

7

1.3 Functions of the Hippocampal Subregions

The hippocampus proper is a laminar structure with distinct anatomical connections flowing

from the most medial layer outwards, and is the central component of the larger hippocampal

formation. The entorhinal cortex serves as the primary cortical input, and passes information to

the HPC through the DG. The DG then projects to the CA3, which in turn projects onto the CA1.

These three layers form what is traditionally referred to as the trisynaptic circuit. The CA1 in

turn sends efferent connections from the hippocampus proper towards the rest of the

hippocampal formation and neocortex (Andersen, Bliss, & Skrede, 1971; Friedman et al., 2002).

Each of these sub-regions has unique anatomical, structural and functional properties, and is

suspected to process mnemonic information passing through the hippocampus in different ways

(Kesner & Rolls, 2015).

The DG has traditionally been characterised as performing pattern separation on incoming inputs

prior to encoding, orthogonalizing similar stimuli to make the outputs more dissimilar than the

inputs (Kesner, 2013; Sahay, Wilson, & Hen, 2011). This allows animals to distinguish between

previously experienced objects and places, even when the stimuli being compared are very

similar. Although the precise nature of how this pattern separation function is implemented is

still under debate, empirical evidence supports the idea that the computations performed by the

DG result in pattern separation effects (Aimone, Deng, & Gage, 2011; Hunsaker & Kesner,

2013; Johnston, Shtrahman, Parylak, Gonçalves, & Gage, 2016).

The DG sends the majority of its excitatory efferents to the CA3, a layer of pyramidal cells with

recurrent collaterals, projecting back on to its neighbouring principal neurons within the region

(Amaral & Witter, 1989; Rebola, Carta, & Mulle, 2017), allowing it to form a recurrent network

with itself. The unique architecture of this subregion is speculated to form attractor networks,

allowing the CA3 to perform pattern completion, a process where by it can react to partial or

degraded input from the direct projections it receives from the entorhinal cortex and retrieve the

whole representation previously constructed by experience (Kesner & Rolls, 2015; Knierim &

Neunuebel, 2016). Therefore, if a part of an object or event is re-encountered, an animal that has

encoded the event will be able to retrieve a more complete representation.

8

The CA1 is traditionally considered to be the output region for the HPC, recapitulating the

computations of the CA3 and projecting towards the subiculum, entorhinal cortex, and

neocortex. This region has been primarily implicated in performing mismatch detection between

internally stored representations and information about external stimuli, which it receives

through direct projections from the entorhinal cortex (Friedman et al., 2002; Hasselmo, 2005).

However, the CA1 has also been theorized to play a substantial role in olfactory representation,

temporal order processing, recall of long term memories (Hoge & Kesner, 2008; Kesner & Rolls,

2015; Li et al., 2017), and even forms of pattern completion depending on the task demands

(Guzowski, Knierim, & Moser, 2004). The presence of entorhinal inputs into the CA1 also

highlights the fact that although the HPC was considered a linear closed circuit for a long time,

each region receives inputs and sends outputs to multiple regions (Risold & Swanson, 1997;

Witter, 2007; Yeckel & Berger, 1990)

In contrast to the work described above, much of the work on the vHPC has treated it as a unitary

structure, with lesion studies damaging the whole HPC rather than in parts (Moser et al., 1995;

Schumacher et al., 2016). This changed in recent years, as studies have begun to examine the

specific properties of the vHPC subfields and their functional contributions to affective

behaviours. For example, the HPC is known to both control and be affected by stress (Conrad,

2008), and chronic stress in the vCA3 actually increases the number of apical dendrites, in

contrast to the decrease seen in the dCA3 (Pinto et al., 2015). The vCA1, in contrast to the

dCA1, shows considerable activation to anxiogenic situations, and responsive cells project to a

range of targets, the most prominent being the mPFC (Ciocchi et al., 2015). In one of the few

studies directly comparing the effects of disruption to different vHPC subfields, it was found that

temporary inhibition of the vCA3 and vCA1 during approach-avoidance conflicts lead to

opposing behavioural effects, with the vCA3 necessary for inhibiting approach, and the vCA1

necessary for potentiating approach towards conflicting conditioned cues (Schumacher,

Villaruel, Riaz, Lee, & Ito, 2017).

One region that has been relatively understudied thus far in the vHPC literature is the DG, at

least compared to the vCA3 and vCA1. Like the vHPC, the vDG appears to be distinct from the

dDG. Though the two halves of the DG have similar first order input regions, these projections

appear to originate from non-overlapping portions of those regions, and second order inputs to

9

the vDG appear to be more closely related to affective processes than the dDG’s inputs (Ohara,

Sato, Tsutsui, Witter, & Iijima, 2013). When the vDG of rats is lesioned, it recapitulates the

effects of complete vHPC damage on the EPM and OF tests (Weeden, Roberts, Kamm, &

Kesner, 2015), though other studies have found contradictory results on the same tests (Kheirbek

et al., 2013). In addition to the effects of whole vDG manipulation, others have looked

specifically at the role that adult born neurons play in vDG processes, an unsurprising topic of

interest given the correlation between neurogenesis in the region and the efficacy of anti-

depressant and anxiolytic medication (Sahay, Drew, & Hen, 2007), as well as the vHPC’s

purported role in emotional behaviours. It has been found that immature adult born neurons in

the vDG are necessary for the anxiolytic/antidepressant effects of fluoxetine (Wu & Hen, 2014).

Additionally, immediate early gene expression indicates that adult born granule cells in the sub

granular zone of the vHPC are preferentially activated during the Morris water maze (Snyder,

Radik, Wojtowicz, & Cameron, 2009), which the authors posit as either as an effect of the

stressful nature of the test or an involvement by the region in contextual fear learning. The latter

implication is interesting, as fear learning in this case requires the animal to distinguish between

safe and dangerous contexts that are not easily distinguishable during learning, and could require

DG mediated pattern separation.

1.4 The Ventral Dentate Gyrus and Learned Approach-Avoidance

Conflicts

The present study examined what specific role the vDG plays in learned approach-avoidance

conflict resolution. Previous studies examined these the effects of temporary pharmacological

inactivations of subregions of the dHPC and vHPC while animals are subjected to a bivalent

approach-avoidance Y-maze paradigm (BAA Y-Maze) (Schumacher et al., 2016). In brief, rats

were trained to associate 3 distinct visuotactile cues with either appetitive, aversive, or neutral

outcomes in a 3-arm Y-maze. Once they demonstrated appropriate discrimination behaviour by

preferentially approaching the appetitive and avoiding the aversive cues, an approach-avoidance

conflict was created by combining the positive and negative associated cues in a single arm. The

various measures taken in this design allows the assessment of how desirable the conflicting cue

arm is relative to neutral arm, how willing the animal is to re-enter the arm following exposure,

and how motivational conflict is perceived. This test also eliminates most spatial aspects during

10

training to prevent learning by place strategies. Additionally, this paradigm can also confirm that

after the conflict test animals still respond to simple valenced stimuli in a normal manner by

directly comparing how much animals prefer an arm of one valence compared to a non-valenced

arm. Additionally the vDG has been shown by various methods to be involved in novelty

detection, anxiety, pattern separation, and locomotion (Bannerman et al., 1999; Hunsaker,

Rosenberg, & Kesner, 2008; Weeden et al., 2015). The role the vDG plays in each of these

processes were assessed by a novelty detection test in a Y-maze, an EPM test, a metric pattern

discrimination test, and a locomotor test. The results indicate that the vDG is a critical actor in

the detection and resolution learned approach-avoidance conflicts.

Methods

2.1 Subjects

Subjects consisted of 38 male Long-Evans rats weighing 350-450g at time of surgery (Charles

River Laboratories, NJ, USA). They were housed in groups of two in a room held at a constant

temperature of 21 degrees, under a 12 h light/dark cycle (lights on at 7:00 A.M). Water was

available ad libitum, while food was restricted to ~18 g of lab chow per day prior to behavioral

testing, sufficient to maintain 85-90% of preoperative body weight. All experiments were

conducted during the light phase and in accordance with the guidelines of Canadian Council of

Animal Care, and approved by the University Animal Care Committee of the University of

Toronto.

2.2 Surgery

Rats were anesthetized with isoflurane (Benson Medical, ON, Canada) and placed in a

stereotaxic frame (Steolting Co, IL) with the incisor bar set at -3.3 mm below the interaural line.

An incision along the midline of the skull was made, and the tissue retracted by small skin clips

to reveal the bregma. Small burr holes were created at the cannulation sites using a dental drill,

and 23-guage stainless steel bilateral cannulae (Cooper’s Needleworks, Birmingham, UK)

implanted into vDG, with the bottom of the cannulae sitting 1mm above the final target

coordinates (AP: -6.5mm, ML: ±4.6mm, DV: -5.5mm). The cannulae were affixed to the skull

11

with dental cement and jewellers’ screws, and stainless-steel stylets were inserted into the guide

cannulae to maintain patency. Surgeries typically lasted 1-2 hours. Rats were then given at least

7 days to recover before the start of behavioural training.

2.3 Radial Arm Maze Apparatus

Behavioral testing for the approach-avoidance conflict task took place in a six-arm radial maze

(Med Associates, VT) placed on a rotatable table elevated 80 cm from the floor. The maze

consisted of six enclosed arms [45.7 cm (L) 3 16.5 cm (H) 3 9.0 cm (W)] stemming from a

central hub compartment with six automatic stainless steel guillotine doors allowing access to the

arms. Arms were enclosed by Plexiglas walls and a removable Plexiglas lid, and contained a

stainless grid floor connected to a shock generator (Med Associates, VT). The end of each arm

contained a receding well consisting of a stainless-steel tray that could be connected to a syringe

pump for the delivery of liquid sucrose. The entire maze was covered in red cellophane paper to

block visibility of extramaze cues, while enabling video recording of behavior via a video

camera mounted above the apparatus. Only three out of six arms (forming a Y maze) were used

at any one time in an experimental session. The maze was wiped down with ethanol solution

after each session to eliminate odor traces, and the maze was randomly rotated left or right by

varying degrees (60, 120, or 180) at the end of the testing day to minimize possible conditioning

to intra-maze cues.

2.4 Habituation

Rats began with 4 habituation sessions. For the first session, animals were initially placed in the

central hub for 1-min adaptation time, after which all three doors retracted, allowing the animal

to explore all arms for 5-min without any cues present. At the end of the trial, the doors

automatically close, with animals returned to the centre manually before removal from the maze.

After the completion of this phase, animals were re-exposed to the maze and run through the cue

conditioning procedure (see relevant section below) without any cues to acclimate the animal

confinement in the maze arms prior to conditioning. The next day, animals were run on their

third habituation phase with visuotactile cues present. The session lasted 6-min total, with 1-min

hub time and 5-min free arm exploration. Cues consisted of either vinyl, duct tape, or wooden

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bars placed along the sides of the arm walls [45 cm (L) 3 4 cm (W) 3 0.5 cm (D)]. Following this

habituation session, cues were designated appetitive, aversive, or neutral, as determined by the

amount of initial exploration time in the habitation session: the most preferred cue was

designated the aversive cue, the least explored the appetitive cue, and the remaining one the

neutral cue. The final habituation session was performed immediately afterwards, consisting of a

2-arm maze configuration, with one arm containing a combination of both ‘appetitive’ and

‘aversive’ cues (an appetitive and aversive cue placed on opposite walls), and a separate arm

containing a neutral cue. This was done to eliminate the chance that any effects seen in the

conflict could be explained by the presentation of a novel combinatory stimulus. The time spent

in each arm was measured for the latter two sessions.

2.5 BAA Y-Maze Cue Conditioning

Animals underwent 12 daily conditioning sessions to concurrently acquire appetitive and

aversive cue conditioning. Each session began with a 30-sec adaptation period in the hub,

followed by a 2-min confinement in each of the 3 arms. Animals had 10-sec to shuttle either into

or out of the arm, after which the experimenter would manually move the animal. This was done

to insure the animal had access to each cue for equal amounts of time. In the arm containing the

appetitive cues, the animals received 4 aliquots of 0.4 ml 20% sucrose solution delivered at 30-

sec intervals. In the arm with the aversive cues, the animals received 4 mild shocks (1 s, 0.25-

0.40 mA) delivered at random intervals. The shock levels were calibrated for each animal to

elicit startle responses without freezing. In the arm with the neutral cue, the animals did not

experience any reward or shock. Previous studies have demonstrated that these specific

schedules of sucrose reward and shock facilitate the development of conditioned approach and

avoidance respectively without inducing generalized fear of the apparatus and freezing responses

to the aversive cue, and results in normal animals spending roughly equal times in the conflict

and neutral arms during the conflict test (Hamel, Thangarasa, Samadi, & Ito, 2017; Nguyen,

Schumacher, Erb, & Ito, 2015; Schumacher et al., 2016). The order of arm presentation was

varied daily to prevent the animals associating the outcomes with the sequence of arm

presentation.

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2.6 Conditioned Cue Acquisition Test and Group Allocation

After every fourth conditioning session, rats underwent a conditioned cue acquisition test to

assess learning of the cue contingencies. Rats could explore the appetitive, aversive, and neutral

cues for 5-min, in the absence of the sucrose or shock. The time spent exploring each of the arms

were recorded by a camera above the maze. Successful acquisition of conditioned cue preference

and avoidance was indicated by (1) more time spent interacting with the appetitive cue than with

the neutral and aversive cues (conditioned cue preference), and (2) time spent interacting with

the aversive cue being shorter than the appetitive and neutral cues (conditioned cue avoidance).

Times from the last acquisition session were analysed and the animals split into drug and saline

groups.

To prevent extinction of the cue contingencies, the animals received a refresher cue conditioning

session on the same day of the acquisition test. Animals underwent 3 cue acquisition tests

overall, after which they were either moved onto the conflict test, or excluded due to poor

learning. Of the 24 animals that survived to the behavioural testing phase, 22 were moved onto

the final phase. Of these, 2 were excluded due to blocked cannulae, 3 showed behaviour outside

of 2 standard deviations from the mean, and 1 was very uncooperative during testing, for a final

group of 16 for the conflict test. Non-learners were used for tests not dependent on BAA Y-Maze

performance. Final group sizes are indicated in the results section of all relevant tests.

2.7 Drug Microinfusion

On the day following the final acquisition test, each animal underwent a saline infusion before

their conditioning session, to minimize the mechanical effects of subsequent drug infusions and

to habituate the animals to the infusion procedure. On the following day, those who had

successfully acquired the cue-outcome associations received bilateral infusions of either a

cocktail of GABA-A receptor agonist muscimol and the GABA-B receptor agonist baclofen

(75ng of each drug dissolved in 0.9% saline, hereafter referred to as BM) or 0.9% saline vehicle

at a volume of 0.3 μl into the region of interest. The drug was infused at a rate of 0.3 μl/min for

1-min via 30-gauge microinjectors projecting 1 mm below the indwelling guide cannulae using

an infusion pump (Harvard Apparatus) mounted with 5μl Hamilton syringes. The micro-injectors

were left in place for a further 1-min to allow the drug to diffuse away from the injector tip.

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During microinfusion animals were held and lightly handled to minimize stress and avoid

injector loss due to movement. A recent study that looked at c-Fos expression following this

same BM infusion procedure found that in neural tissue this results in a 0.3mm radius of reduced

neural activity (Hamel et al., 2017). It is therefore likely that this amount of drug will effectively

reduce neural activity in the vDG within a restricted spatial area. Approximately 10-15 min after

the end of each infusion, the relevant test was administered.

2.8 Mixed Valence Approach-Avoidance Conflict Test

Procedures for this test were identical to the fourth habituation described in section 2.4. During

this session, a state of approach-avoidance conflict was induced in the rats by placing two stimuli

of opposite valences (reward- or shock-associated cue) in one arm, and presenting the neutral cue

in another arm. The time spent in each of the arms and the latency to enter each arm were

recorded by camera for each animal. In addition, the number of entries, the number of retreats

(head only, or half body entries into an arm that did not result in full entries) in the arms were

recorded for each animal.

2.9 Novelty Detection

Following the conflict test, a novelty detection test was administered in a Y-maze decorated with

distinct visual cues lining outside the arm walls, and the lids were clear for visual access to extra-

maze cues. Rats were therefore able to use both intra- and extra-maze cues to detect novelty, and

had access to both spatial and non-spatial information. The test consisted of habituation and test

phases. During habituation, rats were placed at the end of one arm and presented with an

additional arm. Rats were permitted to explore both (familiar) arms for 10-min, and the time

spent exploring each arm was recorded. If the rats showed similar exploration pattern for both

arms, they were tested in the second phase. During the test phase, rats were given access to a

third “novel” arm and to the two familiar arms for 5-min. Time spent exploring each arm was

recorded, and an average for the time spent exploring the two familiar arms was calculated for

comparison with the novel arm.

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2.10 Retraining and Conditioned Cue Preference Test

The day following the conflict and novelty tests, animals received a final cue conditioning

session to prevent extinction to cue valences. On the day following retraining, animals received

another infusion of the same drug they had previously received. 10-15 min following infusion,

animals were exposed to two arms in the maze. One was always neutral, and the other was either

the appetitive or aversive arm. This allowed us to directly compare the animal’s choice between

a simple valenced stimulus and a non-valenced one, in order to assess their preference between

the two. Other than this, the procedure was identical to the fourth habituation described in

section 2.4. After their initial session, they were re-exposed to the same maze 10-15 min later to

assess their preference with the other valence. First valence exposure was counterbalanced

between animals.

2.11 Elevated Plus Maze

The EPM exploits rats’ natural avoidance of bright, open spaces relative to dark ones, and was

therefore used as a test of innate anxiety following preference testing. Previous work has also

indicated that lesioning the vDG shows effects similar to whole HPC lesions (Weeden et al.,

2015). The maze is composed of black Perspex, with a central platform [10 cm (L) × 10 cm (W)]

that connects four arms [40 cm (L) × 10 cm (W) × 22 cm (H)]. Following either BM or saline

infusion, rats were placed in the central platform facing an open arm and allowed to explore for

5-min. Time spent in arms and arm entries were measured. Following the EPM, animals were

also taken off food restriction for their final two tests.

2.12 Metric Pattern Discrimination

Previous research has implicated the DG in metric pattern separation (Hunsaker et al., 2008),

although was only examined in the dDG. On the day following the EPM, rats underwent training

in an additional metric pattern discrimination task, in which they were placed in an open arena

[45 cm (L) × 45 cm (W) × 40 cm (H)], where they freely explored the arena. After an initial 5-

min habituation session to the blank apparatus and the room, the rats underwent 3 sessions

lasting 5-min each. In these sessions, objects were introduced into the arena located exactly 40

cm apart. Exploration of the objects were measured in each phase, with the expectation that

exploratory time would decrease as the objects become familiar to the rats. After the 4th phase,

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rats received intra-cerebral infusions of either BM or saline, followed by 10-min in home cage.

Rats then underwent two more 5-min sessions to explore the objects in their original positions.

For the final phase, objects were moved closer together (20 cm apart), and the rats explored the

arena for a further 5-min. It was expected that the control rats would be able to detect the

changes in the locations of the two objects by reinstating their exploration of the two objects,

while disruption of spatial pattern separation would not allow the rats to recognize any change

has been made and would not increase their exploration.

2.13 Locomotor test

To test for locomotor effects, BM or saline-infused animals were placed in a locomotor chamber

[plastic cage: 47.4 cm (L) × 26.4 cm (W) × 20.5 cm (H)] for a 1-hour session in which they

could freely explore the chamber. Locomotor activity was recorded using a camera and

EthoVision XT software (Noldus Information Technology, ON, Canada), and were measured as

the distance traveled in cm in 5-min bins.

2.14 Histological Procedure

All rats were anesthetized with sodium pentobarbital (2mL/4.5kg, Bimeda-MTC, Cambridge,

ON) and perfused intracardially via the ascending aorta with 0.9% saline, followed by 4%

paraformaldehyde (PFA) solution. Brains were removed, stored in 4% PFA, and transferred to a

20% sucrose cryoprotectant solution before sectioning. Coronal sections (50-60 μm) of the brain

were cut using a vibratome and stained with cresyl violet, to be viewed under the microscope for

the verification of cannulae placements.

2.15 Data Analysis

All data was analyzed using R. A 2-way analysis of variance (ANOVA) was conducted on the

time spent from each of the conditioned cue preference/avoidance tests with Drug Condition

(BM infusion; Saline infusion) as the between-subject factor and Arm (Appetitive, Aversive,

Neutral Cue) as the within-subject factor. All data (time spent, number of entries, number of

retreats) from the conflict test day was subjected to a 2-way ANOVA with Drug Condition as the

between-subject factor, and Arm (Combined Cue; Neutral Cue) as the within-subjects factor.

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Identical analyses were run for the initial conflict habituation, as well as the appetitive and

aversive preference tests. EPM arm open arm entries and time in open arms were analyzed with

an ANOVA (Drug between-subject factor). Total time spent exploring objects in the MPD was

measured by the a 2-way ANOVA with Drug as the between-subject factor and Session as the

within-subject factor. Difference scores between the time exploring the objects in test session

minus time exploring during the final habituation session were used to compare pattern

discrimination by drug group. Finally, the distance travelled during the locomotor test was

analyzed with a 2-way ANOVA with Drugs as between-subject factor and time Bin as within-

subject factor.

Results

3.1 Histological Verification

Inactivation sites ranged from roughly -6.04 to -6.8 mm posterior to bregma (Paxinos & Watson,

1998) (Figure 1). One animal’s infusion site could not be verified, and was excluded from

analyses.

3.2 Habituation

BM and saline group numbers were n = 8 and n = 8, respectively. The final habituation session

prior to training mimicked the approach-avoidance conflict test to show that the groups did not

show any initial bias towards the arms (Figure 2). Analysis did not show any effect of arm or

drug condition (Arm: F (1,28) = 1.499, p = 0.23; Drug: F (1,28) = 0.058, p = 0.81), nor any

interactions (Arm x Drug interaction: F (1,28) = 0.007, p = 0.93). Therefore, it is unlikely that

the effects seen could be accounted for by an inherent preference for the conflict or neutral

stimuli by either group.

3.3 Conditioned Cue Acquisition

BM and saline group numbers were n = 8 and n = 8, respectively. When animals were divided

into either BM or saline groups, analysis on their final acquisition test indicated a significant

effect of arm (Arm: F (2,42) = 59.906, p < 0.001), but not by drug condition or any interaction

(Drug: F (2,42) = 0.277, p = 0.60; Arm x Drug interaction: F (1,42) = 1.20, p = 0.31) (Figure 3).

Therefore, in both groups animals successfully learned to preferentially approach the appetitive

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arm over the neutral (BM: F (1,12) = 12.44, p < 0.01; saline: F (1,12) = 27.17, p < 0.001), and

avoid the aversive arm more than the neutral (BM: F (1,12) = 15.49, p < 0.01; saline: F (1,12) =

5.881, p < 0.05).

3.4 Mixed Valence Approach-Avoidance Conflict Test

BM and saline group numbers were n = 8 and n = 8, respectively. ANOVA of overall time spent

in arms indicated a significant interaction between the arm and drug condition (Arm x Drug

interaction: F (1,28) = 4.761, p < 0.05) (Figure 4). Planned comparisons demonstrated this effect

was driven by the drug group preferring the conflict arm relative to the neutral arm (F (1,14) =

10.22, p < 0.01). In contrast, the saline group showed no preference for either arm (F (1,14) =

0.017, p = 0.90). The results suggest that under circumstances of conditioned conflict, an inactive

vDG leads to increased approach towards conflict stimuli.

In addition, ANOVA showed trending effects toward a drug by arm interaction when looking at

the number of retreat behaviours observed (Arm x Drug interaction: F (1,24) = 3.857, p = 0.06),

which appears to be driven by the BM group making less retreats from the conflict arm than the

saline group (F (1,12) = 5.042, p < 0.05) (Figure 6). However, no significant main effects or

interactions were observed for the number of full body entries into either arm (Arm: F (1,24) =

2.843, p = 0.11; Drug: F (1,24) = 0.482, p = 0.49; Arm x Drug interaction: F (1,24) = 0.797, p =

0.38) (Figure 5).

3.5 Novelty Detection

BM and saline group numbers were n = 10 and n = 10, respectively. Novelty habituation showed

no significant effects of drug or arm (Arm: F (1,36) = 0.081, p = 0.73; Drug: F (1,36) = 0.081, p

= 0.78), or any interaction (Arm x Drug: F (1,36) = 0.617, p = 0.44). This indicates that during

habituation, neither group showed any preference for either of the arms (Figure 7). In the novelty

test, analysis of variance showed a significant effect of arm (Arm: F (1,36) = 63.618, p < 0.001),

but no effect of drug or interaction (Drug: F (1,36) = 0.318, p = 0.58; Arm x Drug interaction: F

(1,42) = 0.147, p = 0.70). Pairwise comparisons showed that both drug and saline groups

preferred to spend time in the novel arm relative to the start and familiar arms (BM: F = (1,18) =

36.88, p < 0.001; saline: F = (1,18) = 27.38, p < 0.001), suggesting that novelty detection was

preserved in the drug group (Figure 8).

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3.6 Conditioned Preference

BM and saline group numbers were n = 7 and n = 7, respectively Preference testing indicated

that neither the drug or saline groups showed any changes in preference when presented with

either the appetitive or aversive arms alone vs. the neutral. ANOVA in the appetitive preference

test showed an effect of arm (Arm: F (1,24) = 28.6185, p < 0.001), but not for drug or any

interaction (Drug: F (1,24) = 0.8997, p = 0.35; Arm x Drug interaction: F (1,24) = 0.1036, p =

0.75), driven by a preference by both groups for the appetitive arm (BM: F = (1,12) = 11.01, p <

0.01; saline: F = (1,12) = 23.46, p < 0.001) (Figure 9). The results were the same for the aversive

preference omnibus test (Arm: F (1,24) = 12.183, p < 0.01; Drug: F (1,24) = 0.805, p = 0.38;

Arm x Drug interaction: F (1,24) = 0.070, p = 0.79), but was driven for preference for the neutral

arm (BM: F = (1,12) = 6.844, p < 0.05; saline: F = (1,12) = 5.686, p < 0.05) (Figure 10). This

suggests that inactivation of the vDG does not impair the association between cue and outcome

per se, nor act to potentiate approach towards simple appetitive or aversive stimuli.

3.7 Elevated Plus Maze

BM and saline group numbers were n = 8 and n = 11, respectively ANOVA did not indicate

significant effect of condition when comparing the overall time in the open arm of the EPM in

drug group compared to the saline group (Arm: F (1,17) = 1.333, p < 0.26) (Figure 11).

Similarly, no effects were observed for the number of entries (Arm: F (1,17) = 0.221, p < 0.64)

(Figure 12). Therefore, it does not appear that vDG inactivations had any effect on innate anxiety

in this test.

3.8 Metric Pattern Detection

BM and saline group numbers were n = 8 and n = 12, respectively ANOVA found a significant

effect of session (Session: F (1,115) = 28.46, p < 0.001), but no effect for drug or interaction

(Drug: F (1,115) = 0.937, p = 0.34; Session x Drug interaction: F (1,115) = 0.013, p = 0.91).

Effect of session appears to be driven by a gradual decrease in time spent exploring objects in the

SPS, with a slight increase in the final session when objects are moved together (Figure 13). Test

minus final habituation difference scores were not different between the groups (F (1, 18) =

2.342, p = 0.14) (Figure 14). These results indicate that the vDG is not necessary for metric

pattern discrimination.

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3.9 Locomotion

Group numbers for the BM and saline groups were n = 7 and n = 7, respectively ANOVA results

showed a main effect of time bin and drug (Bin: F (1,164) = 60.620, p < 0.001; Drug: F (1,164 =

7.865, p < 0.01), but no interaction (Bin x Drug: F (1,176) = 0.271, p = 0.60). However, post-hoc

analysis could not find any effects that survived correction. These results suggest that

inactivation of the vDG might attenuate spontaneous locomotion (Figure 15).

4 Discussion

4.1 The Ventral Dentate Gyrus is Involved in Approach-Avoidance

Conflicts with Conditioned Cues

The results of the conflict test demonstrated potentiated approach into the conflict arm for vDG

inactivated animals. This appears to be the first observed instance of vDG involvement in

learned approach-avoidance conflicts. These patterns cannot be explained as potentiated

approach towards stimuli associated with appetitive or aversive outcomes, since the inactivated

group demonstrated normal approach and avoidance of cues during preference testing.

Therefore, it appears the vDG is critical for normal approach-avoidance behaviours, but not

during the presentation of simple affective stimuli. The effects are also unlikely to be due to

impaired novelty or increased locomotion, which were found to be normal and attenuated in the

BM group, respectively. Finally, the BAA Y-Maze paradigm precluded the use of spatial

strategies, and therefore the results cannot be the result of damage to spatial navigation systems

or the use of spatial reasoning.

The results are also consistent with the results of inactivation of both the entire vHPC

(Schumacher et al., 2016) and the vCA3 (Schumacher et al., 2017). Given the established

anatomical and functional coupling of the DG and CA3, these results suggest that the regions

could act together in mediating behaviours seen in learned approach-avoidance conflicts. In

contrast, the results are the opposite of those seen with vCA1 inactivation, where animals show a

preference for the neutral arm. This possibly suggests the vDG and vCA3 might act

independently of the vCA1 in approach-avoidance conflicts, with their combined activity needed

for normal approach behaviour in learned conflict scenarios.

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4.2 The Results cannot be explained by Novelty or Pattern Separation

Deficits

Novelty testing revealed no abnormal behaviour by the vDG inactivated animals, as all

successfully discriminated between familiar and novel arms. Therefore, the results cannot be

explained through abnormal novelty processing. This stands in contrast to the results of the

previous study mentioned above (Schumacher et al., 2017), where it was found that inactivation

of both vCA3 and vCA1 resulted in novelty detection deficits. Given that the function of pattern

separation (a primary DG function) is to ensure new patterns to be learned by the CA3 are not

encoded in the same neural location as similar previously stored information, it is likely that the

novel arm patterns encountered by the animal (combined spatial and non-spatial stimuli) where

distinct enough from the previously encountered patterns to not need additional

orthogonalization during encoding. However, functions typically ascribed to the CA3 and CA1

(pattern completion and mismatch detection respectively) might be needed in the vHPC to show

typical effects during this test, which would allow the animal to store arm patterns and detect

mismatches between expected arm patterns. In this case, the data suggests that vDG mediated

pattern separation does not appear to be necessary for normal performance in this test.

In addition, the vDG does not appear to be required for metric pattern discrimination, which has

been shown to be sensitive to dDG manipulation (Hunsaker et al., 2008). This suggests that at

the level of fine discriminations of changes to spatial relations between objects, the vDG is not

an important actor. This raises the question of what exactly the role of the vDG might be when it

comes to both pattern separation and spatial processing. In terms of the former, while some work

has been done on olfactory and reward pattern separation by the vDG (Kirk, Redmon, & Kesner,

2016; Weeden, Hu, Ho, & Kesner, 2014), the precise nature of its involvement is still unclear,

and cannot readily explain the results seen in these experiments, as olfaction was not a major

factor in any tests, and reward value was not systematically varied in a way necessary to elicit

pattern separation (Hunsaker & Kesner, 2013). For spatial processing, while it has been

speculated that the vHPC as a whole might exert effects on larger spatial areas than the dHPC

(Jung et al., 1994; Poppenk, Evensmoen, Moscovitch, & Nadel, 2013), the role of the vDG in

this model is not immediately obvious, and should be explored in more detail in future studies.

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4.3 Inconsistent EPM Results

Inactivation of the vDG appears to have had no effect on the amount of time in the open arms or

open arm entries in the EPM, in contrast to tests of innate anxiety during inactivation of the

whole vHPC or vCA3 (Kjelstrup et al., 2002; Schumacher et al., 2017) and even studies on the

vDG (Weeden et al., 2015). This is surprising given the tight functional coupling between the

DG and CA3, as well as the fact that the DG projects directly to regions that have been found to

inhibit approach into the open arms under normal conditions (Yamaguchi, Danjo, Pastan, Hikida,

& Nakanishi, 2013). However, this is not the first time suppressing vDG function has shown no

effects on the EPM, as another study found that optogenetic inhibition of vDG granule cells had

no noticeable effect (Kheirbek et al., 2013). The same study also found that optogenetic

stimulation could drive open arm exploration, though these results are difficult to interpret, since

large scale activation of the region is biologically implausible given the low basal rate of activity

by DG granule cells.

It is unclear why these findings diverge so sharply. It is possible that this present study

had an unusual number of outliers, as the standard deviation of the BM group was quite high (SD

= 45-sec, on a mean of 85-sec in the open arms), though this does not explain why Kheirbek et

al. found the same results. It has also quite possible the lesioning technique used by Weeden et

al. exerted a more powerful effect than the pharmacologic inactivation of this study or the

optogenetic inhibition of Kheirbek et al. Finally, it is worth considering the possibility that

inhibiting the vDG does not exert powerful effects on innate anxiety, and the effects of earlier

studies were false positives. Currently, the evidence is not strong enough to make a conclusion.

4.4 Locomotion results Unexpected

The results of the locomotor test were surprising, given that previous work on the vHPC either

found increased locomotion with inactivation/damage (Lipska, Jaskiw, Chrapusta, Karoum, &

Weinberger, 1992) or no effect (Bannerman et al., 1999; Schumacher et al., 2017). Previous

work suggested that increases in locomotion might be related to food search in the presence of

cues associated with food (Tracy, Jarrard, & Davidson, 2001). However, in this case animals

were well fed, having been taken off food restriction for at least 24 hours prior to locomotion

testing, and had access to food at least 5 minutes before the start of testing, so motivation for

food should have been low. Additionally, the animals were tested in a novel room without any

23

cues associated with the delivery of food. Therefore, it is unlikely that changes to energy

conservation/balance could explain the results. This effect does not appear to show up at any

particular time point during testing, as no single time bin survived post-hoc analysis. Further

work should be done to investigate these findings.

Limitations and Future Directions

This series of experiments highlights and clarifies several important processes the vDG

participates in. Most notably, the vDG appears to be a critical actor in vHPC mediated approach-

avoidance processing during learned conflicts, with inactivation of the region recapitulating the

effects seen with whole vHPC inactivation. Given the similarity of these effects to those seen

with vCA3 inactivation, as well as the documented anatomical coupling between the regions, it is

possible that they work as a subsystem within the HPC to drive avoidance under normal

approach-avoidance conflict conditions. It is unclear whether that the vDG also mediates

avoidance of situations that naturally give rise to anxiety, as the results of this study are

inconsistent with those of others (Weeden et al., 2015). Finally, it appears that inactivation of the

region has some effect of locomotion, but unlike its dorsal counterpart is not involved in spatial

pattern separation.

The results offer insights into internal workings and functions of the vHPC. It now clear that

each component of the ventral tri-synaptic circuit has some role in approach-avoidance conflicts

with both innate and learned valenced stimuli, though the effects vary for each region. However,

much work remains to be done on how these functions are implemented by the neural

architecture of the HPC. Only the results of the novelty detection experiment were easily

explainable in terms of functions commonly ascribed to the HPC subregions. Currently it is

unclear how the tri-synaptic circuit that performs (in linear order) pattern separation, pattern

completion, and mismatch detection would explain the observed results here and in other studies

on both learned and innate tests of approach-avoidance conflict. In fact, an alternative could be

that the effects observed might occur through means other than the traditional circuit

(Schumacher et al., 2017). The vDG, vCA3, and vCA1 all send out unique efferent projection

patterns, though some have a more diverse set of target regions than others. It appears that in at

least one case, subregions of the HPC can target different parts of a single structure. Both the

vCA3 and vCA1 send projections to different areas in the lateral septum (caudal lateral septum

24

and rosto-ventral lateral septum, respectively (Risold & Swanson, 1997)). The lateral septum

(LS) controls the medial septum through GABAergic projections, which in turn controls

hippocampal theta rhythm (Korotkova et al., 2017). In addition, the LS also projects to the

hypothalamus, and manipulations of the region have been shown to affect the same anxiety tests

vHPC is involved in (Degroot & Treit, 2003; Menard & Treit, 1996; Yamaguchi et al., 2013).

While vHPC-LS interactions have been studied (Parfitt et al., 2017), the specific subregions of

the LS were not specifically investigated. Additional regions of interest include the mPFC,

amygdala, and nucleus accumbens, and the VTA, each of which have been implicated in

approach and avoidance behaviours to some degree and are known to have functional

connections with the vHPC (Ciocchi et al., 2015; Lisman & Grace, 2005; Padilla-Coreano et al.,

2016; Parfitt et al., 2017).

Applied more broadly, these results are consistent with literature that emphasizes the

hippocampal formation’s participation in evaluating complex stimuli, without any particular

regard to mnemonic processes (Bannerman et al., 2014; Lee, Yeung, & Barense, 2012). More

specifically, it has been observed that subregions of the dHPC are involved in conflicts where

target spatial positions are marked by a stimulus with a double in an incorrect position

(Bannerman et al., 2012) an effect that is not observed when the visual beacons are dissimilar. It

has also been found that neuropsychiatric patients with damage to the medial temporal lobe show

deficits perceiving subtitle differences between two stimuli when they share many overlapping

features (Lee et al., 2005), even when the stimuli did not have to be remembered. A common

aspect of these studies, as well as the current one, is that all the tasks where the HPC was

involved the participant encountered stimuli that created some sort of conflict that needed to be

resolved. On this interpretation, the dHPC appears to be more involved in conflicts involving

spatial information, while the vHPC is more closely associated with resolving types of affective

conflict (Ito & Lee, 2016), and exert their effects through connections to and from dissimilar

regions.

An interesting aspect of all the specific approach-avoidance conflicts reviewed here, both with

conditioned stimuli and stimuli that elicit innate reactions from the animal, is that the conflict

scenario is novel in some way. Even though this study attempted to minimize novelty effects by

exposing the combined stimuli to the animals prior to conditioning, the exposure of the

appetitive and aversive cues together following conditioning still creates a novel juxtaposition of

25

affective associations that was not present prior to the testing experience. Likewise, the EPM and

light-dark box also create conflict situations most laboratory animals have no prior exposure to,

given their relatively safe and controlled daily experiences. Since the HPC is typically linked to

learning in the presence of novel stimuli, it is worth asking to what extent the vHPC

manipulation in this and other studies would have on well learned conflicts, where the animal is

exposed to a familiar situation that initiates an established approach-avoidance conflict. Current

theories of how the HPC mediate anxiety and approach-avoidance conflicts make no particular

distinctions between novel and well learned conflicts (Gray & Mcnaughton, 2000; Ito & Lee,

2016). Some ideas about the answer to this question could be inferred from patterns of HPC

activity in human participants playing games meant to evoke approach-avoidance conflicts,

which suggest that despite these games becoming familiar over many trials, the HPC is still

involved in well learned conflict scenarios (Bach et al., 2014). Further studies will be needed to

explicitly confirm if this is the case.

In conclusion, evidence strongly supports the idea that the vDG critical for the detection and

resolution of learned approach-avoidance conflicts, and these outcomes are not explainable as

disruption of abnormal approach towards stimuli associated with single valences. This furthers

the growing literature on the internal workings of the vHPC subfields, supporting the notion that

each contributes to specific behavioural processes. In addition to the conflict results, vDG

inhibition showed unique novelty detection and locomotor effects compared to vCA3 and vCA1

inhibition. Given the inconsistent results of the EPM test with existing literature, further

investigation is warranted as to how important the vDG is for mediating innate anxiety reactions.

Finally, the vDG does not appear to be involved in the same type of pattern separation as the

dDG, as indicated by the results of the MPD test.

26

Figures

Figure 1. Placements of injector tips. Red circles are BM infusion sites; black crosses are saline

infusion sites.

27

Figure 2. Time spent in the conflict and neutral arms in the 4th habituation session.

Figure 3. Time spent in each arm during the final cue conditioning acquisition test.

0

20

40

60

80

100

120

BM Saline

Tim

e (s

ec)

Conflict Neutral

0

20

40

60

80

100

120

BM Saline

Tim

e (s

ec)

Chart Title Appetitive Aversive Neutral

**

***

***

28

Figure 4. Time spent in each arm during the mixed valence approach-avoidance conflict test.

Figure 5. Number of entries during mixed valence approach-avoidance conflict test

0

20

40

60

80

100

120

BM Saline

Tim

e (s

ec)

Conflict Neutral**

0

1

2

3

4

5

6

7

8

9

10

BM Saline

En

trie

s

Conflict Neutral

29

Figure 6. Number of retreats during mixed valence approach-avoidance conflict test.

Figure 7. Time spent in start and familiar arms during novelty habituation.

0

0.5

1

1.5

2

2.5

3

BM Saline

Ret

reat

s

Conflict Neutral

*

0

20

40

60

80

100

120

140

160

180

200

BM Saline

Tim

e (s

ec)

Start Familiar

30

Figure 8. Time spent in familiar arms and novel arms during novelty test.

Figure 9. Time spent in appetitive and neural arms during final preference test.

0

20

40

60

80

100

120

BM Saline

Tim

e (s

ec)

Familiar Novel

**

0

20

40

60

80

100

120

140

160

BM Saline

Tim

e (s

ec)

Appetitive Neutral

*****

31

Figure 10. Time spent in aversive and neural arms during final preference test.

Figure 11. Time spent in the open arm of elevated plus maze.

0

20

40

60

80

100

120

BM Saline

Tim

e (s

ec)

Aversive Neutral

* *

0

20

40

60

80

100

120

BM Saline

Open

Arm

(se

c)

32

Figure 12. Number of entries into the open arm of elevated plus maze.

Figure 13. Combined time exploring both objects during metric pattern discrimination.

0

1

2

3

4

5

6

7

BM Saline

Op

en A

rm E

ntr

ies

0

50

100

150

1 2 3 4 5 6Obje

ct E

xp

lora

tio

n (

sec)

Session

BM Saline

33

Figure 14. Difference in time spent exploring objects in the MPD test between final test and last

training session.

Figure 15. Distance travelled with each 5-min time block during locomotor test.

0

5

10

15

20

25

30

35

BM Saline

Tim

e E

xp

lori

ng (

sec)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1 2 3 4 5 6 7 8 9 10 11 12

Dis

tan

ce (

cm)

Time Bin

BM Saline

34

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