The Impact of Cognitive and Physical Enrichment on
Contextual Fear Memory Retention & the Contribution of
Oligodendrogenesis and Myelination
by
Jordan Mak
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Physiology
University of Toronto
© Copyright by Jordan Mak 2021
The Impact of Cognitive and Physical Enrichment on Contextual Fear Memory Retention
& the Contribution of Oligodendrogenesis and Myelination
Jordan Mak
Master of Science
Department of Physiology
University of Toronto
2021
Abstract
Memories are encoded in patterns of neural activity deemed “engrams” and are recalled when this network is reactivated. Physical and cognitive enrichment have been shown to increase neurogenesis and myelinogenesis which can alter the stability of the engram. We housed mice in both physical enrichment and a combination of physical plus cognitive enrichment and found that both caused forgetting of a contextual fear memory if housing occurred after conditioning, but improved memory recall if done beforehand. However, combining physical and cognitive enrichment caused a greater change in freezing behaviour relative to physical enrichment alone, only when housing was done after conditioning. Mice in which oligodendrogenesis was halted exhibited a trend of freezing less than mice with functioning oligodendrocytes regardless of enrichment, though this was not considered statistically significant. These results are consistent with the theory that enrichment exerts its effect on memory retention by altering engram stabilization during memory consolidation.
ii
Acknowledgements
First and foremost, I would like to thank Dr. Anne Wheeler for all of her guidance, support, and mentorship as my supervisor. I cannot stress enough just how much your patience and kindness contributed to my success during this degree. You have always been there to further explain concepts, troubleshoot any issues, and provide input about future directions. I am always in awe at your level of excellence as a scientist, and I do not think I would have enjoyed my time during my MSc if it had not been for you and the lab environment you have fostered.
I would also like to thank Dr. Lisa Gazdzinski, who was responsible for the vast majority of my training during my time in the lab. I am extremely appreciative of how you were patient with all of my questions and mistakes. You have been such an amazing role model with your knowledge, willingness to help, and overall efficiency with juggling a Cirque du Soleil level of tasks (I’m still not 100% certain you are not a robot…). It has also been a joy to get to know your family, as I always look forward to our lab socials to see what shenanigans your kids get into. I still do not fully understand your wizardry with our lab equipment, but I commend you for being the only one the cryostat respects and listens to.
My project would not have been possible without the contributions of my advisory committee, Dr. Brian Nieman and Dr. Paul Frankland. From the beginning of our first meeting, I felt like we were on the same team and working together to shape this project to be the best it could be. Your advice and recommendations were second to none, and I always felt encouraged
iii
after our meetings. Thank you for helping me to develop as a scientist and honing my ability to think critically.
I would also like to thank Percy Azzopardi, Nancy Simpson, and Igor Vukobradovic at The
Centre for Phenogenomics. Percy, thank you so much for accommodating my complicated and hectic cage washing schedule with the custom cages. It was a pleasure working with you and your team. Nancy, thank you for helping me to set up more complicated mouse orders and answering my questions when I had other facility related questions. It was always reassuring when I would walk by your office knowing you had my back. Lastly, Igor I am so appreciative for all of your help with the behavioural testing. You were always supportive and ready to help whenever I needed it, and I enjoyed seeing you around the facility and joking around.
Another huge “thank you” goes to my lab mates – both past and present. One of the best parts of coming to SickKids everyday was to see you guys. Perhaps my greatest contribution to the lab is the collection of Oreos kept above my desk. I’m proud to announce that we have successfully sampled all of the novelty flavours available in Canada and have even branched into flavours exclusive to the States. The friendships I have forged with you guys are very dear to me, and I look forward to continuing them even after I graduate.
Lastly, I would like to thank my family and friends who were always behind me supporting me throughout this rollercoaster of a journey. You helped keep me on track and were there to prop me up during the rough patches. Though finishing my degree during a global pandemic was not how I envisioned my degree ending, being home with all of you really made this special and I hope I have made you proud.
iv
Table of Contents
Table of Contents ...... v
List of Abbreviations: ...... viii
List of Figures: ...... x
Chapter 1
1 Introduction ...... 1 1.1 Overview of Memory Encoding, Recall, and Forgetting ...... 1
1.1.1 Memory & Engrams ...... 1 1.1.2 Forgetting & Transience ...... 3 1.1.3 Recent vs. Remote Contextual Fear Memory...... 6 1.2 Myelin’s contribution to memory retention ...... 8
1.2.1 Function of Myelin ...... 8 1.2.2 Development of Oligodendrocytes and Myelin ...... 10 1.2.3 Adult Oligodendrogenesis, Myelin Plasticity, and Memory Retention ...... 12 1.3 Physical & Cognitive Enrichments’ Effects on Memory Retention ...... 14
1.3.1 Brain Changes Associated with Physical & Cognitive Enrichment ...... 14 1.3.2 Physical & Cognitive Enrichments’ Effect on Memory Recall ...... 18 1.4 Rationale ...... 19
1.5 Objectives & Hypotheses ...... 20
Objective 1: ...... 20 Objective 2: ...... 20
Chapter 2
2 Materials & Methods ...... 21 2.1 Experimental Designs ...... 21
2.1.1 Housing Before/After Conditioning Timeline & Details ...... 21 2.1.2 Recombination Efficiency & Toxicity Assessment ...... 22 2.1.3 MyRF:NG2-ERT:Cre Mice Enrichment Timeline & Details ...... 23
v
2.2 General Methods ...... 24
2.2.1 Mice ...... 24 2.2.2 Tamoxifen & EdU Administration ...... 25 2.2.3 Housing Conditions ...... 25 2.2.4 Contextual Fear Conditioning ...... 27 2.2.5 Perfusions ...... 29 2.2.6 Immunohistochemistry ...... 29 2.2.7 Statistical Analysis...... 30
Chapter 3
3 Results ...... 33 3.1 Freezing when Enrichment Occurs Before & After Conditioning ...... 33
3.1.1 Enrichment After Conditioning Causes Forgetting of Contextual Fear Memories . 33 3.1.2 Timing of Enrichment Relative to Conditioning Causes Different Changes to Memory Retention ...... 34 3.1.3 No Sex Effect When Comparing After, Before, or Combined Groups ...... 36 3.2 Recombination Efficiency ...... 37
3.2.1 Cell Counts of Newly Proliferated Cells (EdU+) ...... 38 3.2.2 Mouse Strain ...... 38 3.2.3 Tamoxifen Dosage ...... 38 3.3 MyRF Mice Experiments ...... 40
3.3.1 Freezing Behaviour Differences Between EE and SE ...... 40
Chapter 4
4 Discussion ...... 42 4.1 Objective 1 – Effect of Different Types of Enrichment on Contextual Fear Memory
Retention & the Importance of Timing of Conditioning ...... 42
4.1.1 Effect of Cognitive Enrichment After Conditioning on Contextual Fear Retention 42 4.1.2 Comparison of the Effects of Enrichment at Different Timepoints Relative to Conditioning...... 43 4.1.3 Potential Mechanisms Underlying Changes to Memory Retention ...... 44 4.2 Objective 2 - MyRF Mouse Model & Specific Contributions of Myelin to Contextual Fear
Memory Recall ...... 48
vi
4.2.1 Efficiency of Recombination in the MyRF Mouse Model ...... 48 4.2.2 Evidence of Myelin’s Contribution to Memory Retention Modulation ...... 49 4.3 Limitations & Future Directions ...... 51
4.3.1 Housing Conditions & the Isolation of Cognitive Enrichment’s Impact ...... 51 4.3.2 1 Day vs 28 Day Delay Between Conditioning & Testing...... 51 4.3.3 Tamoxifen Injection Start Time & Halting of Oligodendrogenesis ...... 52 4.3.4 Novelty of Enrichment & Type of Memory ...... 53 4.3.5 Implications for Pathologic Myelin Diseases & Military PTSD Research ...... 54
References ...... 56
Copyright Acknowledgments ...... 64
vii
List of Abbreviations:
ACTH Adrenocorticotrophin Hormone
AMPA Receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
CFC Contextual Fear Conditioning
DAPI 4′,6-diamidino-2-phenylindole
EdU 5-Ethynyl-2´-deoxyuridine
EE Environmental Enrichment
HPA-Axis Hypothalamic–Pituitary–Adrenal Axis
LTP Long-Term Potentiation
MRI Magnetic Resonance Imaging
MTT Multiple Trace Theory
MWF Myelin Water Fraction
MyRF Myelin Regulatory Factor
NG2 Neuron/Glial Antigen 2
NK Cells Natural Killer Cells
viii
OPC Oligodendrocyte Precursor Cell
PB Phosphate Buffer
PBS Phosphate Buffered Saline
PDGFrα Platelet-derived Growth Factor Receptor α
PFA Paraformaldehyde
PTSD Post-traumatic Stress Disorder
RW Running Wheel
SE Standard Environment
SMSC Standard Model of Systems Consolidation
TCP The Centre for Phenogenomics
ix
List of Figures:
Figure Title Page
1.1 Overview of engram formation and subsequent recall 2
1.2 Overfitting versus generalization of memories 5
1.3 Theories of memory consolidation 7
Outline of saltatory conduction of an action potential along an 1.4 9 axon
1.5 Developmental timeline of oligodendrocytes and myelin 12
2.1 Enrichment after and before conditioning study designs 22
2.2 Recombination efficiency study design 23
2.3 MyRF EE experiment study design 24
2.4 Diagram of different housing conditions 27
2.5 Contextual fear conditioning protocol 28
Reductions in freezing behaviour when enrichment occurs after 3.1 33 fear conditioning Increased freezing behaviour if enrichment is shifted to occur 3.2 34 before conditioning
3.3 No sex differences in freezing behaviour 36
Averaged counts of EdU+ cells in the corpus callosum from 3.4 38 different mice
x
Freezing behaviour exhibited by MyRF mice injected with 3.5 40 tamoxifen in either SE or EE cages
xi
Chapter 1
1 Introduction
1.1 Overview of Memory Encoding, Recall, and Forgetting
1.1.1 Memory & Engrams
Memories are extremely important for understanding our surrounding environments and helping us make sound decisions based on previous experiences. Memories are stored in patterns of neural activity deemed “engrams”, and are recalled when these populations of neurons are reactivated in a similar fashion1. These patterns are stabilized to ensure that they can be reliably reactivated so the memory can persist, while memories are forgotten when these patterns are disrupted and cannot be reactivated as faithfully2. The plasticity of the brain is essential for network formation, stabilization, and destabilization as changes occurring can strengthen or weaken neuronal connectivity in a wide variety of ways. Previous literature has focused on how synaptic factors such as long-term potentiation3 and neurogenesis4–6 impact network stability and memory retention. Memory retention refers to the duration and accuracy at which memories can be recalled for as time passes and is directly affected by the stability of the associated engram.
1 | P a g e
Figure 1.1. Overview of engram formation and subsequent recall: Engrams refer to patterns of neural activity which encode a memory. Recall of the memory then requires reactivation of this pattern. Image used from Josselyn et al. 20157.
Contextual fear memories are established when a fearful experience becomes associated with the specific context in which it occurred. The sights, sounds, and smells of the location alone can then trigger memory recall of the experience to induce fear behaviours.
Within mice, the primary behaviour associated with fear is freezing, where mice will cease all movement other than breathing for extended periods. By measuring how long mice freeze for when placed back in a context where a fearful experience was conditioned, the degree to which the mouse remembers that experience can be assessed. One common way to form contextual fear memories in mice is through contextual fear conditioning (CFC) where mice are placed in a training box with specific olfactory cues where they receive footshocks. Mice have been shown
2 | P a g e
to have stable CFC memory recall that persists even with delays upwards of 30 days between training and testing dates4,8.
1.1.2 Forgetting & Transience
While engram networks can be strengthened and stabilized to ensure the persistence of memories, the opposite can also occur to cause forgetting. When networks are unable to reactivate in the exact pattern, the associated memory is unable to be recalled as accurately and can eventually become forgotten. One such method is to decrease the connection strength between neurons in a network. Depotentiation via the endocytosis of GluA2 AMPA receptors of previously strengthened synapses causes destabilization of networks as connection strength between neurons is degraded9. Another way to change the physical layout of the network is to either add or remove synapses. Adding and removing new connections to networks through neurogenesis, and Rac-mediated dendritic spine shrinkage has been shown to cause forgetting10,11. Anything that disrupts the reactivation of the engram would potentially contribute to reduction of memory retention.
Forgetting has a tendency to be considered in a negative light, and that the optimal course of action is to limit and prevent forgetting as much as possible. However, this is certainly not the case as the ability to forget is crucial for a healthy brain to adapt to an ever-dynamic world. Firstly, forgetting is beneficial to allow for cognitive flexibility and the ability for reversal
3 | P a g e
learning. Once information has been learned about the best way to interact with our surroundings, if the information no longer causes the same consequences or a better approach is discovered, then the brain needs to be able to incorporate or replace the previously learned memory with this new information2. For instance, if mice are trained in a Morris water maze task, they learn to find a hidden platform in a certain region based on contextual cues. If experimenters reduce a mouse’s ability to forget, the mouse takes a lot longer to recognize if the platform has been moved, and will take a lot longer to learn the new location as they will continue to search in the previously learned area12. However, forgetting and transience are not an all or nothing approach. Most of the time there is partial/graded forgetting, meaning that memories are not 100% erased and completely lost13. Instead, memories can have selected portions forgotten while the rest is retained, can become tied to specific cues where it is a lot more difficult to remember without them, or can be forgotten for a period of time before being spontaneously recovered13.
Forgetting can also be beneficial by preventing overfitting of a memory and allowing for generalization of the important information gained. When information is learned, it can be used to form a model to predict outcomes and ultimately make decisions. Overfitting refers to when this model is too focused on specific details of the training data, meaning that the model is only accurate within those very particular conditions14. By stripping the memory down to its most crucial components, less detail allows for this information to be applied in different situations outside of the context it was originally learned in15. Forgetting can help tune memories so that they strike an optimal balance of detail retained so that the general
4 | P a g e
information is accurate but is not constrained to only being useable in very specific circumstances16. At the end of the day, the optimal amount of forgetting is dictated by how often the surrounding environment is changing. If the environment is very dynamic, then forgetting helps to relearn more quickly in order to adapt to any changes. If the environment is stagnant, then the persistence and stabilization memories is preferred since information learned is more likely to still accurately predict outcomes in the future. Regardless, the ability to forget helps to tune memories which can optimize the ability to continue learning and better apply information that was previously learned in either new ways, or in novel situations.
Figure 1.2. Overfitting versus generalization of memories: Overfitting happens when a model becomes too specific to the data it was trained on (blue dots) by incorporating a lot of specific
5 | P a g e
parameters, and therefore cannot predict new data (green dots) well. This would be similar to remembering every pattern of soccer ball we had ever seen, and then evaluating if a new object was also a soccer ball by comparing it to the specifics of each individual representation.
However, generalizing would mean forgetting most of the details from previously seen soccer balls, and only remembering the general concept of a sphere made up of hexagons and pentagons. This would allow identification of a much wider array of potential soccer balls.
Image used from Richards & Frankland 20172.
1.1.3 Recent vs. Remote Contextual Fear Memory
How memories are stored and how accurately they can be recalled changes as the length of time relative to memory formation gets longer. Within hours of memory formation, synaptic circuits are locally strengthened in a process known as synaptic consolidation, whereas system consolidation occurs over a longer period of time where brain regions involved in memory become gradually reorganized, which can involve a rearrangement in the circuits that support recall17. There are two different models of how memories are consolidated known as the Standard Model of Systems Consolidation (SMSC) and Multiple Trace Theory (MTT) respectively. SMSC postulates that memories are stored in both the hippocampus and the cortex, but it takes longer for the cortex to be fully able to support the memory, so the hippocampus is required to recall the memory at early stages and acts as an index to teach the cortex18. After some time, the cortex becomes fully able to house the memory on its own, and the hippocampus is no longer required for memory retrieval18. Supporters of this model cite
6 | P a g e
studies that claim to find that remote memories can be recalled without a functioning hippocampus17. Opposed to this is the MTT, which states that memories are broken down into episodic and semantic variants. The episodic version is stored and recalled using the hippocampus, whereas the generalized semantic version is stored and recalled via the cortex18.
Therefore, depending on what type of memory is being recalled, both the hippocampus and cortex are required to recall different versions of the memory depending on how rich in detail it is18. Though the hippocampus has been found to be co-active with many other memory regions even when mice have recall tested 36 days after conditioning8, it has also be shown that episodic memories can still be recalled even with lesions to the hippocampus17.
Figure 1.3. Theories of memory consolidation: Standard consolidation theory postulates that memories completely transfer to being stored in the cortex and become independent of the hippocampus. Multiple trace theory believes that generalized, semantic memories become stored separately from the hippocampus, but detail-rich, episodic memories still require input
7 | P a g e
from the hippocampus when recalled at remote timepoints. Image used from Akers &
Frankland 200919.
1.2 Myelin’s contribution to memory retention
1.2.1 Function of Myelin
When neurons receive sufficient stimulation, signals are transmitted down the axon to the nerve terminal. These action potentials consist of positively charged Na+ ions moving from the axon hillock to the nerve terminal via an electrochemical gradient. The region closest at the axon hillock has a large influx of positive Na+ ions which flow towards the nerve terminal, as regions in this direction have a lower concentration of Na+ ions, and therefore are also less positively charged.
Myelin is an insulating layer of lipids and proteins that wraps around the axons of neurons in discrete segments which help keep the positively charged Na+ ions inside. This insulation means that Na+ ions less able to flow out towards the less positively charged area outside of the axon, and therefore are more likely to travel down the axon towards the terminal. In between the myelin segments are short unmyelinated sections knowns as the nodes of Ranvier. Here, voltage gated channels open to allow an influx of more Na+ ions into the axon, which then travel down the next section of the myelinated axon toward the nerve
8 | P a g e
terminal. This process is known as saltatory conduction (Figure 1.4) and speeds up signal transmission speed20. Building off this, it is no surprise that both a greater surface area coverage and a thicker myelin sheath further increases conductance speeds.
Figure 1.4. Outline of saltatory conduction of an action potential along an axon: Saltatory
conduction allows Na+ ions to flow toward the nerve terminal via the electrochemical
gradient within each myelinated segment of the axon. Upon reaching an unmyelinated node
of Ranvier, voltage gated sodium channels open to allow an influx of Na+ ions, which then
9 | P a g e
flow down the subsequent section of the axon. Image modified from Purves & Williams
200120
1.2.2 Development of Oligodendrocytes and Myelin
Studies have found that neuronal activity promotes oligodendrogenesis from a self- renewing pool of oligodendrocyte precursor cells (OPCs), and that these newly formed oligodendrocytes (OLs) mature over several weeks21 and myelinate active neurons22. How active a neuron is dictates the extent to which the axon is ensheathed and how stabilized the myelin is23,24. However, only ~20% of these newly formed OLs survive, meaning 80% of these developing OLs die before producing long lasting myelin25. Even for the OLs that do successfully develop and begin myelinating, not all myelin extensions are maintained as they are selectively pruned based on how active their associated axon is following initial myelination26. Recent literature has concluded that new myelin is de novo and comes from these newly matured oligodendrocytes, rather than pre-existing OLs27,28. Using electron microscopy, it was discovered that the amount of myelinated axons increased after spatial learning
(representative of new myelin from newly matured OLs covering unmyelinated axons), but the thickness of existing myelin did not change27. The projection of myelin appears to occur during a 5 hour window after differentiation, after which the sheath can be further modified to adjust thickness and coverage, but the OL does not generate any more new myelin projections, only occasional retractions29. In both the human and mouse brain, oligodendrocytes begin developing in utero, but widespread myelination begins after birth as a huge spike of innate myelination that is independent of neuronal activity and is region-dependent30,31. Myelination
10 | P a g e
in the rodent brain peaks at post-natal day 20, whereas humans have the majority of their tracts significantly myelinated between the ages of 3-5, though myelination continues to occur well into adulthood31. In fact, major cognitive milestones correlate with myelination of certain regions later in life such as the left frontal lobe being positively correlated with working memory capacity, and the left temporal lobe with improved reading ability32.
In general, oligodendrocytes born during development are very stable with little turnover (1/300 per year), as the vast majority have been shown to survive throughout adulthood33. Conversely, the myelin sheath produced by oligodendrocytes is slowly, yet constantly exchanged over time33. All myelin is entirely replaced throughout life, though it is not known if this involves retraction and replacement of sheaths, or if the stable sheath is molecularly exchanged while staying in spot33. However, oligodendrogenesis and subsequent de novo myelination still occurs in the adult brain and is quite dynamic depending on the surrounding events being experienced.
11 | P a g e
Figure 1.5. Developmental timeline of oligodendrocytes and myelin: The process of oligodendrocyte production and maturation, ultimately leading to myelination. Image modified from Sampaio-Baptista & Johansen-Berg 201726.
1.2.3 Adult Oligodendrogenesis, Myelin Plasticity, and Memory Retention
Though oligodendrocytes are very stable once produced and rarely turnover, myelination in the adult brain is dynamic and myelinating oligodendrocytes can be produced throughout life as required. As previously stated, active neurons promote the proliferation of
OPCs in the nearby area and induce oligodendrogenesis to ultimately preferentially myelinate the associated active axons22. This holds true in the adult brain throughout life.
In terms of pre-existing OLs and the associated myelin projections in the adult brain, they can be remodelled as activity levels of both individual neurons and regions of networks change. At the level of the oligodendrocyte, no new myelin projections are generated, but sheaths can be retracted if there is a sufficient decrease in activity for the axon that is
12 | P a g e
covered29. However, there can be substantial changes in the myelin sheath itself regarding the coverage and thickness of the sheath. Sheath length is able to increase or decrease based on the activity of the associated axon, but this has been found to be a rare occurrence and primarily happens in younger brains28. Interestingly, if sections of the myelin sheath are ablated, neighbouring sheaths can extend to cover the gap, and even split afterwards to return the myelination pattern back to its original state34. On the other hand, optogenetic stimulation of neurons results in increased wrapping of the myelin which thickens the sheath22.
Relatively recently, recruitment of new OLs and the production of new myelin has been found to be implicated in both learning and memory. This concept makes sense considering that myelin directly impacts the signal transmission speed along neurons, and therefore plays a large role in network synchrony. The ability to form new myelin was shown to be required to learn a new, complex motor skill, where mice learning to run on a wheel with unevenly spaced rungs were unable to run as quickly if they had their myelination halted35. Furthermore, forming new myelin is also required for recall of a remote, but not recent, contextual fear memory21. Transgenic mice that had their myelination halted had reduced freezing when tested
30 days after fear conditioning compared to non-transgenic mice, but had the same amount of freezing if tested 24 hours after conditioning21. Similarly, transgenic mice with halted oligodendrogenesis and myelination had impaired spatial learning in the Morris Water Maze and reduced freezing when tested 28 days following contextual fear conditioning27. It was determined that disrupting oligodendrogenesis and subsequent myelination impaired consolidation of the spatial memories, making them less accessible at remote timepoints.
13 | P a g e
1.3 Physical & Cognitive Enrichments’ Effects on Memory Retention
Physical enrichment is an environment that promotes exercise and provides greater opportunity to engage in motor stimulation. For mice, this involves placing a running wheel in the home cage so that the mice can run freely at any time. Similarly, cognitive enrichment is considered to be an environment that facilitates mental stimulation through problem solving and exploratory behaviour. When an environment has elements of both physical and cognitive enrichment, it is deemed environmental enrichment.
1.3.1 Brain Changes Associated with Physical & Cognitive Enrichment
Body-Wide Effects
Physical and cognitive enrichment have both been shown to cause a multitude of physiological changes that have a body-wide impact including effects on the immune system, and the HPA-axis. Mice housed for 6 weeks in enrichment had increased immune responses compared to mice in standard cages as measured by the activity of natural killer (NK) cells in their spleens36. Natural killer cells are innate lymphocytes that do not require prior exposure to antigens, and release cytotoxic granules to destroy malignant cells37. This study also found that living in enrichment altered the stress response by elevating the baseline serum corticosterone concentration in enriched mice. However, after experiencing a stressful event, corticosterone
14 | P a g e
levels were elevated in control mice but not enriched mice, which was also reflected by lower stress behaviours in the enriched mice36. Similarly, another study found that a Huntington’s disease model in female R6/1 mice had a greater release of corticosterone from their adrenal cortical cells following stimulation by adrenocorticotrophin hormone (ACTH)38. If mice were housed in an enriched environment, this greater release of corticosterone was no longer present both in vivo and in vitro when an artificial ACTH stimulus was given38.
Neuronal Changes
Specifically, regarding the brain, most literature investigating enrichment has focused on how it alters the functioning of neurons and synapses. One major mechanism is by adding new neurons through neurogenesis to introduce new connections. Adult neurogenesis is known to occur in the dentate gyrus of the hippocampus and the subventricular zone, along the walls of the lateral ventricles. Both physical and cognitive enrichment have been found to promote neurogenesis in both of these regions, increasing both the proliferation of neuronal progenitors and their survival39–41. It appears that physical enrichment causes greater proliferation of neuronal precursor cells, while cognitive enrichment promotes their survival41. Interestingly, combining physical and cognitive enrichment together in the form of environmental enrichment has an additive effect on hippocampal neurogenesis, where the amount of new neurons produced is greater than when doing physical enrichment on its own41. Perhaps more importantly, these new neurons in the dentate gyrus have greater excitability (more likely to fire)42 and are innately more plastic (more easily undergo LTP)43.
15 | P a g e
Enrichment has also been shown to strengthen the connectivity between active neurons through long-term potentiation (LTP)3. LTP was enhanced in the Schaffer collateral pathway of the CA1 in the rodent hippocampus following 8 weeks of environmental enrichment3. Neurons in the CA1 region have also been found to have an increased number of dendritic spines from the Schaffer collaterals following enrichment, and that these dendrites have a thicker neck diameter and shorter length44.
Non-neuronal influences
While the effect of enrichment on synaptic factors has been documented, alterations in non-synaptic components are not well elucidated. As stated above, hippocampal neurogenesis is increased following enrichment, but this also recruits other cells such as microglia and T- lymphocytes to promote survival of the newly born neurons45. In fact, mice with compromised immune systems lacking T-cells had reduced neurogenesis in the dentate gyrus which could not be rescued with environmental enrichment interventions45. However, transferring splenocytes containing mature T-cells to these mice restored neurogenesis in the hippocampus45.
Environmental enrichment also induces metabolic changes in the brain where mice that have lived in EE have increased metabolic activation in the prefrontal and frontal cortices, and decreased activation in the baso-lateral amygdala and hippocampus46.
16 | P a g e
Furthermore, both physical and cognitive enrichment induce myelination by causing proliferation of oligodendrocyte precursor cells (OPCs), maturation of OPCs to mature oligodendrocytes, and ultimately the production of new myelin21,22,41. One theory is that enrichment promotes neuronal activity, resulting in the recruitment of new oligodendrocytes and myelin. It was shown that artificially using optogenetics to activate motor neurons in the premotor cortex caused OPC proliferation and maturation in the surrounding area and thickening of myelin along those axons22. Steadman et al., 202027 found that this myelination is de novo and comes from newly matured oligodendrocytes rather then additional myelin being produced by pre-existing OLs. MRI analysis of environmental enriched brains has shown increases in both white and grey matter volume relative to mice housed in standard cages in a number of regions related to sensorimotor function and spatial navigation including the hippocampal formation and sensorimotor cortex47. Increases in grey matter is indicative of increases in neurons and oligodendrocytes as these cell bodies primarily make up these regions.
Increases in white matter reflects a greater amount of myelin whether on pre-existing axons or newly formed ones.
As discussed before, increasing oligodendrogenesis and subsequent myelin production would also result in different axons becoming myelinated, which would alter network synchrony. A mathematical model has predicted that conduction delay of 1ms would significantly affect signal amplitude and phase coherence by shifting the phase by 30°48.
17 | P a g e
Another network model using large-scale primate white matter suggests that white matter plasticity is sufficient for self-organization of brain oscillations49.
1.3.2 Physical & Cognitive Enrichments’ Effect on Memory Recall
When assessing behavioural differences after living in enrichment, most studies focus on contextual fear and spatial memories, as these are hippocampus dependent. When mice are housed in physical enrichment before being contextually fear conditioned, they freeze significantly more than their standard cage counterparts when tested 1 day after training indicating improved memory retention 50. However, this effect is no longer present if the delay between conditioning and testing is 7 or 21 days later 50. On the other hand, if physical enrichment occurs after contextual fear conditioning has already occurred, it decreases memory retention as seen through reduced freezing levels10,51. However, if there is a delay of
28 or 56 days after conditioning before physical enrichment is initiated, it no longer causes forgetting51. These results have been interpreted that enrichment induces changes that impact memory consolidation, and therefore memory recall. Neurogenesis has been thought to be one of the main factors driving the differences in memory retention from living in the different enriched environments, hence why a lot of studies look at behaviours involving the hippocampus. If a memory has already been formed, then adding new neurons to a network disrupts the connectivity within engrams and makes it less likely to be reactivated properly10,12,51
18 | P a g e
When mice live in this environmental enrichment paradigm before contextual fear conditioning, comparable to physical enrichment, it improves memory retention and mice freeze more after being conditioned and tested3. However, it has yet to be tested how initiating environmental enrichment after a memory has already been formed affects memory retention.
This would inform how environmental enrichment, and more specifically cognitive enrichment, influences retention of a pre-existing memory.
1.4 Rationale
It has been discovered that physical and cognitive enrichment induce many similar changes in the brain including increases in neurogenesis, oligodendrogenesis, and myelination.
Neurogenesis in particular has been studied and found to be a main contributor behind physical enrichment’s impact on pre-existing contextual fear memories by disrupting the engram to cause forgetting. While cognitive enrichment induces similar changes, it is unknown if or how it alters the retention of memories after engram formation has occurred. Even though both forms of enrichment share many potential mechanisms to modulate memory retention, it is not known if they both influence memories in the same ways, and if they differ in the magnitude of change induced. The contribution of oligodendrogenesis and myelination facilitated by enrichment has not been explored to a great degree, and it is not well known how it contributes to forgetting, or how it impacts memories in general.
19 | P a g e
1.5 Objectives & Hypotheses
This study focuses on investigating 2 objectives and hypotheses:
Objective 1: Assess how physical and cognitive enrichment impact contextual fear memory retention if initiated AFTER conditioning, and if this differs from enrichment BEFORE conditioning.
• Hypothesis 1: Housing mice in both physical and cognitive enrichment environments in
the interval between conditioning and testing will hinder their ability to recall the
contextual fear memory. In contrast housing in physical and cognitive enrichment
environments before conditioning will result in improved memory recall.
Objective 2: Specifically investigate the contribution of new myelin to disrupted fear memory retention.
• Hypothesis 2: Blocking the ability to form new myelin after conditioning will reduce the
enrichment-induced impairment of memory retention, resulting in partially rescued
freezing levels.
20 | P a g e
Chapter 2
2 Materials & Methods
2.1 Experimental Designs
2.1.1 Housing Before/After Conditioning Timeline & Details
Housing AFTER Conditioning
To investigate the impact of different forms of enrichment on the recall of a pre-existing contextual fear memory, normal C57/B6 mice were conditioned and then housed in either running wheel (RW, n = 18), environmental enrichment (EE, n = 18), or standard environment cages (SE, n = 27) for 28 days. For SE mice, they were transferred into a fresh standard cage following conditioning to control for the effect of being moved into a new cage. After 28 days of the respective housing, mice were tested and then perfused the following day (Figure 2.1). 8 mice in each housing group received 5-Ethynyl-2´-deoxyuridine (EdU) water for 7 days starting immediately after conditioning.
Housing BEFORE Conditioning
To assess if the timing of the housing intervention relative to memory formation mattered, normal C57/B6 mice were housed in either running wheel (n = 17), environmental enrichment (n = 18), or standard environment cages (n = 18) for 28 days before being contextually fear conditioned. SE mice were transferred into a new SE cage at the same time
21 | P a g e
their enriched counterparts started their housing paradigms to account for cage change effects.
Mice were then tested 1 day after conditioning and perfused 1 day after testing (Figure 2.1). 8 mice in each housing group received EdU water for 7 days starting immediately after conditioning.
Figure 2.1. Enrichment after and before conditioning study designs: Timeline of enrichment relative to conditioning based on experiment type. In both experiments, mice were housed in enrichment for 28 days.
2.1.2 Recombination Efficiency & Toxicity Assessment
Prior to starting experiments with the MyRF mice, the dosage of tamoxifen and mouse strain were evaluated to determine if the dosage could be lowered to reduce mortality while maintaining the same level of recombination efficiency. Dosages of 180mg/kg and 100 mg/kg were tested in both MyRF-NG2 and MyRF-PDGFrα strains. The two mouse strains insert the cre- recombinase gene in either the NG2 or PDGFr푎 loci which both result in the cre expression
22 | P a g e
being localized to the OPC population. 6-week-old females homozygous for floxed MyRF were injected with tamoxifen over 3 days, followed by 7 days of EdU to label new, proliferating cells.
Mice were perfused 21 days after the cessation of EdU water and their brains were collected for immunohistochemistry. Brains were stained for EdU (newly proliferated cells), CC1 (mature oligodendrocytes), and DAPI (cell nuclei). EdU+ cells were quantified using ImageJ to assess which experimental parameters best prevented the maturation of new OLs.
Figure 2.2. Recombination efficiency study design: Timeline of the experiment involving transgenic mice in either SE or EE cages to assess the contribution of myelin in causing behavioural differences between groups.
2.1.3 MyRF:NG2-ERT:Cre Mice Enrichment Timeline & Details
MyRF mice were fear conditioned and then immediately injected with tamoxifen (~2 mins later, 180mg/kg dosage), followed by 2 more injections over the following 2 days. Mice were put in either EE (n = 24) or SE (n = 29) cages immediately after the first tamoxifen injection for 28 days before being tested. In both housing conditions, all mice were administered EdU water for 7 days starting 24 hours after the final tamoxifen injection.
23 | P a g e
Figure 2.3. MyRF EE experiment study design: Timeline of the experiment involving transgenic mice in either SE or EE cages to assess the contribution of myelin in causing behavioural differences between groups.
2.2 General Methods
2.2.1 Mice
Experiments were conducted using 8-week-old mice kept in ventilated cages with 1-4 cage mates. Mice had free access to food and water while kept in standard laboratory conditions (21 ± 1°C, 50 ± 5% humidity, and 12 hr light/dark cycle) at The Centre for
Phenogenomics (TCP). All procedures were performed in compliance with the Animals for
Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care. All procedures conducted on animals at TCP were reviewed and approved by the TCP Animal Care
Committee.
Two strains of mice were used - C57/B6 mice (Jackson Laboratories, Bar Harbor, ME) and transgenic MyRF:NG2-ERT-Cre;F1 (MyRF) mice (Ben Barres, Stanford, CA) from a C57/B6 x
24 | P a g e
129 background. These MyRF mice have floxed MyRF alleles between loxP DNA recognition sites, meaning they can be removed by cre DNA recombinase. The MyRF mice have tamoxifen- inducible cre recombinase sequences inserted into the neuron/glial antigen 2 (NG2) locus which allows the cre to be localized to the OPC population. When tamoxifen is administered to these mice, it enables the cre to move into the nucleus to excise the MyRF alleles and thus inhibit production of MyRF in OPCs. This ultimately prevents OPC differentiation into OLs, which ceases further myelination but leaves pre-existing OLs and myelin unaffected.
2.2.2 Tamoxifen & EdU Administration
30mg aliquots of tamoxifen were dissolved in 0.1ml of 100% ethanol and 0.9ml of sunflower oil, and then injected intraperitoneally once a day for 3 consecutive days at a dosage of 180mg/kg to induce recombination. EdU was administered via drinking water for 7 days to label proliferating cells by integrating into newly synthesized DNA. EdU water contained
0.2mg/mL EdU and 1% dextrose and was remade after 3-4 days.
2.2.3 Housing Conditions
Standard Enrichment Cages (SE)
Standard enrichment (SE) cages consisted of a ventilated, conventional cage
(Techniplast, Buguggiate, Italy) containing a food hopper and a nesting packet. 4-5 mice were housed together in a single, sex-separated cage and bedding and nesting material was changed weekly.
25 | P a g e
Running Wheel Cages (RW)
Fresh standard cages were equipped with a non-metred running wheel (Bio-Serv,
Flemington, NJ) which mice could voluntarily run on at any time during the duration of this housing condition. The running wheel was shared amongst the mice living in the cage, and the bottom of the running wheel also doubled as a housing dome. Bedding and nesting materials were changed weekly.
Environmental Enrichment Cages (EE)
Environmental enrichment (EE) cages consisted of a double decker cage (Techniplast,
Buguggiate, Italy) containing 3-tiered maze layouts that were designed and previously used in
Scholz et al., 2015. Food was placed at the top, and water at the bottom of the maze layouts, and a non-metred running wheel (Bio-Serv, Flemington, NJ) was located on the second floor of the maze which mice had free access to. The bedding and configuration of EE cages were changed every 7 days following the same layout timeline over 28 days (layouts A → C → F → G as used and described in Scholz et al., 201547).
26 | P a g e
Figure 2.4. Diagram of different housing conditions: (A) Icons depicting the three forms of cages used – Standard Environment (SE) acting as controls, Running Wheel (RW) for physical enrichment, and Environmental Enrichment (EE) which combined physical and cognitive enrichment together. (B) An example layout of one of the three-tiered mazes used in the EE cages. The layouts and associated diagram is borrowed from Scholz et al. 201547.
2.2.4 Contextual Fear Conditioning
Mice were brought to the TCP Neurobehaviour Core anteroom to acclimate for 30 mins prior to both conditioning and testing sessions. The contextual fear conditioning behavioural test assesses the ability to recall a contextual fear memory. Conditioning and testing sessions were done using the Video Freeze® fear box apparatus and software (Med Associates Inc,
Fairfax, VT) in the Neurobehaviour Core of TCP. Prior to conditioning, the fear box was wiped
27 | P a g e
with 70% isopropyl alcohol to act as an olfactory cue. Mice were placed in the box and left for 2 minutes before receiving a the first of 3 unsignaled foot shocks of 0.75mA with a 2 second duration (Figure 2.4). After a shock, 58 seconds passed before the next shock was administered.
After the final shock, mice were left in the fear box for 1 minute before being removed, resulting in a total session time of 5 minutes.
Figure 2.5. Contextual fear conditioning protocol: Contextual fear conditioning timeline and shock settings
Testing consisted of placing the mice back in the fear box with the olfactory scent (70% isopropyl) for 5 minutes with no shocks being delivered. Fear memory recall was assessed by measuring the amount of time the mice spent freezing, which is a known fear response that indicates the mice remember being shocked in that context. A reduction in time spent frozen indicates forgetting of the contextual fear memory. The amount of freezing was measured by the Video Freeze® software which has been shown to be consistent with manual scoring 52.
Mice were considered frozen if ceasing all movement (other than breathing) as assessed by changes in pixel composition on screen (motion threshold = 18) for a minimum of 30 frames (1 second).
28 | P a g e
2.2.5 Perfusions
Brains were collected and prepared for future neuroimaging experiments that are not included in this thesis. Mice were anesthetized using ketamine/xylazine (150mg/kg, 10mg/kg) and perfused through the heart with phosphate-buffered saline (PBS) and heparin, followed by paraformaldehyde (4% PFA) and 2mM ProHance (Bracco Diagnostics Inc., Milan, Italy). Skulls were detached from the bodies, and the skin, lower jaw, eyes, and ears were removed. The remaining skull and brain were postfixed in 4% PFA and 2mM ProHance at 4°C for 24 hours, and then transferred and kept in a PBS, 0.01% sodium azide, and 2mM ProHance solution for at least 1 month before being imaged.
2.2.6 Immunohistochemistry
Prior to cryosectioning, brains were removed from the skulls and placed in 15% sucrose for 24 hours followed by 30% sucrose and sodium azide for at least 48 hours. Cryosectioning occurred at -19°C to produce 40μm coronal sections that were immediately mounted on slides.
For the purpose of assessing which mouse line and tamoxifen dosage resulted in the highest recombination efficiency, the number overlapping EdU+ (newly proliferated cells) and CC1+
(mature oligodendrocyte marker) were counted. The corpus callosum was used as the region of interest because it is a large, white matter structure, which means that it is an area rich with oligodendrocytes and myelin. The corpus callosum is important for interhemispheric communication in the brain, and has been implicated in memory storage before53–55.
29 | P a g e
Staining began with antigen retrieval in sodium citrate buffer (pH 6) for 40 minutes, followed by a 15-minute wash in 0.1% Triton X-100 (Bio Basic Inc., Markham, Canada). EdU click-it was then used to stain EdU+ cells. Afterwards, non-specific binding sites were blocked with 5% normal donkey serum for 1 hour and sections were incubated overnight at room temperature with anti-CC1 (1:200, mouse polyclonal, Millipore, Burlington, MA), while omission of the primary antibody was used as the negative control. After 24 hours, a Cy3 conjugated donkey anti-mouse secondary antibody (1:200, Jackson ImmunoResearch, PA) in 1% normal donkey serum was applied to the sections for 2 hours in the dark. Sections were then stained with DAPI (1:10,000 in RO water) for 10 minutes, washed for 10 minutes with 0.1M PB (no salt), and then mounted onto slides for storage at 4°C.
2.2.7 Statistical Analysis
Linear mixed effects models were used to examine the effects of the different experimental conditions on freezing during the CFC test. The provided output from these models includes the intercept (β) which represents the mean value of freezing for the reference group, the unstandardized beta coefficients (Δβ) that indicate how much a change in the independent factors changes the dependent variable, and the p-value (p) that indicates statistical significance as calculated using Satterthwaite’s method. A random cage effect was included in each of the models to account for any variance caused by being in individual cages.
30 | P a g e
The percentage of time frozen during the first 3 minutes of testing was used to calculate differences as to avoid incorporating changes in extinction behaviour.
Testing for Effect of Housing Type in After & Before Conditioning Experiments
Group differences in freezing behaviour for both the enrichment before/after experiments were analyzed by analyzing the main effects of housing type (eq. 1).
(1) Freezing % = βSE + ΔβEE + ΔβRW + γCage
Testing for Effect of Housing Type in MyRF EE Experiments
Data from the MyRF EE experiments were analyzed by assessing the main effects of housing type and cre status to assess their influence on freezing behaviour (eq. 2). The interaction term between housing type and cre status was not significant, so it was removed, and the main effects were reported.
(2) Freezing % = βMyrfSE-Cre- + ΔβMyrfEE + ΔβCre+ + γCage
31 | P a g e
Testing for Sex Differences
The after and before conditioning experiments were separately evaluated by examining the interaction between housing type and sex (eq. 3) to assess if there were any sex differences in the freezing behaviour and if it depended on the housing type. We looked at both the main effect and interactions containing sex to see if they were significant.
Freezing % = βSE-SexF + ΔβEE + ΔβRW + ΔβSexM + ΔβEE-SexM + ΔβRW-SexM + γCage (3)
The MyRF EE experiment was analyzed in a similar manner but also included cre status as a factor as seen in equation (4). The 3-way interaction, 2-way interactions, and the main effects involving sex were all evaluated for significance.
Freezing % = βMyrfSE-SexF-Cre- + ΔβMyrfEE + ΔβCre+ + ΔβSexM + ΔβMyrfEE-Cre+ + (4) ΔβMyrfEE-SexM + ΔβCre+-SexM + ΔβMyrfEE-Cre+-SexM + γCage
32 | P a g e
Chapter 3
3 Results
3.1 Freezing when Enrichment Occurs Before & After Conditioning
3.1.1 Enrichment After Conditioning Causes Forgetting of Contextual Fear Memories
A linear mixed effects model accounting for random cage effects revealed that mice housed in both EE (β = -53%, p = 2 x 10-6) and RW (β = -20%, p = 0.006) cages froze significantly less than mice housed in SE housing if enrichment is initiated after conditioning (Figure 3.1).
Interestingly, though not surprising, EE cages that combined cognitive and physical enrichment caused greater forgetting than physical enrichment alone, as mice housed in EE cages froze significantly less than RW mice (β = -33%, p = 0.0004). Cognitive enrichment seems to have an additive effect when combined with physical enrichment to further disrupt the contextual fear memory engram and cause greater forgetting.
33 | P a g e
Figure 3.1. Reductions in freezing behaviour when enrichment occurs after fear conditioning:
If enrichment is initiated after conditioning, EE and RW mice freeze significantly less than SE mice, and EE mice also freeze significantly less than RW mice. Results are shown as medians with first and third quartiles, and whiskers indicating minimum and maximum values. If plots contain outliers, whiskers are drawn as 1.5 * IQR
3.1.2 Timing of Enrichment Relative to Conditioning Causes Different Changes to
Memory Retention
To examine if the effect of enrichment on reduced freezing during testing was due to non-specific effects of enrichment in the month leading up to the test, we housed mice in
34 | P a g e
enrichment for 28 days before conditioning. Not only was freezing not reduced in these conditions, but it was actually enhanced, as mice housed in EE (β = 26%, p = 0.0001) and RW (β
= 21%, p = 0.002) cages before conditioning froze significantly more than their peers in SE cages
(Figure 3.3). However, mice housed in EE cages froze the same amount as those in the RW cohort (β = 5%, p = 0.4), meaning there was no longer an additive effect when combining cognitive and physical enrichment together in the EE cages before conditioning.
Figure 3.2. Increased freezing behaviour if enrichment is shifted to occur before conditioning:
In contrast to the effect of enrichment after conditioning, EE and RW mice freeze more than
35 | P a g e
SE mice if enrichment is conducted before conditioning. Results are shown as medians with first and third quartiles, and whiskers indicating minimum and maximum values. If plots contain outliers, whiskers are drawn as 1.5 * IQR.
3.1.3 No Sex Effect When Comparing After & Before Conditioning Groups
Two linear mixed effects models that included sex and random cage effects were run to assess if there were sex differences in freezing behaviour as visualized in Figure 3.3. For enrichment after conditioning, there was no main effect of sex (β = -6%, p = 0.5) or any interactions between housing type and sex when comparing SE-EE (β = 14%, p = 0.4), SE-RW (β
= -4%, p = 0.8), or EE-RW (β = -18%, p = 0.3). Enrichment before conditioning had similar results with no significant main effect of sex (β = -5%, p = 0.6), or any interactions between housing type and sex when comparing SE-EE (β = -0.02%, p = 0.999), SE-RW (β = -3%, p = 0.8), or EE-RW
(β = -3%, p = 0.8). Since there were no significant sex differences, sexes were pooled and sex was excluded from the final models. This matches literature which has also found that there are no sex differences in freezing behaviour following contextual fear conditioning21,51,.
36 | P a g e
Figure 3.3. No sex differences in freezing behaviour: There were no sex differences affecting freezing behaviour between groups or between the timing of housing. Results are shown as medians with first and third quartiles, and whiskers indicating minimum and maximum values.
If plots contain outliers, whiskers are drawn as 1.5 * IQR.
37 | P a g e
3.2 Recombination Efficiency
3.2.1 Cell Counts of Newly Proliferated Cells (EdU+)
To investigate the contribution myelin has, we used transgenic MyRF mice and first assessed if we could lower the tamoxifen dosage to reduce toxicity in our mice while maintaining an adequate level of recombination efficiency. Since tamoxifen administration in
MyRF mice should excise their MyRF gene, thereby preventing OPC maturation into new OLs, recombination frequency would be indicated by a lower number of these newly generated cells as labelled by EdU. Figure 3.4 reveals that NG2 mice given the 180mg/kg dosage of tamoxifen seem to have the best recombination efficiency.
3.2.2 Mouse Strain
Figure 3.4 reveals that NG2 mice had better recombination efficiency compared to
PDGFrα mice as PDGFrα appeared to have non-specific recombination occurring where cre- negative mice also had lower EdU+ counts. NG2 was then selected as the mouse strain to use for future MyRF mice experiments.
3.2.3 Tamoxifen Dosage
The dosage of 180mg/ml resulted in better recombination efficiency compared to the
100mg/ml dosage. Within NG2 mice, cre-positive mice given the 100mg/kg dosage had higher
38 | P a g e
amounts of EdU+ cells present compared to mice given the 180mg/kg dose, though we did not test for statistical significance.
Figure 3.4. Averaged counts of EdU+ cells in the corpus callosum from different mice: 3 counts from different regions of the corpus callosum in the same section were averaged together to generate one single value. EdU+ cells represent newly proliferated cells, which are assumed to be oligodendrocytes in the corpus callosum.
39 | P a g e
3.3 MyRF Mice Experiments
3.3.1 Freezing Behaviour Differences Between EE and SE
A linear mixed effects model estimating freezing behaviour and accounting for random cage effects revealed that there was no interaction between housing type and cre status (β =
-3%, p = 0.8), and no main effect of cre status (β = -4%, p = 0.6), meaning that halting myelin in these mice did not cause any changes in freezing behaviour. However, the effect of housing type was still approaching significance as mice in EE froze less than mice in SE (β = -12%, p =
0.07). There were also no interactions between housing type, cre status, and sex (β = -16%, p =
0.4), cre status and sex (β = 1%, p = 0.9), or housing type and sex (β = 6%, p = 0.7). There was also no main effect of sex (β = 12%, p = 0.3) so sex was not included in the final model.
40 | P a g e
Figure 3.5. Freezing behaviour exhibited by MyRF mice injected with tamoxifen in either SE or EE cages: Mice housed in EE cages froze significantly less than mice in SE cages. However, cre-status had no significant effect, as both cre-positive and cre-negative mice froze the same amount within the same housing condition.
41 | P a g e
Chapter 4
4 Discussion
4.1 Objective 1 – Effect of Different Types of Enrichment on
Contextual Fear Memory Retention & the Importance of Timing of
Conditioning
4.1.1 Effect of Cognitive Enrichment After Conditioning on Contextual Fear Retention
While the effect of physical enrichment after conditioning on contextual fear memory retention has been previously investigated10,51, this appears to be the first study to assess how cognitive enrichment contributes to altering contextual fear memory retention after conditioning has already occurred. It is not surprising that animals housed in environmental enrichment after conditioning had reduced fear memory retention similar to animals housed in physical enrichment. It would be expected that both housing types would elicit similar changes in freezing behaviour since both forms of enrichment cause similar physiological and neurological changes. However, it is interesting to see that cognitive enrichment has an additive effect when combined with physical enrichment, as mice in environmental enrichment cages froze significantly more than mice housed solely in physical enrichment. This finding aligns with
Fabel et al., 200941 which found that cognitive and physical enrichment induce different, yet
42 | P a g e
complimentary changes to neurogenesis which result in an additive effect when combined together. Neurogenesis as a mechanism causing increases in forgetting is discussed further below.
4.1.2 Comparison of the Effects of Enrichment at Different Timepoints Relative to
Conditioning
The fact that environmental enrichment causes opposing changes in fear memory retention depending on when it occurs relative to conditioning makes sense since physical enrichment alone follows that same pattern. Again, since physical enrichment reduces freezing if done after fear conditioning, but increases freezing if beforehand, then it makes sense that environmental enrichment has the same effect since it includes physical enrichment, and because cognitive enrichment induces similar brain changes by impacting neurogenesis and oligodendrogenesis, albeit via different methods. Therefore, it is expected to see that these two housing paradigms facilitate a similar change in freezing behaviour when done at the same time relative to conditioning and the subsequent engram formation.
43 | P a g e
4.1.3 Potential Mechanisms Underlying Changes to Memory Retention
Potential Contribution of Neuronal Factors
Hippocampal neurogenesis has already been found to contribute to forgetting when induced by voluntary exercise10,51, which aligns with literature since the hippocampus is a major brain region responsible for encoding and storing contextual memories2,18,19. Therefore, since cognitive enrichment also increases hippocampal neurogenesis, it is plausible that living in EE cages disrupts the engram in a similar fashion, resulting in hampered contextual fear memory recall. Disruption could arise in the form of new connections being created that become integrated with neurons in the engram. On top of this, adult-born neurons have been found to be more excitable with higher firing rates42, and more likely to undergo long-term potentiation43. Though newly formed neurons are hyperexcitable themselves, the addition of these new neurons actually supresses overall activity in the dentate gyrus by recruiting interneurons56,57. These two characteristics combined makes it more likely that new neurons may form connections with those in the engram, while the overall strengthening of the engram is reduced due to the mature neurons in the engram being inhibited and less active. Therefore, the connectivity pattern that is required to recall the contextual fear memory would not stabilize properly, resulting in a new pattern that does not match the engram as accurately.
Since this neurogenesis is initiated immediately following engram formation, it would be disruptive to the ever important period of stabilization required for consolidation, and explains why enrichment does not hinder memory recall if done after a long enough delay after the engram has already been stabilized as seen in Gao et al, 201651.
44 | P a g e
On the other hand, housing mice in enrichment before conditioning occurs causes them to freeze more when tested. Neurogenesis and other synaptic changes certainly contribute to this change in behaviour, and the onset of these explain the opposite effect it has when compared to enrichment after conditioning. Whereas new neurons start being generated after the engram has been formed if enrichment comes after conditioning, new neurons generated through enrichment before conditioning are available to be recruited for the engram. Coupled with the fact that new neurons are more excitable42 and more likely to undergo LTP43, this would mean that engrams including these neurons would have increased connectivity and become stabilized to a greater degree. In fact, neurons with increased excitability are preferentially recruited during engram formation58. Though the pre-existing neurons in the dentate gyrus are less active following an influx of new neurons, this may improve engram stabilization by further exaggerating the difference in excitability between old and new neurons, leading to greater usage of new neurons. It has been speculated that this helps pattern separation by reducing the number of overlapping neurons shared between different memories that can cause interference59.
There is also an additive change in fear expression when physical and cognitive enrichments are combined within EE cages, as a previous study has found that cognitive and physical enrichment promote hippocampal neurogenesis in different ways. Physical enrichment induces greater proliferation of neuronal precursors, whereas cognitive enrichment (in the
45 | P a g e
form of mazes and toys) increases the number of neurons that survive to be long-lasting41. Mice that lived in a combination of physical and cognitive enrichment ultimately had an increased number of hippocampal neurons compared to mice only living in physical enrichment41. Hence, it makes sense that combining the two forms together within EE would have an additive effect on behaviour since running increases the number of precursors available, and then the cognitive enrichment causes more of those accessible precursors to be maintained for long- term. Our study builds off this to show how this change in hippocampal neurogenesis impacts contextual fear expression.
It is curious as to why EE does not provide a greater improvement in memory compared to physical enrichment alone when conducted before conditioning. Perhaps the beneficial effects of hippocampal neurogenesis before conditioning experiences a ceiling effect and being in physical enrichment for a month reaches the maximum amount of improvement. Therefore, even though EE may have greater overall potential to impact memory performance, it may not cause a difference in freezing behaviour because memory retention has already improved to its upper limit.
Potential Contribution of New Oligodendrocytes
As previously stated, both physical and environmental enrichment have been shown to promote oligodendrogenesis and facilitate the formation of new myelin. Stabilization of the engram depends in part on myelination, as remote contextual fear memories are unable to be
46 | P a g e
recalled if oligodendrogenesis is blocked during the consolidation period27. Therefore, it can be assumed that myelination patterns following learning play a large role in assuring proper stabilization of the associated engram. If oligodendrogenesis is induced following engram formation, there may be excessive myelination within the engram where different sections receive uneven amounts of this new myelin. Therefore, the original ratios of myelin would not be maintained across all regions and some connections would become quicker than they originally were relative to the rest of the network. Ultimately this would disrupt network synchrony as some regions of the engram would now be transmitting parts of the signal faster than before, degrading the original, precisely coordinated pattern that was meant to be preserved.
In terms of enrichment improving memory retention if done prior to conditioning, facilitating the maturation of OPCs into oligodendrocytes that can begin myelination speeds up the process by which the engram can become myelinated. If enrichment occurs before conditioning, by the time conditioning happens the brain will already have oligodendrocytes immediately ready to begin myelinating the new neurons that will be recruited in the contextual fear engram. Since myelination has been found to be de novo27 and therefore requires new oligodendrocytes to be produced, having the OLs accessible ahead of time in preparation for engram stabilization may contribute to the improvement in memory recall.
47 | P a g e
4.2 Objective 2 - MyRF Mouse Model & Specific Contributions of
Myelin to Contextual Fear Memory Recall
4.2.1 Efficiency of Recombination in the MyRF Mouse Model
Immunohistochemistry was conducted to evaluate the number of EdU+ cells in different regions of the corpus callosum. The corpus callosum is a large white matter tract, meaning the vast majority of the cell bodies found here belong to myelinating oligodendrocytes. Since EdU becomes integrated into newly synthesized DNA, it labels newly proliferated cells and allows us to get a sense of newly formed oligodendrocytes. This allowed us to compare how well different tamoxifen dosages and mouse lines excised MyRF alleles to ultimately halt further oligodendrocyte production and subsequent myelination. Our results found that the NG2-cre line with a dosage of 180mg/kg had the best recombination efficiency. This is the same mouse line used in other studies exploring the effect of halting myelination21,27. Using the lower dosage of 100mg/kg resulted in slightly higher EdU+ cell counts in NG2-cre mice, and therefore was not chosen to proceed with. When it came to the PDGFrα strain, there seemed to be non- specific recombination occurring where cre-negative mice also had decreased amounts of EdU+ cells. In addition, this PDGFrα strain was bred from a hemizygous for floxed MyRF x homozygous for floxed MyRF background, and we received genotyping results claiming the one of the offspring contained no floxed MyRF. Therefore, we decided not to proceed with the
PDGFrα line due to these issues. All in all, the model using the NG2-cre strain with a 180mg/kg
48 | P a g e
tamoxifen dosage has been found to successfully halt further myelination from occurring by preventing OPC proliferation, but leaving pre-existing OLs and myelin intact21,27.
4.2.2 Evidence of Myelin’s Contribution to Memory Retention Modulation
We hypothesized that the reduction in contextual fear memory recall was partially caused by an increase in myelination occurring. The idea was that an increase in myelination following engram formation would cause some connections to become myelinated, and therefore faster, resulting in desynchrony of the network. Hence, we hypothesized that specifically halting myelination from occurring would somewhat counteract the effect of living in EE housing, though not fully since neurogenesis would still be happening as well. However, while in the midst of running said experiments, two papers came out which shifted what we expected to discover from this part of the project. First, it was shown that the ability to form new myelin was required to maintain a remote memory as transgenic MyRF mice were injected with tamoxifen either 5 days or 3 weeks prior to being fear conditioned21. Once contextually fear conditioned after their oligodendrogenesis and myelin production had been halted, the
MyRF mice froze when tested 1 day later, but no longer froze 30 days later21. This result suggests that myelin is required for the storage of remote, but not recent, memories. This can be further explained since the process of myelination takes days to assess axonal activity and adapt accordingly, so though the initial myelin projections would have begun by 24 hours, it makes sense that myelin does not have an immediate impact during synaptic consolidation as it
49 | P a g e
takes longer to develop and make a significant impact26. Furthermore, a second study found that injecting tamoxifen immediately after spatial memory training resulted in the memory still being accessible 1 day later, but not 30 days later27. This led the authors to attribute this effect to myelin’s contribution being critical during memory consolidation. This was further supported when tamoxifen was injected 25 days after training (just 3 days before testing), meaning myelination would be halted after the consolidation period had been completed.
When mice were tested, their spatial memory was not impacted, providing further proof that myelin’s role comes during memory consolidation.
Therefore, the results from our experiments followed suit where administering tamoxifen to halt myelination after conditioning does not result in a difference in freezing behaviour if tested 1 day after conditioning21. We would assume that if we were to add a longer delay between conditioning and testing, we would see memory deficits and a drastic reduction in freezing. Even though the difference is not significant, we are beginning to see this trend where cre-positive mice who have had their myelination halted, do freeze less than their cre- negative counterparts regardless of housing type. Though we did not find that halting myelination rescued freezing behaviour for enriched mice, we did see the beginning of a trend that supports that myelin contributes to memory consolidation and the storage of remote memories.
50 | P a g e
4.3 Limitations & Future Directions
4.3.1 Housing Conditions & the Isolation of Cognitive Enrichment’s Impact
Experiments were limited by the fact that EE cages combined both cognitive and physical enrichment, so we were unable to specifically elucidate the role of cognitive enrichment on its own. One inherent problem is that using larger cages to allow for mazes/toys also provides more space to allow for more movement in the cage. The inclusion of toys also promotes playful behaviour which further increases levels of exercise. Future studies should create cages that isolate cognitive enrichment by controlling for amount of exercise. One such method may be to house mice in the EE cages with the reconfigurable maze and no running wheel. However, one set of cages would have the maze layout changed weekly, whereas the other set would remain in the same configuration for the entire duration of the study.
4.3.2 1 Day vs 28 Day Delay Between Conditioning & Testing
The reason for having the shorter delay between conditioning and testing in the
“before” timeline is that this experiment was primarily done to show that living in enrichment does not innately reduce memory recall and the elicited fear responses. We focused on controlling the amount of time spent in enrichment (28 days) to prove that enrichment itself was not causing forgetting of the contextual fear memory. This discrepancy accounts for the difference in the SE control groups’ freezing levels where the “before” group (~32%) froze less
51 | P a g e
than the “after” SE group (~72%). While “before” SE mice were still freezing more than the level at which unconditioned mice freeze (0-5%4,21,50), the reduction relative to the “after” SE group can be attributed to the concept of fear incubation which states that fear responses to cues and context are time-dependent and increase as time passes60,61. Our results align with literature that use a 1 day delay between training and testing where standard housed mice have reductions in the amount of time frozen for50,61.
4.3.3 Tamoxifen Injection Start Time & Halting of Oligodendrogenesis
We elected to start injecting tamoxifen immediately following conditioning in order to not begin halting myelination until after the memory had been formed and to avoid the presence of aversive side effects during conditioning due to toxicity of tamoxifen.
Unfortunately, tamoxifen does take up to 3-4 days to reach maximum recombination efficiency, so there is still partial ability for OPCs to proliferate and differentiate into OLs during those first few days. Tamoxifen levels in the brain peak 8 hours after injection, and reach 20-40% recombination efficiency depending on brain region62. In Steadman et al., 202027, which used the same transgenic NG2-cre mouse line, they found that there was 70% recombination efficiency 4 days after tamoxifen injection. Until a quicker method is developed, this issue is inevitable for experiments analyzing how oligodendrogenesis and myelination impact memories after they have been formed. Even if a smaller dosage is achieved to limit the toxic side effects so that behaviour is not affected during training (which we were unable to do
52 | P a g e
without also decreasing recombination efficiency), the initial injection would still need to occur after conditioning was finished in order to not have partial recombination overlapping with memory formation itself.
4.3.4 Novelty of Enrichment & Type of Memory
Another avenue to explore would be how novel enrichment needs to be from the subject’s usual lifestyle to cause a significant difference. For instance, if a mouse were already exercising regularly, would increasing the amount of activity by 10% still increase hippocampal neurogenesis and result in a change in freezing? How different does the new housing condition need to be in order to still generate a significant difference? If there is a relationship between the novelty of enrichment, it can also be assessed if it is linear, exponential, etc. This also brings up the question of the duration of effect of enrichment. Does enrichment indefinitely increase neurogenesis and myelination and increase the baseline levels, or does the brain eventually accept the new conditions as a new baseline and revert to a reduced, “normal” rate of neurogenesis/oligodendrogenesis?
Furthermore, we only tested the retention of a contextual fear memory which is an aversive experience. Would enrichment have the same effect on memory recall if the memory were appetitive? If so, it would be interesting if appetitive memories were more resistant to being forgotten following enrichment. To test this, a similar experiment could be run except
53 | P a g e
using appetitive context conditioning to form this different type of contextual memory with a different connotation associated with it.
4.3.5 Implications for Pathologic Myelin Diseases & Military PTSD Research
Lastly, these results bring up questions regarding the fact that exercise and enrichment help promote both neurogenesis and myelination. It is reasonable to believe that there may be beneficial applications for those with neurodegenerative diseases. For instance, it has been shown that exercise has beneficial effects for those suffering with multiple sclerosis (MS), which is when the immune system attacks and degrades myelin in the central nervous system.
Unfortunately, the MS population is less likely to engage in exercise despite the proven advantages, mainly due to the physical limitations that come with having MS63. Since physical exercise may be difficult for those with MS, perhaps focusing on cognitive stimulation could be a viable alternative since it also promotes oligodendrogenesis and myelination.
On the other hand, these results may have implications for those suffering from post- traumatic stress disorder (PTSD). In fact, one study specifically elevated hippocampal neurogenesis using memantine to treat PTSD-like behaviour in mice64. Mice were put through a social defeat stress paradigm that created PTSD-like behaviour, but increasing neurogenesis with memantine caused forgetting of the traumatic memory and reduced chronic, anxiety-like behaviour64. Another study found that in veterans exposed to combat in Afghanistan and Iraq,
54 | P a g e
PTSD was correlated with higher myelin water fraction (MWF) values, which acts as a metric for myelin content65. Since both neurogenesis and myelin have an impact on PTSD development and maintenance, using enrichment to try to treat these underlying mechanisms may prove useful.
However, we also found that enrichment can actually improve how well contextual fear memories are remembered, which may contribute to why soldiers are more prone to developing PTSD during their military service. By constantly doing physical activity while also problem solving and being on alert in a novel environment, this environment is similar to our
“before” conditioning mice which had improved retention of a contextual fear memory. This enrichment would likely contribute to the development of PTSD in soldiers by enhancing fearful experiences and solidifying the context associated with them. Furthermore, this builds upon why we also need to evaluate how novel enrichment needs to be to still cause a discernible effect on memory. Ramping up exercise and cognitive stimulation may help out the normal citizen with lessening the severity of PTSD, but these treatments may not be as applicable to military personnel if they are simply elevating a soldier’s level of enrichment back up to the same level they were living in during active duty. If it is found that maintaining the same level of enrichment before and after the conditioning event only improves memory and does not cause forgetting, these treatments would not be expected to be as effective at treating PTSD in veterans.
55 | P a g e
References
1. Dudai, Y. Memory from A to Z: Keywords, Concepts, and Beyond. (Oxford University
Press, 2002).
2. Richards, B. A. & Frankland, P. W. The Persistence and Transience of Memory. Neuron 94,
1071–1084 (2017).
3. Duffy, S. N., Craddock, K. J., Abel, T. & Nguyen, P. V. Environmental enrichment modifies
the PKA-dependence of hippocampal LTP and improves hippocampus-dependent
memory. Learn. Mem. 8, 26–34 (2001).
4. Akers, K. G., Arruda-Carvalho, M., Josselyn, S. A. & Frankland, P. W. Ontogeny of
contextual fear memory formation, specificity, and persistence in mice. Learn. Mem. 19,
598–604 (2012).
5. Huang, Y. Q. et al. Effects of voluntary wheel-running types on hippocampal
neurogenesis and spatial cognition in middle-aged mice. Front. Cell. Neurosci. 12, 1–9
(2018).
6. Kempermann, G. et al. Why and how physical activity promotes experience-induced
brain plasticity. Front. Neurosci. 4, 1–9 (2010).
7. Josselyn, S. A., Köhler, S. & Frankland, P. W. Finding the engram. Nat. Rev. Neurosci. 16,
521–534 (2015).
8. Wheeler, A. L. et al. Identification of a Functional Connectome for Long-Term Fear
56 | P a g e
Memory in Mice. PLoS Comput. Biol. 9, (2013).
9. Hardt, O., Nader, K. & Wang, Y. T. GluA2-dependent AMPA receptor endocytosis and the
decay of early and late long-term potentiation: Possible mechanisms for forgetting of
short-and long-term memories. Philos. Trans. R. Soc. B Biol. Sci. 369, (2014).
10. Akers, K. G. et al. Hippocampal Neurogenesis Regulates Forgetting During Adulthood and
Infancy. Science (80-. ). 344, 598–602 (2014).
11. Hayashi-Takagi, A. et al. Labelling and optical erasure of synaptic memory traces in the
motor cortex. Nature 525, 333–338 (2015).
12. Epp, J. R., Mera, R. S., Köhler, S., Josselyn, S. A. & Frankland, P. W. Neurogenesis-
mediated forgetting minimizes proactive interference. Nat. Commun. 7, 5–12 (2016).
13. Nørby, S. Varieties of graded forgetting. Conscious. Cogn. 84, 102983 (2020).
14. Hawkins, D. M. The Problem of Overfitting. J. Chem. Inf. Comput. Sci. 44, 1–12 (2004).
15. Kumaran, D., Hassabis, D. & McClelland, J. L. What Learning Systems do Intelligent
Agents Need? Complementary Learning Systems Theory Updated. Trends Cogn. Sci. 20,
512–534 (2016).
16. Sekeres, M. J. et al. Recovering and preventing loss of detailed memory: Differential rates
of forgetting for detail types in episodic memory. Learn. Mem. 23, 72–82 (2016).
17. Frankland, P. W. & Bontempi, B. The organization of recent and remote memories. Nat.
Rev. Neurosci. 6, 119–130 (2005).
57 | P a g e
18. Yassa, M. A. & Reagh, Z. M. Competitive trace theory: A role for the hippocampus in
contextual interference during retrieval. Front. Behav. Neurosci. 7, 1–13 (2013).
19. Akers, K. G. & Frankland, P. W. Grading gradients: Evaluating evidence for time-
dependent memory reorganization in experimental animals. J. Exp. Neurosci. 2009, 13–
22 (2009).
20. Purves, D. & Williams, S. M. Neuroscience. (Sinauer Associates, 2001).
21. Pan, S., Mayoral, S. R., Choi, H. S., Chan, J. R. & Kheirbek, M. A. Preservation of a remote
fear memory requires new myelin formation. Nat. Neurosci. 23, 487–499 (2020).
22. Gibson, E. M. et al. Neuronal Activity Promotes Oligodendrogenesis and Adaptive
Myelination in the Mammalian Brain. Science (80-. ). 344, 1252304–1252304 (2014).
23. Hines, J. H., Ravanelli, A. M., Schwindt, R., Scott, E. K. & Appel, B. Neuronal activity biases
axon selection for myelination in vivo. Nat. Neurosci. 18, 683–689 (2015).
24. Mensch, S. et al. Synaptic vesicle release regulates myelin sheath number of individual
oligodendrocytes in vivo. Nat. Neurosci. 18, 628–630 (2015).
25. Williamson, J. M. & Lyons, D. A. Myelin dynamics throughout life: An ever-changing
landscape? Front. Cell. Neurosci. 12, 1–8 (2018).
26. Sampaio-Baptista, C. & Johansen-Berg, H. White Matter Plasticity in the Adult Brain.
Neuron 96, 1239–1251 (2017).
27. Steadman, P. E. et al. Disruption of Oligodendrogenesis Impairs Memory Consolidation in
Adult Mice. Neuron 105, 150-164.e6 (2020).
58 | P a g e
28. Hughes, E. G., Orthmann-Murphy, J. L., Langseth, A. J. & Bergles, D. E. Myelin remodeling
through experience-dependent oligodendrogenesis in the adult somatosensory cortex.
Nat. Neurosci. 21, 696–706 (2018).
29. Czopka, T., ffrench-Constant, C. & Lyons, D. A. Individual oligodendrocytes have only a
few hours in which to generate new myelin sheaths invivo. Dev. Cell 25, 599–609 (2013).
30. Bechler, M. E., Byrne, L. & Ffrench-Constant, C. CNS Myelin Sheath Lengths Are an
Intrinsic Property of Oligodendrocytes. Curr. Biol. 25, 2411–2416 (2015).
31. Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M. & Noble-Haeusslein, L. J. Brain
development in rodents and humans: Identifying benchmarks of maturation and
vulnerability to injury across species. Prog. Neurobiol. 106–107, 1–16 (2013).
32. Nagy, Z., Westerberg, H. & Klingberg, T. Maturation of white matter is associated with
the development of cognitive functions during childhood. J. Cogn. Neurosci. 16, 1227–33
(2004).
33. Yeung, M. S. Y. et al. Dynamics of oligodendrocyte generation and myelination in the
human brain. Cell 159, 766–774 (2014).
34. Auer, F., Vagionitis, S. & Czopka, T. Evidence for Myelin Sheath Remodeling in the CNS
Revealed by In Vivo Imaging. Curr. Biol. 28, 549-559.e3 (2018).
35. McKenzie, I. A. et al. Motor skill learning requires active central myelination. Science (80-.
). 346, 318–322 (2014).
36. Benaroya-Milshtein, N. et al. Environmental enrichment in mice decreases anxiety,
59 | P a g e
attenuates stress responses and enhances natural killer cell activity. Eur. J. Neurosci. 20,
1341–1347 (2004).
37. Abel, A. M., Yang, C., Thakar, M. S. & Malarkannan, S. Natural killer cells: Development,
maturation, and clinical utilization. Front. Immunol. 9, 1–23 (2018).
38. Du, X. et al. Environmental enrichment rescues female-specific hyperactivity of the
hypothalamic-pituitary-adrenal axis in a model of Huntington’s disease. Transl. Psychiatry
2, (2012).
39. Hüttenrauch, M., Salinas, G. & Wirths, O. Effects of long-term environmental enrichment
on anxiety, memory, hippocampal plasticity and overall brain gene expression in C57BL6
mice. Front. Mol. Neurosci. 9, 1–11 (2016).
40. Sakalem, M. E. et al. Environmental enrichment and physical exercise revert behavioral
and electrophysiological impairments caused by reduced adult neurogenesis.
Hippocampus 27, 36–51 (2017).
41. Fabel, K. et al. Additive effects of physical exercise and environmental enrichment on
adult hippocampal neurogenesis in mice. Front. Neurosci. 3, 1–7 (2009).
42. Schmidt-Hieber, C., Jones, P. & Bischofberger, J. Enhanced synaptic plasticity in newly
generated granule cells of the adult hippocampus. Nature 429, 184–187 (2004).
43. Ge, S., Yang, C., Hsu, K., Ming, G. & Song, H. A Critical Period for Enhanced Synaptic
Plasticity in Newly Generated Neurons of the Adult Brain. Neuron 54, 559–566 (2007).
44. Hosseiny, S. et al. Differential neuronal plasticity in mouse hippocampus associated with
60 | P a g e
various periods of enriched environment during postnatal development. Brain Struct.
Funct. 220, 3435–3448 (2015).
45. Ziv, Y. et al. Immune cells contribute to the maintenance of neurogenesis and spatial
learning abilities in adulthood. Nat. Neurosci. 9, 268–275 (2006).
46. Leger, M. et al. Environmental enrichment improves recent but not remote memory in
association with a modified brain metabolic activation profile in adult mice. Behav. Brain
Res. 228, 22–29 (2012).
47. Scholz, J., Allemang-Grand, R., Dazai, J. & Lerch, J. P. Environmental enrichment is
associated with rapid volumetric brain changes in adult mice. Neuroimage 109, 190–198
(2015).
48. Pajevic, S., Basser, P. J. & Fields, R. D. Role of myelin plasticity in oscillations and
synchrony of neuronal activity. Neuroscience 276, 135–147 (2014).
49. Noori, R. et al. Activity-dependent myelination: A glial mechanism of oscillatory self-
organization in large-scale brain networks. Proc. Natl. Acad. Sci. U. S. A. 117, 13227–
13237 (2020).
50. Kohman, R. A. et al. Voluntary wheel running enhances contextual but not trace fear
conditioning. Behav. Brain Res. 226, 1–7 (2012).
51. Gao, A. et al. Elevation of hippocampal neurogenesis induces a temporally graded
pattern of forgetting of contextual fear memories. J. Neurosci. 38, 3190–3198 (2018).
52. Anagnostaras, S. G. et al. Automated assessment of Pavlovian conditioned freezing and
61 | P a g e
shock reactivity in mice using the VideoFreeze system. Front. Behav. Neurosci. 4, 1–11
(2010).
53. Paul, L. K., Erickson, R. L., Hartman, J. A. & Brown, W. S. Learning and memory in
individuals with agenesis of the corpus callosum. Neuropsychologia 86, 183–192 (2016).
54. Miu, A. C. et al. Behavioral effects of corpus callosum transection and environmental
enrichment in adult rats. Behav. Brain Res. 172, 135–144 (2006).
55. Zaidel, D. W. The case for a relationship between human memory, hippocampus and
corpus callosum. Biol. Res. 28, 51–57 (1995).
56. Ikrar, T. et al. Adult neurogenesis modifies excitability of the dentate gyrus. Front. Neural
Circuits 7, 1–15 (2013).
57. Drew, L. J. et al. Activation of local inhibitory circuits in the dentate gyrus by adult-born
neurons. Hippocampus 26, 763–778 (2016).
58. Park, S. et al. Neuronal Allocation to a Hippocampal Engram. Neuropsychopharmacology
41, 2987–2993 (2016).
59. Gonçalves, J. T., Schafer, S. T. & Gage, F. H. Adult Neurogenesis in the Hippocampus:
From Stem Cells to Behavior. Cell 167, 897–914 (2016).
60. McAllister, D. E. & McAllister, W. R. Incubation of fear: An examination of the concept. J.
Exp. Res. Personal. 2, 180–190 (1967).
61. Tsuda, M. C., Yeung, H. M., Kuo, J. & Usdin, T. B. Incubation of fear is regulated by TIP39
peptide signaling in the medial nucleus of the amygdala. J. Neurosci. 35, 12152–12161
62 | P a g e
(2015).
62. Jahn, H. M. et al. Refined protocols of tamoxifen injection for inducible DNA
recombination in mouse astroglia. Sci. Rep. 8, 1–11 (2018).
63. Motl, R. W. et al. Exercise in patients with multiple sclerosis. Lancet Neurol. 16, 848–856
(2017).
64. Ishikawa, R., Uchida, C., Kitaoka, S., Furuyashiki, T. & Kida, S. Improvement of PTSD-like
behavior by the forgetting effect of hippocampal neurogenesis enhancer memantine in a
social defeat stress paradigm. Mol. Brain 12, 4–9 (2019).
65. Jak, A. J. et al. PTSD, but not history of mTBI, is associated with altered myelin in combat-
exposed Iraq and Afghanistan Veterans. Clin. Neuropsychol. 34, 1070–1087 (2020).
63 | P a g e
Copyright Acknowledgments
This thesis contains third party copyrighted material for which permission has been granted. Figure 1.1 was taken from Josselyn et al., 2015 and permission granted from publisher
Springer Nature. Figure 1.2 was taken from Richards & Frankland, 2017 and permission granted from publisher Elsevier. Figure 1.3 was taken from Akers & Frankland, 2009 and permission granted from publisher Elsevier as licensed under CC BY 3.0. Figure 1.4 was taken from Purves
& Williams, 2001 and permission granted from publisher Oxford University Press. Figure 1.5 was taken from Sampaio-Baptista & Johansen-Berg, 2017 and permission granted from Elsevier as licensed under CC BY 4.0. Figure 2.4 was taken from Scholz et al., 2015 and permission granted from Elsevier.
64 | P a g e