The role of perineuronal nets in spatial memory

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The role of perineuronal nets in spatial memory

by

Katrine Haram Olsen

Thesis for the degree of

Master of Science in Molecular Bioscience

(Main field of study in physiology and neurobiology)

Program for Physiology and Neurobiology Department of Bioscience

Faculty of Mathematics and Natural Sciences University of Oslo

Juni 2016

Teacher Education Program

Department of Teacher Education and School Research Faculty of Educational Sciences

Universitetet i Oslo

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Abstract

The interaction between the hippocampus and parahippocampal region is essential for the processing and storage of episodic memories. Spatial memory is a form of episodic memory, which entails the details of the what, when and where components of experienced events. The medial entorhinal cortex (MEC) is anatomically located as an interface between neocortex and hippocampus and it is considered to be a hub in the distributed network for spatial navigation. The area of interest for spatial memory is the medial entorhinal cortex(MEC), which is found to be essential for spatial navigation and memory.

Stable spatial representations are essential in order to recall and discriminate between similar memories.

Recently it has been proposed that a specialized form of condensed extracellular matrix molecules called perineuronal nets (PNNs) stabilize neural connections and restrict adult brain plasticity. In the adult neocortex, PNNs tightly enwrap the parvalbumin-positive (PV+) neurons, a major subclass of interneurons found in cortex including the MEC.

This tightly encasing structure of PNNs restricts plasticity in adult animals, but can be dissolved by the enzyme chondroitinase ABC (chABC). The role PNNs have for acquiring and maintaining spatial memory have never been investigated. I investigated the effects of degradation of the PNNs in the MEC for acquisition, consolidation and plasticity of spatial memory. This was done by performing microinjections of chABC into the medial entorhinal cortex of rats, and testing their performance in a Morris watermaze.

The results from this study suggest that degrading PNNs in MEC does not affect spatial learning in the watermaze task. During the acquisition phase, all groups performed well and showed a steep learning curve. By the end of the training week they had in general halved the amount of time to find the escape platform (escape latency). However, the chABC treated group needed one more day that the other groups to acquire an escape latency below 60 seconds. This behaviour was also seen during the second trial week, by the group treated with the enzyme between the two trial weeks.

Treatment with chABC prior to a remote memory test did not appear to affect recall of spatial memory. The three groups did not show significant differences when remote memory was tested. Finally, I tested the rats ability to learn a new platform location when the salient cues in the room was changed. The daily probe trials revealed that the group treated with chABC prior to trial week two took quickly to the new platform location in the trials. Both the sham treated animals and the latter group showed erratic behaviour judged by the latency measure from the probe trials. The conclusion from my study is limited by the low number of animals in each group, and future studies will test

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Acknowledgements

The work done in relation to this thesis was performed at the Program of Physiology and Neurobiology, department of Biosciences, University of Oslo, under the supervision of associate professor Dr. Marianne Fyhn, associate professor D. Torkel Hafting and doctorate Ane Charlotte Christensen. This thesis is also a part of the teacher education program, department of teacher education and school research, University of Oslo.

Thanks to my supervisors Marianne and Charlotte for taking on a short term teacher student and letting me be a part of a very new and exciting project. Having the experience of being a part of a research project from the very start has been a thrilling and sometimes confounding ride, but has also given me valuable life experiences. Thanks to Torkel for help with everything MATLAB, for your time and effort put into developing the script, and all the quick replies when something did not go according to plan. Thanks to all of you for your guidance and input on the writing process. I want to especially thank Charlotte for letting me look over you shoulder for every little thing done in surgery, care and management of my lovely research animals. The support and encouragement from you have been both inspiring and invaluable. Thanks to Mattis Brænne Wigestrand for helping me with statistics. A big thank you to everyone in the Fyhn-Hafting group for all the little tidbits of help along the way. It has in so many ways been beneficial to me in completing this task.

I would also like to thank the wonderful student association Realistforeningen, for both time well spent, and well wasted. The fun times are memories that I will take with me for life, and the welcoming atmosphere and people will always bring about a smile as I think back to my grand days at Uni. When spending time in RF I have been able to meet some outstanding individuals, among them Eivind Storm Aarnæs, and Sigmund Slang, which I would like to thank for your help with the finer points of writing in LaTex. I would also like to send out my boundless appreciation to Simen Tennøe for your enormous patience when introducing me to LaTex and in giving me the tools to complete this project, as well as your always helpful replies to my occasional, small panic attacks. And to Resmije Gjonbalaj I am forever grateful for keeping me company during those long working days, for all our lunches and walks. Thanks for keeping me sane and keeping me focused.

Lastly I would like to thank my family, mom and dad, for motivating and guiding me through the rough patches, for making me laugh, and telling me like it is when my head was way up in the clouds. Your love and support means everything to me. Thanks to my brother for always being an inspiring beacon for me to follow, and try to outshine. Even though your interest in my project may have purely been about my research animals, having you along for the ride has been both fun and helpful.

Contributions: Due to the time limitations on this project, and the complexity of the surgical procedures, they were performed by Ane Charlotte Christensen and Kristian Lensjø. Christensen also performed the histology procedures.

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List of Figures

1.1 An axonometric drawing of a typical watermaze setup and the Atlantis

platform (Morris, 2015) . . . 4

1.2 Schematic representation of the molecular composition of PNNs on the surface of a neuron. . . 5

1.3 WFA stained PNNs . . . 6

1.4 Potential mechanisms of chABC treatment . . . 8

2.1 Dorsal view of the rat skull . . . 12

2.2 Example of swim path . . . 17

3.1 Light microscopy images of DAB stained sagittal sections . . . 20

3.2 Schematic figure of the C-6-S antigen labeling . . . 21

3.3 Swimtrack for rat 1 . . . 22

3.4 Escape latency for the acquisition week . . . 23

3.5 Escape latency for all animals during acquisition week . . . 24

3.6 Results from probe-trails without platform present. . . 25

3.7 Quadrant measurements for the acquisition week . . . 26

3.8 Remote memory assessed by time-in-quadrant analysis . . . 27

3.9 Latency for the second and third testing week . . . 27

3.10 Escape latency for the second testing week . . . 28

3.11 Quadrant measures from the second testing week . . . 30

3.12 Escape latency for the third testing week . . . 31

3.13 Swimtrack for rat 8 from testing week two and three . . . 32

3.14 Quadrant measurements from testing week three . . . 33

A.1 Spread of chABC in the MEC of the chABC group. Stubs colored from DAB solution. . . 48

A.2 PNN stub coloring for rat 5 . . . 49

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List of Tables

2.1 Stereotaxic coordinates . . . 13 2.2 Drop points. The table shows the drop points for all trial days. . . 15

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Chapter 1 Introduction

1.1 Memory, development and plasticity

Neuroplasticity is defined as the ability of the nervous system to respond to extrinsic or intrinsic stimuli by a reorganization of its function, structure, or connections (Ruge et al., 2012). When we rehearse with a musical instrument or practice a neat trick for use on the football field, neuronal circuits are being formed. This leads to better performance and autonomy, which means less waste of energy. The phenomenon of neuroplasticity leads to abilities such as habituation, sensitization, drug tolerance and can even influence the recovery from certain brain injuries. Neuroplasticity is often divided into two groups, structural and functional neuroplasticity. Structural neuroplasticity involves changes in number and location of synapses, neuronal migration and also neurogenesis. Functional neuroplasticity fall under the purview of learning and memory. During these processes permanent changes occur in synapses between neurons due to structural adjustment or intracellular biochemical processes (Demarin et al., 2014).

During fetal development, the structural changes involve neurogenesis and migration of neurons. In contrast, in the adult brain the level of plasticity is more limited and new long range projections are seldom formed. Before reaching adulthood the brain has a very high degree of plasticity, often referred to as critical periods. Critical period (CP) refers to the period during which exposure to a certain environment is necessary for the development of minimal normal function (Yang and Tang, 2011). During these periods the animal learns skills or traits that are indispensable to the survival of the animal. Following the critical period, plasticity is reduced or sometimes even absent, meaning that the animal may be unsuccessful at developing these skills later in life.

1.2 Plasticity in learning and memory

The synaptic plasticity and memory hypothesis states that: Activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both

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necessary and sufficient for the information storage underlying the type of memory me- diated by the brain area in which that plasticity is observed (Martin et al., 2000). The SPM hypothesis has been developed over several decades and through hundreds of papers and has as such, become widely accepted. Synaptic plasticity refers to different forms of activity-dependent modification of synaptic efficiacy, which includes aspects of long-term potentiation (LTP) and and long-term depression (LTD) (Schinder and Gage, 2004). In both LTP and LTD, neuronal activity affects the molecular composition at the synapse.

This leads to a change in the efficacy of signal transduction between the connected neurons, LTP causes enhanced signal transduction, while induction of LTD leads to reduced signal transduction (Martin et al., 2000). The long-lasting changes in synaptic efficacy that these two phenomena produce are thought to be important mechanisms for memory formation (Martin et al., 2000; Whitlock et al., 2006).

In 2010, H¨ubener and Bonhoeffer found that the formation and elimination of dendritic spines are the basis for structural remodeling of networks. This process takes place in the cortex as a response to exposure and learning (H¨ubener and Bonhoeffer, 2010). Recent studies, such as (Yang et al., 2009; Xu et al., 2009), indicates that learning a motor task resulted in a rapid increase in spine turnover. This implies that the formation and loss of synaptic connections with dendritic spines may be important during learning and memory encoding. CP level plasticity cannot be matched later in life (Wiesel and Hubel, 1963;

Sato and Stryker, 2008), though through synaptic modification in the form of LTP and LTD it is possible, to some extent, even in adulthood (Bliss and Lømo, 1973). Adult- born neurons in the hippocampus remain susceptible to change, though the ability of the central nervous system (CNS) for repair and regeneration is relatively poor.

1.3 The medial entorhinal cortex and spatial naviga- tion

Spatial memory is a part of episodic memory, which is the memory of personal experiences including the what, where, when and other contextual information of experienced events.

Several studies, including Squire (1992), conclude that the hippocampal formation and the parahippocampal areas are important in encoding, consolidation and recall of episodic memories. To ensure that this process occurs throughout life there needs to be continous functional changes in this circuitry. In essence, there is a need for the hippocampus and parahippocampal areas to inhabit a high degree of plasticity. As reviewed by Van Strien et al. (2009), the entorhinal cortex (EC) is of particular interest to study with regards to spatial memory, particularly due to its role in the processing of spatial memories and navigation. The EC is viewed at a nodal point between the association cortices

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attributed the ’where’ component as it appears to mainly process spatial information. The importance of the entorhinal cortex in spatial memory is further emphasized by lesion and behavior studies by Steffenach et al. (2005). This study concludes that the dorsolateral band is necessary for storage, consolidation and retrieval of spatial information. In addition, studies conducted by Burwell and Hafeman (2003), and Fyhn et al. (2004), suggests that the dorsolateral band of the EC may contribute directly to the computation of spatial information. Testing spatial memory in animals is easily done in the watermaze task initially developed by Richard Morris.

1.4 Morris watermaze

The Morris watermaze (MWM) was developed by Richard G. Morris in 1984, and is widely used to study spatial memory and learning. The maze was designed as a method to assess spatial, or place, learning(Vorhees and Williams, 2006). The MWM is not a maze in the traditional sense, i.e. it is not a labyrinth. It is an open circular pool, that is close to featureless internally and approximately half-way filled with water, as can be seen in figure 1.1. The ’maze’ part of the task is that the animal must search in order to find the platform hidden below the surface of the water, and upon locating and climbing the platform, escaping the task. The water in the pool has been rendered opaque by adding either non-fat milk or non-toxic tempera paint. By rendering the water opaque the animals cannot see the platform, and due to the nature of the task, the animal cannot rely on scent to locate the platform. Therefore, the animals must rely on external cues to memorize the location of the platform. As the task becomes familiar to the animals, the time used to locate and climb onto the platform should decrease.

There are several variations of the task, but the most basic involves a circular pool, sized according to the animals used, with a hidden platform. The environment can be altered to investigate working memory, reference memory and task strategy. While the basic task specifically tests spatial memory, there are several components of the task that affects the animal. There is a degree of stress involved, the animal is unused to swimming, and it must understand the rule of the task. The rule being; in order to escape, one must find a hidden platform, and stay on the platform in order to be picked up.

Once an animal has learned the location of the hidden platform, it is possible to change the location of the platform and/or alter the external cues for additional spatial memory tasks.

The figure 1.1 shows a typical watermaze setup, with an overhead video camera and a rat swimming to find the hidden platform. In the figure the watermaze has a baseboard.

In the setup used in this project, the watermaze had a frame below it, enabling the water level to be about waist height for easy release and pick up of test animals. The Atlantis platform, also shown in the figure, depicts the platform in the bottom and top position.

The Atlantis platform facilitates the use of probe trials, by having the platform at the bottom of the pool the swimming rat is not able to bump into it by chance. When the probe trials come to an end, one can simply raise the platform and conduct the trials.

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Figure 1.1: An axonometric drawing of a typical watermaze setup and the Atlantis platform (Morris, 2015)

A typical watermaze setup with an open circular pool, as featureless as possible internally and approximately halfway filled with water. The figure also shows an overhead camera and a rat swimming to find a hidden platform. On the right is the Atlantis platform in both the top and bottom position. The Atlantis platform provides an advantage during probe trials, where the platform needs to be unavailable.

1.5 Perineuronal nets

The extra cellular matrix composition in the central nervous system differs from the extra cellular matrix found in the rest of the body. Proteoglycans (PGs) are together with hyaluronic acid the major components of the macromolecular part of the extracellular space in the CNS (Yamaguchi, 2000). PGs are proteoglycans extensively modified by glycosamoniglycan (GAG) chains by glycosylation. Due to the many varieties of chains that PGs can carry, PGs are categorized into three groups, keratan sulfate PGs (KSPGs), heparin sulfate PGs (HSPGs) and chondroitin sulfate PGs (CSPGs). Of these three the CSPGs have been of particular interest, due to the fact that they are prominent in the central nervous system as well as the role this subclass plays in injury repair and plasticity in the CNS.

Perineuronal nets (PNNs) are reticular structures that surround the cell body of many neurons, and extend along their dendrites. They are considered to be a specialized extracellular matrix in the central nervous system (Wang and Fawcett, 2012). The PNNs wraps tightly around the neurons in a lattice-like formation (see figure1.2), and consists of several of the same components that are used in cartilage, such as hyaluronic acid

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Figure 1.2: Schematic representation of the molecular composition of PNNs on the surface of a neuron.

This is a proposed structure of the PNNs, where hyaloronic acid synthase (HAS) synthesize and secrete hyaloronic acid (HA), which binds to the different members of the lectin family through link proteins. The C-terminus of the lecticans bind to Tenacin-R, while the N- terminus binds to HA, allowing for supramolecular aggregates to form (Kwok et al., 2011).

et al. (2009), shows that CSPGs in the form of perineuronal nets are the molecular mechanism for closing a postnatal critical period, and that the degradation of perineuroral nets make memories susceptible for erasure.

Parvalbumin-positive interneurons in the medial entorhinal cortex

Cortical neuronal network operations depend critically on the recruitment of gamma- aminobutyric acid positive (GABAergic) interneurons and the properties of their inhibitory output signals (Bartos and Elgueta, 2012). These interneurons, found in MEC layers II and III, can have local projections or long-ranging projections to the hippocampus. One of the main types of GABAergic interneurons in the MEC are fast-spiking basket cells expressing the calcium-binding protein parvalbumin (PV⁺ cells)(Sik et al., 1995; Wouterlood et al., 1995).

The GABAergic neurons that express the calcium binding protein parvalbumin (PV⁺) seem to play a particularly important role in the regulation of plasticity. Perineuronal nets are mainly associated around these GABAergic neurons. In the mammalian visual cortex, the PV⁺ cells mature at the onset of the critical period (del Rio et al. (1994);

Pizzorusso et al. (2002)). Donato et al. (2013) found that low PV⁺ expression enhanced synaptic plasticity, memory consolidation and retrieval, whereas high PV⁺ expression

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Figure 1.3: WFA stained PNNs

Perineuronal nets (green) in rat medial entorhinal cortex (MEC) stained with Wisteria floribunda agglutinin (WFA) enwraps parvalbumin positive (PV⁺) cells (red) stained with rabbit anti-parvalbumin (Christensen, 2014).

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produced the opposite result. Studies of the role of the inhibitory activity of PV⁺ cells in CP plasticity models points to an interplay between PV⁺ cells and the perineuronal net as key players for the reduced plasticity in the adult brain.

1.5.1 The PNNs and plasticity

The PNN is an important regulator of CNS plasticity, both during development and into adulthood (Wang and Fawcett, 2012). A study from Hockfield et al. (1990) concludes that the formation of PNNs coincides with the end of the critical period. Though as proven by Pizzorusso et al. (2006) and several other studies, the assembly of PNNs are activity-dependent and a lack of stimuli can prolong the critical period. It has also been suggested that the hyaloronan component of PNNs is a keyplayer in the regulation of short-term memory (John et al., 2006).

Following the critical period, the ECM in the CNS are continuously modified by a group of enzymes called matrix metalloproteinases (MMPs). MMPs constitute a large group of endoproteases that are capable of cleaving all protein components of the extracellular matrix, as well as activating or inactivating other signaling molecules, such as growth factors, receptors and adhesion molecules (Verslegers et al. (2013)). The MMPs are linked to CNS plasticity mechanisms, playing a crucial role in the remodeling of synapses and dendritic spine dynamics (Reviewed by Huntley (2012)). Huntley also found evidence that learning and memory tasks, such as fear conditioning tests which induces LTP, also brings about increased levels of MMPs.

Chondroitinase ABC (chABC) is a bacterial enzyme isolated from the bacterium Pro- teus vulgaris and can be used to artificially degrade PNNs. ChABC works by cleaving the chondroitin sulfate GAG-side-chains of the CSPGs into disaccharides (Br¨uckner et al., 1998). Studies performed by Pizzorusso et al. in 2002 and 2006, have shown that treatment with chABC can induce CP-level plasticity in adult rats in experiments exploring the possibility of ocular dominance(OD) shifts. Further studies by Carulli et al.(2010) supports the postulate that PNNs are involved with the closure of the critical period.

The increase of plasticity after chABC treatment has been shown to be a general concept, as the injection of chABC also affects other systems such as fear conditioning (Gogolla et al., 2009). The enzymatic digestion caused by chABC works quite differently than the proteolytic cleavage of the natural MMPs. Treatment with chABC disrupts the tight organization of ECM molecules, and leads to a loss of the overall PNN structure. A possible mechanism by which PNNs restrict plasticity and how chABC treatment affects PNN structure is shown in figure 1.4. The perineuronal net forms a dense structure around synapses, and when treated with chABC, the PNN component deteriorates and there is a potential for extrasynaptic movement of receptors and neurotransmitters into the extrasynaptic space (McRae and Porter, 2012).

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Figure 1.4: Potential mechanisms of chABC treatment

Schematic depiction of the perineuronal net surrounding a subset of neurons (Left panel).

The potential result after treatment with chABC is a complete disruption of ECM structure (Right panel). Figure adapted from McRae and Porter (2012). HAPLNs - link proteins, HAS - Hyaloronan synthases, CD44 - Receptor for hyaloronan.

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1.5.2 A possible role for PNNs in learning and memory

Perineuronal nets wrap and secure existing synapses and at the same time restrict the formation of new contact points. In 2013, Roger Tsien proposed that PNNs could be the molecular basis for storing long term memories and several studies previously mentioned has shown that PNNs might be involved in acquisition and storing of memories in different areas of the brain (Tsien, 2013). The tight organization of the perineuronal nets inhibit juvenile plasticity in adult animals by obstructing the formation of new synapses. As seen in figure 1.4 the extracellular matrix is closely packed around the synapses, leaving little room for formation of new synapses without prior degradation of the ECM. The highly stable molecular composition of the PNNs makes them suitable to maintain stable, long-lasting neuronal connections. Romberg et al. (2013) found indications that PNNs regulate both memory and experience-driven synaptic plasticity in adulthood.

A large body of evidence suggests that perineuronal nets have a role in CNS plasticity, including the hippocampal formation and parahippocampal areas. However, research regarding PNNs’ role in plasticity with regards to principal neuronal activity in the EC has not been sufficiently studied. In the current project, the potential role PNNs have in spatial learning and navigation in the MEC will be looked into.

1.6 Aims of the study

The main objective of this study was to explore whether the breakdown of perineuronal nets in the entorhinal cortex affects spatial memory and learning. This was achieved by testing if:

1. degrading PNNs in MEC affects acquisition of a platform location in the Morris water-maze task.

2. intact PNNs is essential for maintaining the memory of a platform location.

3. degrading PNNs would affect the animals ability to be flexible, i.e. learn a new platform location with different external cues.

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Chapter 2 Materials and Methods

2.1 Experimental animals

The practical work done was performed at the Department of Biosciences (IBV) at the University of Oslo, Norway. The experiments were approved by the Norwegian Animal Research Committee (FDU). All parties participating in the research holds an animal researcher certificate as required by the Norwegian Food Safety Authority (Mattilsynet).

The housing and treatment of the animals fulfills the requirements set by the European Union and the FDU.

Thirteen male Long Evans rats (aged 3-6 months, bodyweight 400-600g), were used for the experiments. One animal died during anesthesia prior to surgery. The animals were divided into different test and control groups as described below. During the first round of surgeries, four animals were injected with chABC, and four were sham operated with an artificial Cerebrospinal Fluid(aCSF). Another rat was trained and added to the untreated group and these four animals were injected during the second round of surgery with chABC. My project is a pilot project for hypothesis testing and optimization of parameters and the sample size in each test group is therefore low but still sufficient to draw preliminary conclusions.

The animals were housed on a 12h light/dark cycle, where the lights were on from 08.00-20.00. The training and testing of animals were performed during the light cycle, which corresponds to the rats inactive phase. The temperature in the cages were kept at 21+/-0.1°C, with a humidity level of approximately 55%.

2.2 Surgical Procedures

All animals were anaesthetized with isoflurane (Baxter, Oslo, Norway) mixed with air, and then injected with a mix of Ketamine (60mg/kg) and Medetomidine (0.6mg/kg) intraperitonally. Silicon oil was applied to the rats eyes, to prevent them from drying out. Toe pinching reflexes were checked to ensure anesthesia was induced before placing

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the animal in a stereotaxic frame (World Precision Instruments Ltd, Hertfordshire, UK).

The head was immobilized by positioning ear bars in the external auditory meatus. The midline of the skull was aligned with the frame, with the help of the ear bars. Correct positioning of the head is important to facilitate precise and reproducible stereotaxic measurements of coordinates.

Stereotaxic coordinates were found in the atlas of the rat brain by Watson and Paxinos (2007). A flat skull position was obtained by the use of a height-adjustable nose-clamp.

In order to minimize the risk of infections, the surgery procedure were strived to be kept aseptic and sterile. Surgery equipment was heat sterilized (150°C for 90 minutes) or autoclaved (cotton swabs) before surgery. Furthermore, the fur on the animals head was shaved, and the shaved skin was cleaned with ethanol and chlorhexidine.

Figure 2.1: Dorsal view of the rat skull

Dorsal view of the rat skull showing the bregma, lambda and sites for microinjections (marked by the red circles). Modified from Paxinos and Watson (1998).

A longitudinal cut was made in the skin, and skin was moved aside to expose the skull.

Artery clamps were used to keep the skin aside during the surgery. In order to prevent the skull bones from drying out, and to reduce the heat during drilling, the skull was kept constantly moist throughout the operation using sterile saline water (0.9% NaCl).

Stereotaxic coordinates for injection and recording sites were measured relative to lambda (skull landmarks at the intersections of the sagittal and coronal skull sutures shown in figure 2.1). Craniectomies of approximately 2.5 mm in diameter were performed bilaterally using a hand-held Perfecta-300 dental drill (W&H Nordic, Sweden).

After injection, the holes were filled with KWIK-SIL silicone(World Precision Instru- ments Ltd, Hertfordshire, UK). The wound was cleaned with 0.9% NaCl and sutured shut with 5-10 stiches, and antibiotic ointment(Fucidin) was applied. After surgery, all animals were given s.c. injections of Rimadyl (carprofen 5mg/kg) for post-operative analgesia. In addition the animals were given a s.c. injection of Anti-Sedan, to counteract the effects

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Table 2.1: Stereotaxic coordinates

Table with stereotaxic coordinates for chABC injections. AP, anterioposterior relative to the transverse sinus; ML, mediolateral relative to the midline; DV, dorsoventral relative to the dura mater. Each injection site received a dose of 10x32,2nl(total 322nl per site)

ML AP Angle DV Dose

Left/right hemisphere

4.4± 0.1mm 0.6±0.1mm anterior of sinus

10-15 ^◦ anterior of sinus sagittal

2.5±0.1mm 322nL 3.3±0.1mm 322nL 4.7± 0.1mm 0.6± 0.1mm

anterior of sinus

10-15 ^◦ anterior of sinus sagittal

2.5±0.1mm 322nL 3.3±0.1mm 322nL

2.2.1 Injections of chABC and aCSF

Glass pipettes were pulled from borosilicate capillary glass(Sutter Instrument Complany, CA, USA) with an outer diameter of 1.2 mm, using a P-30 pipette puller(sutter instrument company, USA). Taper length was made no shorter than 8 mm to avoid causing tissue damage during injections deeper than 2500µm below dura mater in the DV plane.

The opening of the pipette tip was between 15 and 25 µm. This size range allowed for penetration of the dura mater, but at the same time avoided clogging of the pipettes after two or three tissue penetrations(Lensjø2013).

Pipettes were backfilled with raps oil and assembled into a NanoJect II (Drummond Scientific Company, USA) microinjector, and then filled with chABC or aCSF. Protease free chABC from Proteus vulagaris was purchased from Amsbio (Abingdon, UK) in 10U vials, and diluted in phosphate-buffered saline (PBS) to aliquots of 61 U/ml, which were stored at -20C. in order to visualize the injections, aliquots were further diluted with Fast Green FCF (Sigma-Aldrich Chemie, Munich, Germany) just before samples were loaded into the pipette, at a final concentration of 48 U/ml, in accordance with Pizzorusso and coworkers (2002). The aCSF was purchased from Harvard apparatus (Massachusetts, USA) and diluted with Fast Green FCF in a 4:1 ratio.

The coordinates for the injection sites were taken from previous work performed in the lab, such as the work of Kvello(2014) and C. Christensen(unpublished). The chABC/Fast Green mix was injected into two ML coordinates and two DV coordinates in the MEC of both hemispheres (see table 2.1). The injection was done at approximately 0.6 mm anterior to the sinus edge, as well as at a 15 degree angle posterior-anterior in the sagittal plane. Each site received 10 single injections of 32,2 nl chABC/Fast Green, and the pipette was kept in place for 2-5 min after the last injection in order to let the enzyme diffuse before removal of the pipette.

2.3 Watermaze

In order to let the animals recover from surgery, the experiments were not initiated until a week after surgery. There was two weeks between the end of the training week and at

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the start of the second training week. In this time period, a second group were injected with chABC and termed the chABC2 group.

2.3.1 Behavioral testing

The testing was conducted in a custom made circular white tub, with a diameter of 2 m.

The water temperature was set to 22 +/- 1 °C. The platform was located 50 cm from the wall of the tub, and approximately 2 cm below the surface of the water. The platform diameter was 10 cm, and could be raised or lowered remotely. This allowed for the possibility of a daily probe trial, with the platform unavailable for two minutes before the platform was raised. The water was colored opaque using non-toxic white paint(Tempera, Panduro) to prevent the animals being able to visually locate the platform. If the rat did not find the platform within two minutes, it was guided to the platform and allowed to stay there for 10-15 seconds.

The watermaze was divided into four quadrants. Two principal axes bisect the maze perpendicular to each other, forming a ’+’. These axes mark the four cardinal points:

North(N), West(W), South(S), East(E). The S was chosen to be point closest to the experimenter and N being at the opposite. The platform is positioned in the middle of one of the quadrants. The drop points were chosen according to Vorhees and Williams (2006), in a pseudorandom/semi-random way, such that at least one trial each day is from each of the four positions. The release points can be found in table 2.2. The training weeks consist of five days, with one probe trail and four trials for every session.

The trials were divided into three training sessions. The first acquisition training consisted of day 1-5 with 11 rats, 4 who had undergone surgery with SHAM injections, 4 who had been injected with chABC, and 3 who had not undergone any surgery. The second training week occurred two weeks later, as the previously untreated rats were injected with chABC. One rat who had undergone the same training was added to this group, resulting in a total of 4 animals in each group. The platform position and external cues were the same as in the first training session. The platform was moved by the end of the second week, and the third training session were started the following day. The external cues for the first two weeks consisted of a metal plate located at the northern side of the tub, and the metal door at the southern side of the pool. During these weeks the platform was located in the NE quadrant, while for the third training session it was moved to the SW quadrant. The external cues in the last week were the metal door, and the metal plate at the northern side was covered with a dark brown curtain, and an A3 sized colored picture was put up on the western side of the tub.

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Table 2.2: Drop points. The table shows the drop points for all trial days.

Day Probe Trial Trial 1 Trial 2 Trial 3 Trial 4

1 W N E S W

2 N S N W E

3 S W S E N

4 W E W N S

5 E N S E W

6 W S W N E

7 S N S E W

8 N E N W S

9 E W E S N

10 N S N W E

phate buffered saline solution (PBS). The brains were removed and post-fixed in PFA for a minimum of 24 hours. Preceding the slicing, the brains were incubated at 4^◦C in a 30%

sucrose/PBS solution for cryoprotection until saturated. The brains were flash frozen and mounted in a cryostat (CM1950, Leica Biosystems). Sections of 40 mum were cut in the sagittal plane, and collected with a fine paint brush and put in 1x PBS for immunohis- tochemistry staining. Complete protocols for the staining procedures are found in the appendix.

2.4.1 Staining for chondroitin sulfated ’stubs’

To confirm correct injection and activity of chABC in the entorhinal cortex, staining for chondroitin sulfate (CS) neoepitopes was performed on 40 µm free floating sections.

ChABC degradations leaves the inner six monosaccharides of the CS polymer intact on the protein core, which can be stained with a monoclonal anti-chondroitin-6-sulfate antibody (MAB 2035 Milipore). Sections were blocked with 1.5% BSA (Sigma-Aldrich, USA), 0,3%

Triton 100-X (Sigma-Aldrich, USA) in 1x PBS for one hour at room temperature and then incubated at 4°C overnight in a 1/1000 block solution with the primary antibody. On the second day the sections were incubated in a 1/500 dilution of anti-mouse biotin-conjugated antibody (A-24522, Life) in block solution. The endogenous peroxidase activity was quenched using a 2% H₂O₂ solution, and incubated for one hour in an ABC peroxidase 21 staining kit solution (Thermo Scientific, Rockford, IL, USA). The stainings were visualized using 3,3’-diaminobenzidine (DAB) (Sigma Aldrich CHemie, Munich, Germany)/Tris-HCl solution. The sections were then washed using TNS (6g Trizma + 1L ddH₂O, to pH 7.4 with 1M NaOH) to stop the DAB-reaction, and mounted onto Superfrost plus glass slides using a fine brush and PBS. The slides were then left to dry overnight, before being rinsed in ddH₂O and subsequently dehydrated with 90% EtOH, then 100% EtOH and finally xylene. Sections were mounted with DRX/entellan and left to dry under fume hood overnight.

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2.4.2 Fluorescent double staining of PNNs and PV

⁺

cells

The staining used in figure 1.3 is a double staining of sagittal free floating sections ranging from 25-60 µm. The sections were washed, blocked and incubated as described for the DAB stained section. When double staining for parvalbumin positive (PV+) cells, sections are incubated with WFA and a primary rabbit-anti-parvalbumin (Swant, Marly, Switzerland) in TS-PBS at 4^◦ overnight. On the following day, the sections are washed and incubated with secondary antibodies goat-anti-rabbit Texas Red-X (Invitrogen Life Tech, California, USA) and Streptavidin Alexa 488 fluor conjugate (Invitrogen Life Tech, California, USA). Incubations are done at room temperature in a dark room. Cover slips and FluorSave Reagent (Merck Millipore, Darmstadt, Germany) were used to secure the sections. The protocol can be found in the appendix. (Kvello, 2014)

2.5 Data analysis

2.5.1 Recording set up

A flycaptureC camera mounted on the ceiling above the water maze was used to record the movement of the animals using the tracking software Bonsai. The tracking software produces .csv files that could be analyzed in MATLAB using a script developed by Torkel Hafting for this task.

Figure 2.2 is an example of a figure that is produced after the .csv file from Bonsai tracking software is run through MATLAB. This is one of several figures the software produces and it shows the four quadrants in different colors, with the dotted circle in the north east (NE) quadrant representing the platform. The four colors represents the different quadrants; Turquoise is the north eastern (NE) quadrant; Green represents the south east (SE) quadrant; Blue is the south west (SW) quadrant; Black represents the north west (NW) quadrant. During the first two weeks, the platform in located in NE, and in the third test week the platform is moved to the SW quadrant. This figure is from one of four trials for the first day of testing of rat 8. Rat 8 is one of the four rats injected with chABC prior to the training week. The trial shows that the rat found the platform, but during this trial it barely exceeded the two minute time limit, and as such is also an accurate representative for a probe trial.

2.5.2 Statistical analysis

From the information given from the MATLAB script, statistical analyses were performed

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Figure 2.2: Example of swim path

Rat swimming path in the water maze. The dotted circle in the north east (NE) quadrant represents the escape platform. The four colors represents the different quadrants, Turquoise - North east; Green - South east; Blue - South west; Black - North west. Rat 8, day 1, trial 2.

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relative to variance within groups. ANOVA is a useful tool for comparing three or more means for statistical significance, giving the opportunity to draw valid and incontrovertible conclusions from experiments where observations are subject to erratic variation. Values are reported as single values or as median and/or Mean with standard errors of mean (SEM).

2.5.3 Immunohistochemical analysis

The CSPG stained sections were photographed using an Axiocam HRZ camera connected to an Axioplan 2 microscope (both provided by Carl Zeiss, Oberkochen, Germany). Pho- tos were taken at 5x magnifications as whole-sections photos stiched together using Axio- Vision (Carl Zeiss, Oberkochen, Germany) and the MosaiX Acquisition/Stitchin modules.

Adobe Photoshop2 (Adobe, California, USA) was used for image cropping and minor contrast adjustments.

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Chapter 3 Results

3.1 Histology

Histology was performed to assess the areas affected by the chABC treatment. In order to visualize where the PNNs had been digested we used 3,3’-diaminobenzidine(DAB) to visualize the stubs of the six innermost monosaccharides of the CS polymer left from enzyme treatment (figure 3.1). Light microscopy images of the DAB staining for all animals treated with chABC can be found in the appendix.

Using templates from Paxinos and Watsons (2008), the images of the DAB staining of 4 rats were superimposed to visualize the distribution of affected areas of the chABC treatment (Figure 3.2). The chABC treatment affected the dorsal part of the MEC and extended anterior and dorsal from the MEC. The ventral MEC was affected to a limited extent. The figure is based on histology from the animals that were treated with chABC before the first training week. The areas have been colored in using the sagittal sections stained with DAB solution as a guideline. The coloring has been done in layers, where each layer represents one rat. The areas affected by the chABC treatment lie more in the dorsal part of the brain, and the more ventral part of the entorhinal cortex has been left untreated.

3.2 Watermaze recordings

3.2.1 Acquisition

During the first week of training the animals were introduced to the watermaze task. The trials were conducted to examine the animals ability to learn the watermaze task and to investigate if degradation of perineuronal nets affects acquisition of spatial memories.

Figure 3.3 displays the swim path of rat1 from day 1, 2 and 5 for the first testing week, also termed acquisition week. In accordance with previous studies on the training of rats in the watermaze, the animal spends much of the first trial swimming against and along

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(a) (b)

Figure 3.1: Light microscopy images of DAB stained sagittal sections

Light microscopy images stained sagittal sections showing the extent of chABC activity.

(a) DAB staining of PNN stubs from rat 8, right hemisphere. The DAB staining illustrates the area affected by chABC treatment, by appearing darker than the surrounding area.

(b)Right brain hemisphere from rat 9, where the area affected by the enzyme covers most of the MEC.

an alternate escape route, this being the platform. There was a pronounced decrease in path length across the days, from the drop point to the platform location, and an almost direct route to the escape platform on day 5 of the trial week.

In order to compare acquisition of spatial memory between groups, the average time spent on locating the platform (escape latency) was calculated for each group during the first testing week (figure 3.4). The time spent locating the platform for the four daily trials were averaged for each rat, these numbers were again averaged across the group to produce the graph in figure 3.5.

The graph shows a clear decrease in escape latency of the trials. For all groups the escape latency has more than halved from day one to day five, going from an average of 85, 77, and 77 seconds for the sham, chABC and untreated respectively, to 32, 28, 37 seconds.

The figure shows no discernible difference between the three groups, with the exception being on day two. The sham group follows an expected learning curve comparative to the literature (Vorhees and Williams, 2006), with a rapid decrease in trial duration on the second day and a generally low amount of time spent during the remainder of training week. The untreated group has a similar curve with a slightly more gradual learning curve. During this trial week the animals treated with the enzyme required an extra day in learning. As can be seen in figure 3.5 there are great variations within each group, and that within the group treated with chABC there seems to be quite large differences between the individual animals.

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Figure 3.2: Schematic figure of the C-6-S antigen labeling

The top image illustrates the location of the hippocampus (HPC) and the medial entorhinal cortex (MEC). Schematic figure of the C-6-S antigen labeling, indicating the spread of chABC activity for the right hemisphere for rats 5-9. These are superimposed on illus- trations of brain sections (Paxinos and Watson (2006). The topmost figure is lateral 4.20 mm, second is 4.32 mm, and the bottom figure represent the lateral 4.60 mm section. For each individual rat, the brown color illustrates the area where chABC has digested PNNs.

Where there is an overlap between injections in the rats, the brown color darkens.

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(a) (b)

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Figure 3.3: Swimtrack for rat 1

Examples of swim paths of rat number 1 during the first trial week. Rat 1 is a part of the control group who underwent sham surgery prior to the acquisition week. The red circle is the hidden platform and the black circle is the platform zone. (a) One of the first trials on day one. As the swim path does not connect with the platform, the animal did not find the platform within the trial time of two minutes. (b) Swim path on the second trial day. Rat 1 was among the animals that showed decreased escape latency already on the second trial day. (c) Swim path day 5 is fairly direct at the platform location.

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Figure 3.4: Escape latency for the acquisition week

The y-axis time to find the platfrom (seconds), and the x-axis represents the training days. The average escape latency of the four trials performed each day for each animal was calculated and averaged again to represent the test group (sham operated, N=4, chABC, N=4, untreated, N=3). All groups learned the task and the results were quite similar, with the exception of day two.The error bars shows standard error of the mean (SEM).

Probe trials

Every training day started with a probe trial, without escape platform available, in order to test if the rat remembered and searched for the platform in the right location. All probe trails lasted 120 seconds and since the platform was unavailable, escape latency is not a relevant measure. In order to reveal how well the animal remembered the platform location during the probe trial, we calculated the amount of time spent in each quadrant of the water maze, proximity to the platform location and time to the first entering of the platform zone (latency).

Latency was calculated as the time from the rat was released in the water until it entered the platform zone (a circle slightly bigger than the platform, an area corresponding to 1%

of the area of the watermaze). Over the course of the week all animals decreases the time spent until entering the platform zone (figure 3.6a).

Proximity is a measure calculating average distance of the rat from the center of the platform location across the probe-trail (Gallagher et al., 1993). The fact that the drop points are located at four points of equal distance around the rim of the watermaze, will affect numbers such as path length and proximity. Yet, there is a discernible decrease in the average distance the rat is from the center of the platform as the week progresses (figure 3.6b). The proximity measure coupled with the percentage of time spent in the target quadrants, gives a clear indication that the animals have learned the task.

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(a) (b)

(c)

Figure 3.5: Escape latency for all animals during acquisition week

Escape latency for all animals shown separatly. The escape latency is the time the animal used to find the escape platform. An average of the four trials performed each day on all animals of the the group. Graphs display the escape latency for each individual animal, to illustrate the spectrum of results. The error bars shows standard error of the mean (SEM). (a) Results for rat 1-4, Sham group. (b) Escape latency results for animals treated with chABC (rat 5-8). (c) The results for untreated animals (rat 9, 10 and 12).

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(a) (b)

Figure 3.6: Results from probe-trails without platform present.

(a) Latency to platform zone is calculated as the time from the rat was released in the water until it entered the platform zone (an area corresponding to 1% of the area of the watermaze). At the end of the first training week all groups reach the platform zone in shorter time than the first day. (b) Proximity is a measure calculating average distance of the rat from center of the platform location across the probe-trail. Proximity measure of the first trial week shows that on average the animals spends more time in the platform vicinity at the end of the week than at the start.

Figure 3.7b shows the percentage of time spent in each quadrants for all groups. All groups starts spending almost the same amount of time in the four different quadrants on the first two days. Later there seem to be a preference for the two northern quadrants in all groups. On the final day the three groups spent more than 35% of the trial time in the target quadrant, where the platform was normally located. Figure 3.7d shows that there was a similar increase of percentage time spent in the target quadrant indicating.

With regards to this figure, all groups show no difference in learning of the task.

3.2.2 Long-term memory

Testing of long-term memory were performed approximately two weeks after the end of the first training week. The group that underwent chABC treatment in the first round, have been named chABC1, and the group that received chABC treatment 10 days after the end of the first training week, have been named chABC2. An animal who has undergone training were added to the group being injected with chABC between the first and second week, so that there is 4 animals in each group.

The probe trial of the first day of the second week is what can be considered the remote memory test. The latency numbers found in figure 3.9 shows that the three groups perform equally on day one. The percentage of time spent in the four quadrant do not differ greatly between the groups on the remote memory test (3.8). The time spent in the different quadrant are more equally distributed for day one, though all groups still spend most of the time in the two northern quadrants and particularly in the target quadrant.

The graph displaying the percentage of time spent in the target quadrant for the two days shows that all groups drop somewhat in the percentage, but do not differ greatly from week one to week two, nor between the three groups.

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(a) (b)

(c) (d)

Figure 3.7: Quadrant measurements for the acquisition week

Average amount of time spent in the four quadrants, given in percentages. During training the platform was always located in the target quadrant, North East (NE). All groups developed a preference for the target quadrant compared to the opposite SW.(a) Control group with sham operated rats. (b) chABC treated animals (c) Control group with untreated animals (d) The time spent in the target quadrant over the week is plotted.

Data points represents an average of the three/four animals in each group.

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(a) (b)

(c)

Figure 3.8: Remote memory assessed by time-in-quadrant analysis

(a) The time spent in the different quadrants for all groups on the final day of the acquisition week. (b) Probe trial testing spatial memory two weeks after the last day with training. Percentage of time spent in the four quadrants. (c) A comparison of time spent in the target quadrant on the final day of the acquisition week and the first day of the second testing week. All groups show a decrease in average time spent in the target quadrant, indicating that all groups remembered the platform location. There were no significant differences between the groups.

Figure 3.9: Latency for the second and third testing week

The y-axis time to find the platfrom (seconds), and the x-axis represents the training days.

Latency is calculated as the time from the rat was released in the water until it entered the platform zone (an area corresponding to 1% of the area of the watermaze). The two weeks are represented in the same graph to better observe the different test groups(sham operated, N=4, chABC, N=4, untreated, N=4) response to the change in environment.

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Figure 3.10: Escape latency for the second testing week

The y-axis time to find the platfrom (seconds), and the x-axis represents the training days. The average escape latency of the four trials performed each day for each animal was calculated and averaged again to represent the test group (sham operated, N=4, chABC, N=4, untreated, N=4). The sham and chABC1 group both decrease the already low escape latency. The chABC2 group performs poorer than the two other groups, with an unexplainable increase on day 5.

Measures during training week 2

Figure 3.10 shows the escape latency for the training with the same platform location as in the first week. They start out at almost the same levels as they ended with in the first training week and the during the first days the latency time decrease a bit further before reaching asymptotic levels for the remainder of the week. The sham group had an average of 32 seconds on day five of the first training week, to an 32 seconds average on the first day of the second testing week. This is similar for the chABC1 group, which goes from an average of 28 seconds, to 25 seconds at the start of the second week. The group treated with chABC prior to the remote testing week, chABC2, is the only one that show a decline in performance on the escape latency measure. The animals in the chABC2 group has an average of 37 seconds at the end of the first training week, and starts with an average of 42 seconds in the second training week. This group continues to have a greater escape latency than the other two groups throughout the week.

If the results from training week 2 is representative, it suggest that chABC may have an effect on spatial memory, the ANOVA analysis gave a significant effect of recent drug treatment (F (2, 9) = 7,863, P = 0,0106). Time is considered not quite significant (F (4, 36) = 2,377, P = 0,0701), and the interaction is not significant (F (8, 36) = 0,9547, P

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Probe trials from the second week

The quadrant measure is a calculation of the average time spent in the four quadrants.

Figure 3.11 shows the percentage of time spent in each quadrant for the three groups, as well as the time spent in the target quadrant over the course of the week. The platform location is the same as in the training week, which is located in the NE quadrant. The animals in the sham group develop a larger preference for the target quadrant. They start out at the same percentage as they had by the end of the acquisition week, and by the end of the second week spend more than 50% of the time in the target quadrant. The chABC groups preference falls somewhat in the time between week one and two, lowering to just below 30%. During the week the group spends more time in the two northern quadrants and by day five spend again more than 35% of their time in the target quadrant. The chABC2 group maintains a strong preference for the target quadrant at the start of the second week, but develops over the course of the week, a stronger preference for the north western quadrant.

The graph displaying the percentage of time spent in the target quadrant shows that both the sham and the chABC1 group increase their preference for the target quadrant.

The chABC2 group does not increase the time spent in the target quadrant by the end of the week, and the percentage remains mostly the same throughout the week.

3.2.3 Learning a new platform location

In the third testing week, the platform location was changed from the north east quadrant to the south western (SW). In addition, some external cues were changed and added. A brown curtain covered the metal plate at the northern side of the pool, and an A3 sized colored picture was put up at the western side of the tub. Figure 3.13 depicts the swim path of rat8 during the second testing week and the novel environment testing. The trial shown from day 1, is the animals worst trial of the day, but the escape latency is still almost half the time from the trials of day one in the acquisition week. Figure (b) shows the swim path on day five, where the rat quickly found the platform with an escape latency of merely 10 seconds. On the following day, the platform location was changed to the south west quadrant, and the external cues were changed/added. As expected the path length increases on day six. It can be observed that when the animal did not locate the new platform location on the first few tries, it searches an area in the opposite quadrant(i.e. old platform location). Figure (d) is on the last day of the reversal learning, and shows that the rat has again learned the platform location and that the swim path is a direct route to the platform.

As is expected when the distal cues and platform location are changed in trial week three, the escape latency increases (Figure 3.12). The sham and chABC1 group doubles their escape latency, from 14 and 15 seconds respectively on day five, to 35 and 30 seconds on the first day with the new platform location. The chABC2 group experiences a decrease in escape latency over the same two days, going from 52 seconds to 28. The sham group seems to follow the same learning curve as the previous week, but chABC1 needs another day to adapt to the novel environment.

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(a) (b)

(c) (d)

Figure 3.11: Quadrant measures from the second testing week

This is a measure of average time spent in the four quadrant for the three test groups, given in percentages. The platform location is still in the NE quadrant. The increasing time spent in the target quadrant over the week is projected in (d). (a)Percentage of time spent in quadrants for sham rats over the course of the second week. Day 1 percentages are similar to the groups percentages on day 5 of the acquisition week. By day 5 the animals spend more than 50% of the time in the target quadrant. (b) The chABC animals begin and end the week with a preference for both of the northern quadrants, though with more time spent in the target quadrant. (c) The chABC2 group also display a near equal preference for the two northern quadrants, but unlike the two other groups have a higher preference for the NW quadrant and not the target quadrant, NE. (d)Depiction of percentage time spent in target quadrant. An average of the four animals in each group.

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Figure 3.12: Escape latency for the third testing week

The y-axis time to find the platform (seconds), and the x-axis represents the training days. The average escape latency of the four trials performed each day for each animal was calculated and averaged again to represent the test group (sham operated, N=4, chABC, N=4, untreated, N=4). All animals were successful in learning the new platform location, which can be seen in the decrease in time spent before locating and climbing onto the platform. No deviant behavior can be observed between the groups.

The ANOVA results for the escape latency from the third trial week suggest that treatment with chABC does not have an significant effect on reversal learning. The only significant result from this was time, with a p-value of 0,0183.

Probe trials from the third training week

Figure 3.9 is a depiction of the latency during the probe trials of the remote testing and the new learning. As this is the probe trial, day six is the first time with the new platform location. It is interesting to note that the sham group and the chABC2 groups behave as expected, searching for the platform location in the old location, thus resulting in an increase in the latency. The first group who were treated with chABC does not show increased latency, it is up to speculation if this is due to chance or some differences in swim patterns by animals in this group.

The behavior in general from day six to ten, is somewhat erratic, the sham and chABC2 groups shows the expected decrease on days seven and eight, but for unknown reasons, the latency increases again on days nine and ten. While this effect is seen during the probe trials, it does not appear to have any effect on the escape latency during the training trials.

The percentage of time spent in the quadrants during the probe trials in week three is displayed in figure 3.14. On the first day with a new platform location, day 6, the

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(a) (b)

(c) (d)

Figure 3.13: Swimtrack for rat 8 from testing week two and three

Swimtrack of rat 8 during testing week two and three. (a)Taken from day 1 of trial week 2. (b) The last day of a trial week shows a fairly direct route to the escape platform.

(c)This is a swim path from day 6, the first day of trial week 3, where the platform have been moved and external cues have been added. (d)The swim path have again evolved into a direct route to the escape platform on day 10.

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(a) (b)

(c) (d)

Figure 3.14: Quadrant measurements from testing week three

Quadrant measurements for the three test groups for week three. This is a measure of average time spent in the four quadrants, given in percentages. Over the course of the week all groups increase the time spent in the target quadrant, SW. (a)Sham (N=4) group revert back to almost chance level on day one. By day 5 the animals spend more than 35% in the new target quadrant. (b) The chABC (N=4) animals shift their preference from the two northern quadrants at the end of week two, to the two southern quadrants by the end of week three. (c) The recently chABC treated group (N=4), displays chance level on day 1, and by the end of the week spend more than 35% of the time in the SW quadrant. (d) Display of percentage time spent in target quadrant, an average of the four animals in each group. All groups increase the time spent in the target quadrant.

percentages revert back to chance levels for the sham operated controls. Over the course of the week, the animals develop a preference for the new target quadrant, SW. The more recently chABC treated group, chABC2, display a similar behavior as the sham group.

The chABC animals shift their preference from the two northern quadrants at the end of week two, to the two southern quadrants by the end of week three. During the third testing week, all groups experience an increase in time spent in the new target quadrant.

At the end of the week the groups spend about 35% of the time in the target quadrant, a similar level to that found in the acquisition week.

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Chapter 4 Discussion

4.1 Main findings

The aim of this work was to study if the removal of PNNs affect acquisition, remote memory or the ability to adapt to new environments. The results indicate that the removal of PNNs prior to remote memory testing does not affect the recall of spatial memory.

However, on all days during the second testing week, the chABC2 group performed con- sistently worse than the other two groups and did not develop a preference for the target quadrant in the probe trials.

Performing an analysis of variance on the results from the training week shows time as a significant effect. Time is considered a significant effect during the third testing week as well, when the platform location has been changed and the distal cues have been altered.

The animals are fully capable of acquiring the knowledge necessary to escape the task, regardless of chABC treatment. This is supported by the decrease in escape latency, as well as the preference for the target quadrant the animals developed over the course of the weeks. The results given in this study indicates that the removal of PNNs does not affect acquisition of spatial memory in the medial entorhinal cortex. Being treated with chABC does not appear to have a significant effect on an animals ability to quickly adapt to a novel environment.

4.2 Methodological consideration

The sample size in this project was small, due to the fact that it was a pilot project.

Pilot studies are often recommended by scholars and consultants to address a variety of issues, including preliminary scale or method development. Specific concerns such as item difficulty, item discrimination, internal consistency, response rates, and parameter estimation in general are all relevant (Johanson and Brooks, 2010). Determining the number of animals needed for a study depends on many variables, including an estimation of the standard deviation and a defined significance level (i.e. p-value). The objective of the current pilot study was to identify possible effects of the removal of PNN, and to identify

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possible trend lines. Therefore, we chose to keep a low sample size. However, there are several drawbacks to having a small sample size, this includes a reduced chance of detect- ing significant findings and a reduced ability to generalize the study. The small sample size in this study, made it difficult to find significant differences and draw conclusions.

In the present study we used thin glass pipettes and several small injections at multiple sites for injections instead of Hamilton syringes and one single large injection reported in previous studies. The latter has the danger of causing tissue damage. The histology revealed minimal tissue damage after microinjections of chABC. We did not check the amount of cells affected by the chABC treatment, but a previous study performed in our group by A. Kvello (2014) showed that 1.5µL, per hemisphere, of chABC microinjections in MEC is sufficient to reduce PNNs to a minimum in the area.

The enzymatic digestion by chABC targets chondroitin sulphate proteoglycans of the ECM, leaving behind a 6-monosaccharide stub on the CS chain (Kwok et al, 2011). CSPGs are not only found within the PNNs but in the overall ECM composition of the adult CNS (Kwok et al, 2011), and the chABC treatment causes all extra cellular matrix structures with CSPGs to be digested. Thus, enzymatic treatment disrupts the ECM in general in the injected area. Because of this it is important to note that the results found may not be caused specifically by the disruption of PNNs, but the removal of the ECM in general.

The lack of required pretraining and cross-species utility are among the factors that has made the Morris watermaze an attractive method to test spatial memory and navigation.

Rats and mice being the most common test animals, but using a virtual maze, an adapted version can be used on humans. The MWM also has extensive evidence of its validity as a measure of hippocampally dependent spatial navigation and reference memory (Vorhees and Williams, 2006). However, the Morris watermaze performance is influenced not only by factors such as training procedure and apparatus, but also by the characteristics of the animals undergoing the task (sex, species/strain, age, nutritional state, exposure to stress or infection) (D’Hooge and Deyn, 2001). As is seen in the present study, there is a high degree of individual differences that are difficult to identify and eliminate. Given the sample size in this project it difficult to account for these individual differences.

4.2.1 Probe trials

During the development of the protocol for this project it was decided on having a daily probe trial, with the duration of two minutes, at the start of the day. The length of the probe trial according to Blokland et al. (2004) is most favorable at 30 seconds. According to this study, the preference the rats have for the target quadrant visibly drops after the first 30 seconds. The results from investigating the rats behavior shows a clear preference for the target quadrant at the end of the trial weeks, but as it looks only at the two

The role of perineuronal nets in spatial memory