REPRESENTATION OF OBJECT-IN-CONTEXT WITHIN MOUSE HIPPOCAMPAL NEURONAL ACTIVITY. Herborg Nanna Ásgeirsdóttir. A Thesis Submitted to the Faculty of

REPRESENTATION OF OBJECT-IN-CONTEXT WITHIN MOUSE HIPPOCAMPAL NEURONAL ACTIVITY by Herborg Nanna Ásgeirsdóttir A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Master of Arts Florida Atlantic University Boca Raton, FL August 2013

ACKNOWLEDGEMENTS I would first of all like to thank my advisor, Dr. Robert W. Stackman for believing in me and giving me the invaluable guidance towards to completing this thesis. I would like to thank my committee members, Dr. Robert W. Stackman, Dr. Elan Barenholtz, Dr. Rodney Murphey, and Dr. Robert Vertes for their invaluable input for preparing of this thesis. I would like to thank my family and friends for countless words of encouragement and especially my parents; without them, none of this would have been possible. I would like to thank all my colleagues in Dr. Stackman s lab for always being willing to help. I would like to thank the National Institutes of Health for the support provided by grant NIH: R01MHO86591. iii

ABSTRACT Author: Title: Institution: Thesis Advisor: Degree: Herborg Nanna Ásgeirsdóttir Representation of Object-in-Context within Mouse Hippocampal Neuronal Activity Florida Atlantic University Dr. Robert W. Stackman, Jr. Master of Arts Year: 2013 The rodent hippocampus is critical for processing spatial memory but its contribution to non-spatial, specifically object memory is debated. The cognitive map theory of hippocampal function states that the hippocampus stores relationships of goal locations (places) to discrete items (objects) encountered within environments. Dorsal CA1 place cells were recorded in male C57BL/6J mice performing three variations of the novel object recognition paradigm to define object-in-context representation of hippocampal neuronal activity that may support object memory. Results indicate, (i) that place field stability is higher when polarizing environmental cues are provided during object recognition; (ii) hippocampal place fields remain stable throughout the novel object recognition testing without a polarizing cue; and (iii) time dependent effects on stability when objects were dissociated from the context. These data indirectly support that the rodent hippocampus processes object memory, and challenge the view that iv

object-in-context representations are formed when mice perform novel object recognition task. v

REPRESENTATION OF OBJECT-IN-CONTEXT WITHIN MOUSE HIPPOCAMPAL NEURONAL ACTIVITY List of Figures... viii Part I: Introduction...1 1.1 Why we use Rodents to study Human Memory Systems...1 1.2 Memory Processes...2 1.3 Memory Systems...3 1.3.1 Implicit Memory...4 1.3.2 Declarative Memory...5 1.4 Anatomy of the Hippocampal Circuit...5 1.5 Spatial Memory and the Hippocampus...7 1.5.1 Neuronal Correlates of Spatial Memory...9 1.6 Non-Spatial Memory and the Hippocampus...14 1.7 Object-In-Context Representation...20 1.7.1 Object Related Neuronal Activity...21 1.8 Current Study: Purpose and Hypothesis...23 Part II: Materials and Methods...31 2.1 Subjects...31 2.2 Electrode Construction...31 2.3 Surgery...32 2.4 Habituation to Recording Apparatus...33 2.5 Histology...34 vi

Part III: Place Cell Recordings and Object Discrimination...36 3.1 Recording Materials...36 3.2 Place Cell Criteria...37 3.2.1 Cue Card Rotations...38 3.2.2 Stability Measures...39 3.3 Data Analysis...40 Part IV: Results...43 4.1 Place Cell Recording Results...43 4.1.1 Place Field Rotations Results...47 4.1.2 Object Specific Activity Results...47 4.1.2 Novel Object Recognition Results...48 4.2 Place cell Stability Discussion...49 4.2.1 Use of Objects as Landmark Cues...50 4.2.2 Influence of Delay between Sessions on Place Cell Stability...52 4.2.3 Novel Object Recognition Discussion...54 4.2.4 Correlation between Place Field Stability and Object Memory...55 Part V: General Discussion...56 5.1 Rotations of Internal Spatial Representation...56 5.2 Object Specific Activity...59 5.3 The Cognitive Map...61 5.4 Conclusion...64 Appendix...66 References...79 vii

LIST OF FIGURES Figure 1. Separation of declarative and nondeclarative long-term memories...66 Figure 2. The hippocampal circuit...67 Figure 3. The Where and What pathways...67 Figure 4. Novel Object recognition behavioral testing...68 Figure 5. Novel Object Recognition protocols...69 Figure 6. Objects used to study object memory...70 Figure 7. Interneuron and Pyramidal neuron characteristics...70 Figure 8. Cluster cutting parameters...71 Figure 9. Histological analysis of electrode placement...72 Figure 10. Complex Spike Activity by place cells...72 Figure 11. Stability measures for protocols and delay intervals...73 Figure 12. Regression analysis...74 Figure 13. Place field rotations within symmetrical environment...75 Figure 14. Place Field Examples from the tree different protocols...76 Figure 15. Object related activity within dorsal CA1...77 Figure 16. Novel object recognition discrimination ratio results...78 viii

PART I: INTRODUCTION 1.1 Why we use Rodents to study Human Memory Systems Exploring the impairments of memory following trauma or disease provide an initial discernment of the functions of human memory. Case studies, such as the wellknown example of H.M. who had bilateral medial temporal lobes surgically removed to alleviate his severe epilepsy, have provided invaluable understanding of the anatomical substrates for memory. Despite the extensive knowledge we learn from cases like H.M., we cannot specifically replicate, manipulate or salvage experimentally the specific memory functions lost or obtained in individual human cases. To further our understanding of how healthy human memory functions and to define memory deficits after damage to specific anatomical regions of the brain, non-human studies can be experimentally carried out to enhance our knowledge of the underlying circuits of memory. In order to draw appropriate conclusions from animal models of human memory, we must first ensure that the specific brain region has anatomical and behavioral similarities between the species (Cave & Squire, 1991). Rodents have proven an excellent model to answer questions regarding the molecular, cellular, and behavioral mechanisms that underlie mammalian long-term memory. However, the degree to which such mechanistic findings from rodent studies can be fully applied to human memories and the appropriateness of rodent models to infer knowledge regarding specific memory functions in humans has been questioned. Providing additional support for the 1

resemblance between the rodent and human memory system will bridge the gap in the experimental field towards answering mechanistic questions regarding human memory by utilizing rodent models. 1.2 Memory Processes The main purpose of memory is to be able to store newly acquired information so that the learned material can be utilized later on. Memory consequently involves multiple processes to create the new mental representation of the material, store it within a circuit or a distinct location, and later recall the information when required. These processes are referred to as encoding, consolidation and retrieval of the memory, respectively. Reconsolidation of a memory refers to the process of altering or adding to a previously encoded memory, to render it stronger, weaker, or to alter the meaning of the stored information. Multiple studies, aiming to dissociate the anatomical regions where specific memory processes occur have yielded abundant data with ambiguous conclusion. However, a meta-analysis of 54 Positron Emission Tomography (PET) studies suggested both encoding and retrieval occur bilaterally within the hippocampus in human subjects, a structure that lies deep within the medial temporal lobe. The report stated that encoding of both verbal and non-verbal novel information occurs within the more rostral portion of the hippocampus, whereas the retrieval of that same information seems to be processed in the caudal portion (Lepage et al., 1998). Consolidation of memory processes has proven more difficult to explore using human subjects but animal models have indicated that this process also occurs within the hippocampus and the strength of the memory can be dependent upon emotional arousal related to amygdala activity (McGaugh, 2000). 2

Although H.M. s surgery significantly decreased his seizure activity, he could no longer verbally discuss new memories for more than few seconds. Given the results from Lepage et al., his deficits did not stem from the encoding of new memories since structural Magnetic Resonance Imaging of his brain revealed that most of his caudal hippocampal formation remained intact (Corkin et al., 1997). Seemingly, it appears that H.M. s anterograde amnesia resulted from the inability to consolidate or retrieve specific memories encoded. Further cases have been reported where damage to regions within the medial temporal lobe resulted in severe memory impairment. For example, patient R.B. suffered an ischemic episode following heart surgery that restricted blood flow to the hippocampus, consequently damaging the structure. He demonstrated severe anterograde amnesia following the episode that was later contributed to bilateral lesions of the entire CA1 region of the hippocampus (Zola et al., 1986). Despite no other cognitive impairments observed in the patient, his anterograde amnesia remained severe for the remainder of his life, with extremely minor improvements within a year onset. Together, human case studies have narrowed down the anatomical structures required for specific types of memory. 1.3 Memory Systems Human memory can further be classified according to its duration or by type of function. Short-term memory is different from long-term memory due to its limited amount and relatively short time of storage. Long-term memory refers to memories that are retained for more than a few minutes and after they are consolidated, can sometimes last a lifetime. Furthermore, there are two distinct types of long-term memory; things we 3

learn and can talk about such as facts and events are called declarative memories. Nondeclarative or implicit memory refers to information learned and retained for an extended period of time, but which cannot be consciously recollected or talked about in detail. These different kinds of long term-memory systems will be further discussed in the following sections. 1.3.1 Implicit Memory Memories that guide our behavior without conscious recollection or verbal declaration is referred to as implicit memory, such as that categorized in our stored knowledge of how to ride a bike, or the skills gained during mirror drawing training. These memory processes have been found to be supported by structures outside of the medial temporal lobe and were all spared after H.M. s surgery (Figure 1 A). Nondeclarative memory is used to describe various skillful behaviors such as habits or conditioning. The striatum is believed to be responsible for procedural memory, mainly simple skills and habits whereas the cerebellum, with the support from the amygdala, provides neural substrates for simple classical conditioning. Priming as well as perceptual learning was discovered to be unaffected in amnesic patients such as H.M. and later found to be supported by areas of the neocortex (Squire, 2004). The view that these types of memory are implicit processes, which one does not need to consciously recall has been supported from animal and non-human primate studies (Squire, 1992); nondeclarative memories such as priming are consciously recollected but are expressed through performance (Squire, 2004). 4

1.3.2 Declarative Memory When the term memory is referred to in everyday language, it is usually declarative memory that is implied. Long-term memories we can consciously recollect and discuss in a representational way are believed to be dependent on intact medial temporal lobes and especially the hippocampus (Eichenbaum, 2000; Squire, 1992). Declarative memory is further dissociated into semantic and episodic memory as a way to separate the memories for facts from events that occur in our lives (Figure 1 B). Semantic memory refers to remembered information that is independent of specific episodes, and that may have been acquired through and require effortful, conscious recollection (Eichenbaum, 2000). Episodic memories however, provide the ability to re-experience a past event in the original context, but this process is thought to also require the participation the frontal lobe (Squire, 2004). These episodic memories are highly dependent on the hippocampus and neurophysiological evidence may provide support that context specific processing occurs within the structure. Long-term declarative memory that depends upon the hippocampus and surrounding structures will be the main focus of this thesis. 1.4 Anatomy of the Hippocampal Circuit Long-term declarative memory in humans and other mammals depend critically upon a circuit of interconnected structures within the medial temporal lobe. The hippocampal formation; consisting of the dentate gyrus, CA3, CA1 and the subiculum, contains a complex circuit pathway with input from neocortex through the entorhinal cortex (Figure 2). The formation or encoding of new declarative memory is considered to be dependent on the flow of multimodal sensory experiential information through the 5

hippocampal formation. Associative cortical regions such as the perirhinal cortex (PER) and postrhinal cortex (POR) receive this input and then these structures differentially distribute the information through the parahippocampal region. Specifically, the PER sends projections to, and receives input from the lateral entorhinal cortex whereas the POR sends projections to, and receives input from, the medial entorhinal cortex. The lateral and medial entorhinal cortices are considered to receive input from two distinct pathways, respectively the what and where pathways and the information streams converge in the hippocampus (Figure 3). These pathways have been suggested to receive distinct representational inputs. The lateral entorhinal cortex receives nonspatial information consisting of olfactory, auditory and visual object information whereas medial entorhinal cortex is thought to process spatial and idiothetic information (Hunsaker et al., 2007) before both areas project the information to the hippocampus. The well-characterized trisynaptic pathway through the hippocampal formation (dentate gyrus, hippocampus proper and subiculum) is defined as axonal projections from the medial and lateral entorhinal cortices, which terminate on granule cells of the dentate gyrus. Axons of dentate granule cells, termed mossy fibers, distribute to pyramidal neurons of area CA3 of the hippocampus proper. Axons of the CA3 neurons, termed Schaffer collaterals distribute to pyramidal cells in area CA1 of the hippocampus. CA1 then projects output from the hippocampal formation through the subiculum and to the entorhinal cortex again, completing this prominent circuit within the hippocampal formation (Strien et al., 2009). The entorhinal cortex also receives input from the presubiculum and in turn, projects to the dentate gyrus and CA3 through layer II and CA1 6

and subiculum through layer III (Amaral et al., 2009). These structures and pathways have been identified as key areas for processing episodic and semantic memory. 1.5 Spatial Memory and the Hippocampus It is well established that the human, as well as the rodent hippocampus plays an important role in spatial navigation and learning about new environments. Spatial memory is a form of episodic memory that allows one to associate a location to a specific event. Rodents will quickly learn the location of a specific place where they receive reward or to avoid aversive stimuli by associating the goal location with available environmental cues. Spatial memory in rodents and other smaller mammals can be studied using various methods and the most common tool to study spatial memory in rodents is the Morris water maze. The Morris water maze tests for spatial memory in a circular pool where rodents must learn to locate a submerged platform in a pool filled with opaque water by using external cues. Over several trials, rodents can be trained to find the platform location and later can be tested by removing the platform and measuring times spent searching in the appropriate area of the pool. Morris et al. (1982) showed that rats do not simply recognize the correct location of the platform when they reach it, but learn to efficiently navigate directly to it from a novel start location; essentially creating a short cut to the target location. Morris et al. concluded that efficient performance in the water maze required that the rodent form a hippocampal-dependent spatial or cognitive map of the platform position relative to the surrounding visual cues. When the hippocampus is lesioned prior to training, rats are significantly impaired at learning to use the distal cues to locate the hidden platform (Morris et al., 1982). When a selective AMPA antagonist is chronically infused into the dorsal CA1 of hippocampus rats during 7

training, rats show impaired acquisition as measured by significantly longer escape latencies (Reidel et al., 1999). Together, these data indicate that the hippocampus is required for the encoding and/or consolidation of new spatial memories. When the hippocampus of a successfully trained rat is temporarily inactivated only before the water maze testing session, rats demonstrate a spatially localized searching behavior but not in the appropriate location (Reidel et al., 1999). This indicates that a fully functioning hippocampus is required to retrieve information about a location whereas searching strategies are likely to depend on a different brain region. Furthermore, the hippocampus seems to be functionally dissociated between the dorsal and ventral regions. When various volumes of either dorsal or ventral hippocampus of rats is lesioned, findings demonstrate that mainly the dorsal region is important for spatial learning in the water maze and navigational impairments seem to be positively correlated to dorsal hippocampal volume damaged (Moser et al., 1993). It appears that although the hippocampus is required to encode, consolidate, and retrieve new spatial memories, the dependence of retrieval of remote long-term memory on the hippocampus has been debated. A spatial memory experiment conducted on the case study of patient E.P. revealed that although he was unable to create new spatial memories, he was able to very accurately recall the layout of the neighborhood he grew up in 50 years prior (Teng & Squire, 1999). This finding would suggest that the spatial memory was stored in a secondary region where retrieval was unimpaired, but also raises further questions regarding spatial memory development during childhood. Other tasks have been developed to study spatial memory, both in rodents and other species. The Hebb-Williams maze is designed to test navigational memory in 8

rodents. Despite not having clearly defined components of what kind of spatial memory it tests; a reference or long-term memory is required to acquire the fixed location of the goal position. The Hebb-Williams maze is a task composed of 12 mazes, varying in difficulty where the start and goal positions are kept consistent. Each maze is presented six times in a row and the number of errors made is measured. Depending on how fast errors are reduced over the 6 consecutive sessions, the animal can be said to retain the spatial short-term memory for that maze depending on their learning index (Glasier et al., 1997). Kveim et al. (1964) tested rats with partial or nearly total hippocampal volume loss and found that their cortical lesion counterparts significantly outperformed them. They did however note, that rats with total hippocampal lesions made significantly more errors than the rats with partial lesions of the hippocampus. Further studies demonstrate that temporary inactivation or permanent lesion of the rat dorsal CA1 produces impairments in the Hebb-Williams maze between-days (long-term spatial memory). However, the rats did not demonstrate impairments within-days (short-term spatial memory) when the hippocampus was compromised (Vago et al., 2007). This provides further support for the positive relationship between spatial abilities and proportion of the hippocampus lesioned, as well as indicating functional differences between encoding and retrieval of long-term spatial memories. 1.5.1 Neuronal Correlates of Spatial Memory The discovery of place cells by O Keefe and Dostrovsky in 1971 has provided a potential hippocampal mechanism that supports successful spatial navigation, and an explanation for impaired navigation in hippocampus-lesioned rats. Place cells, recorded from freely-moving rodents, are specific pyramidal neurons within the hippocampal 9

formation that fire at a high frequency corresponding to animal s physical location in space. These cells are silent when the animal is out of the cell s so called place field but generally remain stable (fire at same location at similar Hz) when the animal returns to that specific area (O'Keefe & Dostrovsky, 1971). The well-defined characteristics of hippocampal place cells provide strong support for the role of the hippocampus in spatial memory (O Keefe & Nadel, 1978). For example, it is likely that when the dorsal hippocampus is temporarily inactivated, as in Reidel et al., (1999), the resulting navigational impairments are a consequence of compromised place cell function. Hippocampal place cell recordings are carried out by surgically placing tetrodes into the hippocampus of freely moving rodents, which detect extracellular action potentials when the wires are close to the cell body of a hippocampal principal neuron. The development of the tetrode permits recordings of many individual neurons simultaneously and such ensemble activity recordings have provided support for the view that place cells are a component of spatial mapping within the hippocampus (Wilson & McNaughton, 1993). The cognitive map theory states that place cells provide an internal map of the environment and offer the ability to create novel routes to familiar locations as long as the map remains stable (O Keefe & Nadel, 1978). The proposed theory suggested that the place cell system and the hippocampus work to represent the global record of experiences, including spatial and non-spatial elements (O Keefe & Nadel, 1978). When an animal encounters a novel recording arena; different from a familiar one, the hippocampal representation transforms in an unpredictable manner, creating a new map as a result of differences in the active subset and characteristics of individual cells (Bostock et al., 1991). This process, called remapping, occurs when a separate set of 10

place cells fire within a different environment in a different pattern (Fyhn et al., 2007). For instance, place cells recorded in a cylinder environment remap when the animal enters a different geometrical environment such as a square arena or a linear track. Some of the same cells may contribute to the cognitive map in the square arena as in the cylinder but other cells turn silent. Depending on how long these place fields are able to remain consistent in a given space with repeated exposures could give some insight to how spatial memories develop over time. The stability of place cells in mice over long periods of time has been somewhat debated although long term-place field stability in rats has been demonstrated to last up to 153 days (Thompson & Best, 1990). Recently, new technology has allowed researchers to observe place cell firing within a familiar environment for weeks at a time using Ca 2+ sensitive dyes and a head mounted camera. Ziv et al. (2013) concluded that mouse place cells remain largely stable for over 11 weeks with some overlap and fluctuations during encoding and recollection of the environment. Place cells have been studied extensively and the application of the same methodology to other limbic regions has determined the presence of other spatial correlates, which collectively are likely to support spatial behavior. For example, hippocampal place cell activity is influenced by grid cells within the medial entorhinal cortex (Fyhn et al., 2007; Brun et al., 2008), and by head direction cells found in the postsubiculum (Taube et al., 1990), anterior thalamus (Taube & Burton, 1995; Yoganarasimha & Knierim, 2005), and the lateral mammillary nuclei (Stackman & Taube, 1998). The firing properties of grid cells resemble place cells except that they fire in multiple locations in a hexagonal pattern within a familiar environment. Grid cells 11

have been found to strongly modulate place cells and experiments have been conducted to determine the exact functional pathway between the medial entorhinal cortex and the dorsal hippocampus. Simultaneous recordings from the entorhinal cortex and hippocampal place cells in rats have demonstrated direct input from the medial entorhinal cortex to CA3 and CA1. As hippocampal place cells are only one synapse upstream from the entohrinal grid cells via a direct pathway from the medial entorhinal cortex to CA1, lesions of the entorhinal cortex have been shown to disrupt firing fields of established place cells (Brun et al., 2008). Furthermore, grid cells in the entorhinal cortex depend on the information from intact hippocampus within the complete circuit to maintain stable place fields. When muscimol was infused into the CA1 in rats to temporarily inactivate the hippocampus, the structured spatial firing properties of grid cells was impaired for at least 150 min (Bonnevie et al., 2013). When a rodent is placed in a novel environment; place cells remap, and the activity is accompanied by a corresponding shift in the grid cells, to represent the spatial differences within the environment. Together, grid cells and place cells provide the location-specific information to maintain a stable cognitive map of the environment. Head direction cells on the other hand are controlled by a complex combination of proximal and distal environmental cues and have been shown to mediate the control of distal landmarks of place fields. These cells fire in accordance to the rodent s head direction in the horizontal plane and are independent of the animal s specific location in space. Head direction cells (Stackman & Taube, 1997) and hippocampal place cells (Stackman et al., 2002), are both highly dependent on the vestibular system and repeated disorientation of the animal can promote place cell remapping in a familiar environment 12

(Knierim et al., 1995). The location-specific firing properties of hippocampal CA1 place cells are dependent upon neuronal input to the hippocampus from postsubiculum and anterior thalamic nuclei, presumably head direction cell input (Calton et al., 2003). Therefore, place cells, grid cells and head direction cells all contribute to an animal s perception and recognition of space to create a single representation of location and direction within an environment. Taken together, these recording studies repeatedly demonstrate the clear correlate of hippocampal, entorhinal, postsubiculum, and anterior thalamic neuronal activity to spatial memory. Recording such spatially tuned neurons from rodents engaged in the performance of hippocampal dependent memory tasks might provide further insight into human spatial memory functions. Recordings from the rodent dorsal CA1 of the hippocampus are the ideal location for various reasons. Primarily, this area is the main output of the hippocampus; projecting back to the subiculum as well as to other cortical areas. Secondly, the rodent dorsal hippocampus is positioned close to the skull s surface, allowing for relatively limited cortical damage to surrounding areas (Paxinos & Franklin 2004). Damage to less than 1 mm thick cortex is endured to place the recording wires close to the pyramidal layer within the CA1 of the hippocampus. The area of cortex that is prone to damage from the electrode serves as somatosensory processing area for the lateral torso, a region likely to be required for healthy perception of the rodent s environmental surroundings. Recording from other sites such as CA3, entorhinal cortex and surrounding areas of the medial temporal lobe may result in more damage to cortical areas. 13

1.6 Non-Spatial Memory and the Hippocampus In order to utilize rodent models to study functions of human memory we must determine if the rodent hippocampus plays an important role in non-spatial memory as it does in humans (Cave & Squire, 1991). The novel object recognition task (NOR) tests episodic memory for objects, through a well-established paradigm that can be modified to address specific questions (Antunes & Biala, 2012). The NOR test is typically carried out in a familiar environment, and objects that the animal gets to freely explore for a relatively short period of time, later to be remembered. The initial object session (Sample) is typically carried out with two identical objects in separate locations within a familiar arena. The rodent will usually explore the two objects equally, indicating that they recognize both of them as novel entities and equally interesting. The following session (Test) is conducted in the same environment where one of the previously encountered objects is present, but the other one is replaced with a never before seen object; object locations remain consistent between the sample and test sessions. The animal freely explores both objects and the amount of time the rodents explore each one is carefully recorded. As mice are naturally curious, they tend to explore new objects more than ones they are already familiar with (Ennaceur & Delacour, 1988). A rodent that fully encoded and consolidated a memory of the sample objects should retrieve that object memory during the test session and spend more time exploring the novel object, whereas a rodent that did not successfully encode and consolidate the memory of the sample objects cannot then retrieve that memory and will explore both objects equally during the test session (Figure 4). 14

The novel object recognition task has been widely used to study object memory and over time two main brain regions have been isolated as playing an important role in this form of memory. The perirhinal (PER) cortex has been identified by some researchers as being responsible for object memory in monkeys (Buffalo et al., 1999) and rodents (Winters & Bussey, 2005), whereas others disagree and state that the hippocampus plays that role (Clarke et al., 2010; Cohen et al., 2013; Broadbent et al., 2009). Similar studies have been carried out testing both areas with conflicting conclusions. There is a possibility that these contradicting results are due to differences in experimental protocols but in reality it is likely that both brain regions play an important role in creating and recollecting memories for objects. In the Winters & Bussey (2005) study, rats with lesions and temporary inactivation of the PER cortex explored appropriate objects in a Y-maze NOR task. Rats with lesions of the PER demonstrated equal preference for the novel and familiar objects during the test session, suggesting that the PER cortex encodes and consolidates object memory (Winters & Bussey, 2005). Other studies have provided support for the PER cortex playing an important role in visual object perception and that may provide an explanation for deficits during object recognition in the Winters and Bussey study. It is possible that inactivation of the PER cortex via local infusion of lidocaine, caused the rats to lose the ability to perceive visual information about the objects presented, whereas the actual object memory may have been intact. The what pathway conveys unimodal sensory information (such as object feature information) through the PER cortex, and then projects the object information to the hippocampus. It is important to identify these different processes as it offers difficulties in studying object memory in rodents when cued retrieval is blocked through 15

perceptual impairments. Burke (et al., 2012) recorded extracellular activity in the PER cortex in rats running a circular track with or without 3D objects and found that approximately 38% of neurons within PER respond specifically to objects. Results showed that PER activity was not specific to objects identity and was not modulated by experience, suggesting that neuronal plasticity in the PER cortex is not responsible for object memory, but may be important for object perception. Taken together, the neural substrates that support object memory, and the neuronal representations within the hippocampus and PER cortex that support object memory are not clearly established. Extensive research has also been carried out looking at object memory processes within the hippocampus. When the dorsal CA1 of the hippocampus is temporarily inactivated in rodents during various stages of NOR, after receiving 2 days of context habituation; impairments of object memory are seen; indicating that object memory is processed within the hippocampal circuit (Cohen et al., 2013). However, enhanced object discrimination has also been reported following a hippocampal inactivation (Oliveira et al., 2010). These conflicting results may be contributed to familiarization to the testing environment prior to testing. The increased preference of hippocampal muscimol inactivated mice for the novel object during the test session was only observed when the mice received one habituation session within the testing context. The enhancing effect of intrahippocampal muscimol disappeared when mice were given 5 habituation sessions prior to testing; object recognition task performances was comparable between the muscimol and vehicle treated mice (Oliveira et al., 2010). It is possible that providing either too little information regarding the testing context or providing extensive familiarization, makes it more difficult to explore the true object memory separately from 16

the context. More importantly, differences in object exploration during the sample and test sessions vary greatly between the experiments. Cohen et al. (2013) placed a strict criterion, a minimum 30 second exploration with each object during the 10 min sample session, and excluded mice that did not explore the objects during the 5 min test session. Imposing this criterion reduces the possibility that mice do not obtain enough information about the sample objects to encode the memory fully, and matches all mice for sample session object experience. Interestingly, few published reports of object recognition and hippocampal lesions have employed a criterion on sample object exploration, opting instead for a set time for the sample session (e.g. 15 min). Requiring C57BL/6J mice to explore sample objects for 30 s each, leads to a strong novel object preference during a test session 24 h later (Stackman et al., 2002; Hammond et al., 2004; Cohen et al., 2013). Given the average exploration by C57BL/6J mice of sample objects (22.5 sec (Veh) and 23.33 sec (muscimol)) and test objects (16.7 sec (Veh) and 13.6 sec (muscimol) during the 15 min sessions reported by Oliveira et al. (2010), it is possible that these mice did not sufficiently explore the sample objects fully to encode the object memory. When the rodent hippocampus is permanently lesioned, further conflicting results have been reported regarding object memory. There is a possibility that lesion size may contribute to impairments during object recognition. Ainge et al. (2006) described significant impairments during novel object recognition when the entire hippocampus was lesioned in rats, whereas partial lesions spared the object memory. The size of lesions and temporary inactivation does not seem to positively relate to performance during object recognition. Significant impairments can be seen when 1% of hippocampal volume is temporarily inactivated (Cohen et al., 2013; Hunsaker et al., 2007), whereas 17

permanent damage to more than 50% of the structure can result in unimpaired performance of the task (Mumby et al., 2002; Winters et al., 2004). Approximately half of the studies, carried out to determine whether rodent hippocampal lesions affect performance during novel object recognition have yielded no impairments during the task. The other half has found significant impairments in object memory (Squire, 2007) when the delay interval between sample and test sessions exceeds roughly 10 min (Clark et al., 2000; Mumby et al., 2002). It is beyond the scope of this thesis to evaluate those roughly 200 papers published regarding the matter, but a review by Squire et al. (2007) provides some possible theories. One notion is that memory strength can determine what medial temporal lobe region processes the information. A strong memory is associated with increased activity in the hippocampus, whereas a weaker memory does not require the hippocampus but may be related to surrounding cortical areas. The stronger memory is further determined to be recollection based, where the ability to recall the previously experienced stimuli without it being present at that time is possible. A weaker memory is more related to recognition, where the memory would not have had the ability to be retrieved without the presented stimuli. The theory is supported by human fmri data collected during the encoding and retrieval of memories (Squire et al., 2007). Lastly, the delay interval between the sample and test sessions appears to play an important role in how we interpret object memory dependence. Both permanent lesion studies and temporary inactivation of the rodent hippocampus seem to support the notion that object memory is not hippocampal dependent within 5-10 min of encoding. When the dorsal hippocampus is inactivated immediately before the sample session with fast onset lidocaine and mice are tested 5 min later, they show no impairments; preferring the 18

novel object. However, when tested 24 hours after receiving the lidocaine infusion, mice perform at chance (Hammond et al., 2004). Lesion studies demonstrate similar results. Damage to 75% of hippocampal volume spared object recognition performance when the rats were presented with the test objects either 10 sec or 1 min following the sample session, but impairments were seen following delays exceeding 10 min (Clark et al., 2000). Furthermore, hippocampal lesion rats demonstrated no preference impairments when tested with 1, 2, and 3 min delay intervals between sample and test sessions, whereas they did show deficiencies discriminating between object locations when they had been displaced (Mumby et al., 2002). This indicates that object memory does not become hippocampal dependent immediately; it may require time to consolidate from a weak memory to a strong hippocampal memory, whereas the spatial information regarding that object is processed by the hippocampus more rapidly. Similar to patient E.P., who was able to recall spatial memories acquired 50 years earlier; hippocampal object memory may be relocated to other brain areas when it has been fully consolidated. Rats who received hippocampal lesions 1 day, 4 weeks, or 8 weeks following a sample session did not all perform the same way during the test session. When tested 2 weeks following surgery; only the 1 day and the 4 week consolidation groups were significantly different from their control counterparts. The rats that had their hippocampus intact for 8 weeks after seeing the sample objects performed as well as the control rats. To exclude the possibility of spared hippocampal function, all groups received a second NOR sample session and when tested 3 hours later performed at chance. These data indicate time specific processing for both spatial and object memory within the hippocampus. It is possible that hippocampal dependent memories only require 19

the structure for a time sensitive period, before they become permanently stored in other regions. 1.7 Object-In-Context Representation What is defined as environment is not always consistent. Objects contribute to our surroundings - they may be landmarks where it can be difficult to separate them from the environment (i.e., the context) we encounter them in or they can be context independent, yet recognition seems effortless regardless of the environment. These issues prove difficult to resolve since objects can never be encountered without context. Place cell recordings have demonstrated clear correlates for the specific location of the animal within its context but fewer experimental questions have been answered regarding objectspecific neuronal activity. One of the main critiques of the NOR task is that rodents usually encounter the testing objects in the same arena, and it is truly the object in location or object in context memory that is impaired when the CA1 area of the hippocampus is inactivated. For investigation of the hippocampal dependence on nonspatial object memory, it is optimal that the testing arena be as limited in environmental cues as to avoid hippocampal activation due to the spatial input and to focus the attention on the objects during the sample and test sessions. Studies have shown that changing the location of a familiar object in a well-known arena results in increased exploration (Mumby et al., 2002), indicating that animal s recall that something has changed regarding that object and they explore novelty further. A novel object in a familiar location would also be explored widely whereas that object in a novel location would be explored more extensively (Save et al., 1992; Mumby et al., 2002; Manns & Eichenbaum, 2009). Thus, it has been suggested that the deficits in object recognition 20

tasks reported after temporary or permanent hippocampal lesions actually reflect impaired object-in-place or object-in-context memory. Such memories, being spatial in nature, are thus, accepted as hippocampal dependent. Cohen et al., (2013) examined the hippocampal dependence of object memory independent of context by presenting mice with the same sample session objects each day for 10 min in a distinct and different environment for 3 consecutive days. During a test session presented in another distinct context on the 4 th day, the mice preferentially explored the novel object. When the hippocampus was temporally inactivated by infusing muscimol into the dorsal CA1 of the hippocampus prior to the test session in a novel environment, mice could not perform the task successfully (Cohen et al., 2013). This result indicates that the object memory dissociated from context (i.e., truly non-spatial) is also dependent upon the hippocampus. Results suggests that mice are equally successful at retrieving the memory of the familiar object in an environment they are familiar with, as well as when it is presented in a neverbefore-encountered context when the hippocampus is intact. This may also indicate that although both spatial and object information are processed by the hippocampus, the location of the object is processed independently from the object identity itself. In order to further corroborate these object dissociated of context data, further experiments examining hippocampal neuronal activity need to be carried out. 1.7.1 Object Related Neuronal Activity Previous studies have examined how hippocampal place cells react to 3- dimensional objects placed into a familiar recording arena where novelty discrimination is not specifically recorded and the environment has clear geometrical or environmental cues. Extracellular recordings from dorsal CA1 area of rats running a circular track, with 21

or without objects; yielded no significant place field remapping between sessions, possibly due to the spatial distinctiveness of the track. Place fields close to an object remained stable when the object was exchanged for another one but interestingly some of the CA1 neuron firing patterns were sensitive to a particular object, or to the combination of specific objects in a specific location (Burke et al., 2011). These results suggest that there are cells in the hippocampus that process whole object identity distinct from object location. Cressant et al., (1999) stated that the hippocampal place cell system must be capable of using object identity to anchor the location of place fields. They recorded from the dorsal CA1 in rats in an open field cylinder containing 3 objects positioned on the periphery in an isosceles triangle configuration. Their findings indicate however, that objects only influence place cell stability when placed on or clustered near the periphery of the arena and not centrally placed (Cressant et al., 1997). Furthermore, object location does seem to influence how the information is processed by the hippocampus. Manns & Eichenbaum (2009) found no specific object activity independent of location when recorded from the dorsal CA1, but they were able to detect different activity due to different objects in the same location (Manns & Eichenbaum, 2009). More recently, recordings from dorsal CA1 and CA3 in the hippocampus of a rat during object exploration reveal a new type of place cell called landmark-vector cell (Deshmukh & Knierim, 2013). These pyramidal cells develop fields at a specific direction and distance from objects that are introduced into the familiar arena. However, landmark-vector cells can retain their fields when the object is removed or relocated between sessions or can even follow the object to its new location while also firing at locations where it had been found previously. These results provide further support for the notion that object memory 22

is retained within the hippocampus for some period of time with location as a significant contribution to its identity. 1.8 Current Study: Purpose and Hypothesis Based on the aforementioned studies, additional experimental questions need to be explored to better appreciate the relationship between hippocampal-dependent object memory and the spatial firing properties of hippocampal neurons. The present experiments were designed to examine the influence the performance of the object recognition task has upon hippocampal place cell activity and the degree to which object information is represented within hippocampal neuronal activity. Most of the recording studies to date have been designed to examine the effect objects have on the firing of cells within the hippocampus (Cressant et al., 1997; Manns & Eichenbaum, 2009; Burke et al., 2011; Deshmukh & Knierim, 2013). In contrast, the present studies addressed how established place cells respond as the rodent engages in a novel object recognition task in an environment where spatial cues are limited. The current experiments were designed to address a specific question regarding rodent perception of the environment while undergoing novel object recognition. That is, does a mouse recognize an environment as familiar when the mouse finds it to contain two identical novel objects at the start of the sample session? This question was tested by determining whether place fields of CA1 place cells are stable (i.e., do not change the position of their place field) between an arena habituation session and the following sample session. If found to be stable between the arena habituation session and the sample session, then this would be interpreted as evidence that encountering objects in a familiar environment is not a sufficient change to the environment to warrant remapping of hippocampal place fields. 23

As described above, many argue that the object recognition task requires the rodent to encode a memory of the object-in-context or object-in-place, and that this is the reason the task is hippocampal dependent. Therefore, the present studies permitted testing whether object information is encoded within the context/spatial map (as an object-incontext representation), or encoded independent of the spatial map. If place fields were found to remain stable between the test session and a final empty arena session, then this would be interpreted as evidence for hippocampal representation of objects that is independent from that of the spatial map. It is also possible that two orthogonal representations are elaborated within mouse hippocampal neuronal activity: (i) spatial representation of the recording arena deprived of environmental cues and (ii) a novel spatial representation of objects located in the empty familiar context. The following protocols were used to examine these predictions. Determining that the rodent hippocampus is engaged during novel object recognition due to processing of the object memory, and not only due to spatial processing of the testing arena; provides further support for the importance of the rodent hippocampus for object memory. It is vital to define the specific functions of the rodent hippocampus during Novel Object Recognition testing in order to accurately infer similarities between the human and the rodent hippocampus. Protocol 1: Cue Card Novel Object Recognition. This protocol was used to test the influence of object recognition task performance on hippocampal place cell activity within an arena with features that match those commonly used for recording place cells in rodents. Typically object recognition testing is conducted in an otherwise feature-less high-walled square or cylindrical arena. However, it is well established that a salient 24

polarizing cue within a recording arena exerts stimulus control over the position of a place field (Knierim et al, 1995; Muller & Kubie, 1987) and hippocampal recordings are rarely carried out in the absence of such a cue. Therefore, the cue card protocol entailed testing object memory and hippocampal CA1 place cell activity in mice in a white square arena containing a polarizing cue card covering one wall. The sequence of 10-min recording sessions of the Cue Card protocol was; Arena Habituation 1, Arena Habituation 2, Sample Session, and Test Session (see Figure 5A for schematic). The cue card was present during each recording session. During the sample and test sessions, two previously screened objects were placed into opposite corners within the arena before the mouse was placed in for the 10 min exploration. The Cue Card protocol was designed to determine the influence of object recognition testing under conditions that should limit potential remapping of place fields between sessions. Therefore, it was predicted that place fields observed to remap would be attributed to the presence of the objects or induced by the non-spatial task performance. Specifically, if such remapping were to occur, then they were expected between Arena Habituation 2 and Sample Session, or between Sample Session and Test Session. Protocol 2: Conventional Novel Object Recognition. This protocol was designed to test the influence of object recognition task performance on hippocampal place cell activity within an arena that lacked a polarizing visual cue. That is, the Conventional NOR protocol tested object memory and CA1 place cell activity in mice in an otherwise feature-less high-walled square arena; a method that closely resembles the protocol typically used to test behavior only (Cohen et al., 2013). The objects were placed in the same corners as in the Cue Card protocol prior to the entrance of the mouse during the 25

sample and test sessions. The sequence of 10-min recording session of the Conventional NOR protocol was: Arena Habituation 1, Arena Habituation 2, Sample Session, and Test Session (see Figure 5B for schematic). Without the cue card, place cells were expected to be less stable; a condition that might facilitate object-induced place cell remapping. Determining if place fields remain stable between sessions of the Conventional NOR protocol will provide further support for the notion that novel object recognition impairments following hippocampal inactivation is not solely due to deficiencies in spatial processing, but may be a result of object memory disruption. If place fields remain highly stable during all sessions within the Conventional protocol, then this will be interpreted as stability in the spatial representation, challenging the notion of Object-in- Context representation created when rodents encounter objects within a familiar arena. If stability is low between sessions of the Conventional NOR protocol, then this will be interpreted as evidence that the presence of objects induces place fields to remap. Protocol 3: Drop-In Novel Object Recognition. The final protocol was designed to test place cell stability during novel object recognition using a procedure to further dissociate the object memory from the spatial memory of the empty arena to encourage separate encoding of the two distinct memories. The Drop-in NOR protocol resembles the Conventional NOR in that the arena lacks the polarizing cue card; however, during the sample and test sessions, the mouse first explored the empty arena for 2 min, after which the two objects were introduced into the corners of the arena. The sequence of 10- min recording sessions of the Drop-in NOR protocol was: Arena Habituation 1, Arena Habituation 2, Sample Session, and Test Session (see Figure 5C for schematic). This protocol specifically addressed the question raised above; does the mouse recognize the 26

arena as familiar when it subsequently finds it to contain two objects? Here, the prediction would be that place fields should be stable between the Arena Habituation 2 and the first 2-min of the Sample Session (before the objects are inserted) Thus, by design, the Drop-in NOR protocol should facilitate the mosue recognizing the arena as familiar since it has time to explore the arena before objects are inserved to verify that it matches the retrieved spatial memory of the arena. Further, the Drop-in protocol may facilitate the observation of object exploration triggered remapping of place fields. Specifically, instability in place field between Arena Habituation 2 and Sample Session would be attributed to object-induced remapping of place fields. During the habituation sessions in the Drop-in protocol, the mouse was allowed to explore for 2-min and then hand movements simulating the ones used to place the objects into the arena were performed to familiarize the mouse with the introduction (Figure 5). Determining the effects of separating the retrieval of the spatial representation and the encoding of object memories on place cell stability may provide further support for the rodent hippocampus playing a vital role in object memory, similar to its function in humans. The behavioral protocols used for the current study consisted of two 10 minute arena habituation sessions; where the mice are permitted 10 min of free exploration of the empty square arena, followed by a 10 min sample session and a 10 min test session over a 1 day period. The testing procedures are adapted from the 4-day protocol used for behavioral studies to avoid compromising the exact tetrode location within the dorsal CA1 area due to minor movement of the electrode during plugging and unplugging of the cable. The time duration of the test sessions was also extended from 5 min to 10 min long 27

to ensure similar amount of neuronal sampling during the recordings and this is accounted for during behavioral analysis. To address the issues of time sensitive consolidation of object memory, the delay interval between successive recording sessions was also adapted to suit the requirement of the one-day recordings. It has been suggested that consolidation of object memory requires a certain amount of time to become hippocampal-dependent (Hammond et al., 2004; Clark et al., 2000; Mumby et al., 2002), and since all experiments were carried out in the course of one day, to minimize strain on the electrode; delay intervals between each session of <5 min or 20 min were implemented. Restricting the inter-session delay to <5 min, limits the consolidation of the object memory after the sample session, possibly resulting in non-hippocampal dependent object memory (Hammond et al., 2004). During the 20 min delay between sessions, mice are placed into their home cage for 20 min rest (with cable plugged in). This should provide sufficient consolidation time for the object memory to become hippocampal dependent (Clark et al., 2000). The three novel object protocols were run simultaneously, randomly assigning each electrodeimplanted mouse to one of the three protocols and a delay interval. In addition to the above mentioned comparisons of place field maps within a given protocol, comparisons are also planned to determine place cell stability between the Cue Card and the Conventional protocols to provide insight for the relationship between spatial and object memory processes within the hippocampus. It is hypothesized that stable place fields will be detected within the three different protocol variations of novel object recognition. The presence of a salient spatial cue in the testing arena is expected to result in overall higher stability measures between sessions due to the increased spatial 28

information mice receive from the cue card, compared to two protocols that lack a salient cue. Although, the Conventional NOR protocol is designed to minimize spatial input to the mouse performing the behavioral task, it is predicted that the input the mouse receives from the geometrical cues within the environment is sufficient to support and maintain the formation of a stable cognitive map. Furthermore, the addition of objects into the environment is not expected to induce remapping; which would occur if the mouse perceived itself as being in a novel environment. The Drop-in protocol, where the attempt is to influence the animals to encode the object memory separately from retrieval of the context, is expected to yield place fields with slightly higher variability in stability measures. The overall stability is expected to be identical to the Conventional NOR during the habituation sessions, but it is expected decrease when the object sessions are compared. Delay intervals are not expected to result in varying place cell stability for the habituation sessions as no studies have demonstrated significant changes in place field stability between sessions comparing short delay intervals. However; it is possible that a delay effect will emerge for the place field stability between the sample and test sessions due to object consolidation interference within the hippocampus. During the <5 min delay following the sample session, the object memory should not have become hippocampal dependent and all of the resources within the hippocampus may be devoted to spatial processing. When there is a 20 min delay following the sample session, the object memory is likely to have become hippocampal dependent due to adequate consolidation time, seen by object memory impairments when the hippocampus is lesioned (Clark et al., 2000). This may interfere with the spatial processing within the 29

hippocampus, resulting in lower stability seen during the test session. Additionally, a positive relationship between place cell stability and performance during object discrimination is potential, given the supporting data for object memory processing within the dorsal CA1 area of the rodent hippocampus, the same region recorded from in the current study. Determining whether mice create a novel spatial representation for a familiar context when objects are introduced is an important factor in determining if the rodent hippocampus processes object memory like it does in humans. 30

PART II: MATERIALS AND METHODS 2.1 Subjects Subjects used for hippocampal recordings were male 10-40 week old C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME). Prior to surgery, 8 week old mice were housed 4/polycarbonate cage while they acclimated to the vivarium for 7 days. Mice were housed individually post operatively in a temperature and humidity-controlled vivarium. Room temperature was kept at 22 ± 4 C and 50 ± 5% humidity while maintaining a 12- hour light/dark cycle beginning at 7 AM. All experimental procedures were conducted during the light period. Home cages were maintained on a ventilated rack and mice received ad libitum access to food and water throughout the experimental period. Animal use procedures were carried out in accordance with the guidelines required by the National Institute of Health Guide for the Care and Use of Laboratory Animals. The Florida Atlantic University s Institutional Animal Care and Use Committee obtained approval for all procedures prior to experiments 2.2 Electrode construction A miniaturized custom-built microelectrode was constructed (adapted from Stackman et al., 2002) to carry out extracellular recordings from individual CA1 neurons from the dorsal hippocampus. Each tetrode (4) consisted of 4 twisted Formvar-coated 25 μm diameter Fe-Ni-Cr wires (Stablohm 675, California Fine Wire, Grover Beach, CA). 31

The 16 wires were interfaced to a double-row pin connector array (Mill-Max) and fixed in place using dental acrylic. Two drive screws (14 mm long) were attached to the pin connector to permit advancing the recording tetrode to the desired region after implantation; completing the light weight (<2 g) electrode. Electrode wires were tested by passing current through the array and sterilized with ultraviolet light prior to surgical implanting. Carrying out recordings using a tetrode, where four wires are bundled together into a closely spaced recording tip provides simultaneous capture of each spike, resulting in a higher resolution of putative neuronal sources than would be obtained using a single tip microelectrode (Jog et al., 2002). 2.3 Surgery Mice were surgically implanted with a unilateral microelectrode directly above the CA1 region of the right dorsal hippocampus at 9 weeks of age. Prior to surgery, each mouse was anesthetized by isoflurane (Webster Veterinary, Devens, MA), vaporized at 5% per 1 liter of oxygen in an anesthesia chamber (VetEquip, Pleasanton, CA). Scalp hair was shaved and the mouse securely placed into a stereotaxic apparatus (Model 1900, David Kopf Instruments, Tujunga, CA), and isoflurane reduced to 1.5% for the remainder of surgery to verify appropriate depth of anesthesia. Lidocaine (1%, 0.1ml) was injected under the scalp skin to reduce discomfort and pain responses were regularly checked throughout the surgery duration. Sterile eye lubrication ointment (Pharmaderm, Florham Park, NJ) was carefully applied using a cotton swab and the scalp washed with Betadine scrub, 70% ethanol and Betadine solution. The rostral-caudal incision was made over the midline of the scalp and the underlying periosteum retracted to expose the skull sutures. Distance between the lambda and bregma skull sutures was measured and the skull 32

leveled by adjusting the pitch and roll over head holder on the stereotaxic apparatus. A hole for the electrode wires was drilled above the dorsal CA1 region of the hippocampus at 2.0 mm posterior to bregma, +1.50 mm lateral to the midline and approximately 1.0 mm ventral to the skull surface (Franklin & Paxinos, 2007). Three holes were drilled for the anchor screws (1/8 000-120 jeweler s Small Parts Inc., Miami Lakes, FL). The custom made electrode was lowered with the wires entering the burr hole and resting in the cortex above dorsal CA1, approximately 1.00 mm ventral of the dura. The electrode was fixed in place using dental acrylic (Dentsply International Inc., Milford, DE), making sure to cover the anchor screws and all of skull surface. The scalp incision was sutured around the base of the electrode, covering parts of the dental acrylic using tissue glue (VetBond, St. Paul, MN). When dental acrylic was dry, triple antibiotic ointment was applied to the affected area and the mouse removed from the stereotaxic apparatus. Each mouse was weighed, given an IP injection of 0.8 ml sterile 0.9% saline and a SC 0.5 mg/kg injection of buprenorphine to relieve discomfort. Mice were then placed into an empty polycarbonate cage on a heating pad until they recovered their righting reflex and full mobility before being returned into a clean home cage. Mice received medicated water (3 ml ibuprofen/80 ml water) for the following 48 hours and were carefully monitored for 7 days post-surgery. 2.4 Habituation to Recording Apparatus On the seventh day following surgical implantation, mice were brought into the recording chamber for acclimation to the recording apparatus and white noise generator. Each mouse was then carefully restrained using a white cloth hand towel and a recording cable plugged into the pin connector. The mouse was then placed into a white cylinder 33

arena containing a black cue card, covering approximately ¼ of the internal surface. While each mouse was freely exploring the cylinder arena, waveforms detected by the electrode were screened for hippocampal activity (pyramidal & interneuron waveforms). If no waveforms were detected, the mouse would be restrained again and the microelectrode drive screws turned (1/32-1/4) in a clockwise direction so the wires descend deeper towards the dorsal CA1 layer (1 full turn=75 μm). The mouse would then be returned to the home cage and the process repeated on the following days until hippocampal activity detected. When signals from stable pyramidal neurons containing a place field were detected, behavioral experiments could be conducted while recording extracellular activity from the hippocampus. 2.5 Histology When the electrode had passed through dorsal CA1 of the hippocampus or the mice had reached a certain age where they couldn t be utilized for further recording studies, they were sacrificed by isoflurane overdose (Webster Veterinary). The brains were dissected and placed in 4% paraformaldehyde for few days. Brains were then cryoprotected in 20% then 30 % sucrose in paraformaldehyde solution. When fixed, brains were sectioned into 50 μm coronal sections on a sliding microtome (Leica Microsystems Inc., Bannockburn, IL), freezing the tissue at -19 C with a Physitemp freezing stage. Slides were mounted on subbed glass slides, stained with Cresyl violet Nissl stain and cover slipped securely using histological mounting medium (National Diagnostics, Atlanta, GA). After drying for few days, the slides were cleaned and examined with a Nikon Eclipse 55i microscope to verify electrode placement, indicated 34

by a disruption in the pyramidal cell layer of dorsal CA1. Data for any mouse that was determined to have an inaccurate recording location was excluded from further analysis. 35

PART III: PLACE CELL RECORDING AND OBJECT DISCRIMINATION 3.1 Recording Materials Neuronal activity was monitored from the tetrodes while mice moved freely around a recording arena situated on white acrylonitrile butadiene styrene (ABS) sheet flooring. The arenas used were a white polyethylene cylinder arena (44 cm dia. by 36 cm tall) containing a black cue card (covering ¼ of cylinder internal surface) and a white square arena (37.5 x 37.5 x 50 cm, constructed of white ABS) and were easily exchanged between sessions. For the Cue Card NOR recording sessions, a dark gray ABS sheet was placed on the east facing side on the inside of the white square arena. The cue card covered 34.5 cm of the wall, leaving approximately 1.5 cm white strip on either side. During recording sessions, neuronal activity was processed through the unity gain amplifier with the headstage of the recording cable (LP16CH, TDT, Alachua, FL). Signals were then amplified and band pass filtered (10.000 ± x, filtered at 150-8000 Hz, and digitized at 40kHz) using a 16-channel Multichannel Acquisition Processor (Plexon Inc., Dallas, TX). Spike waveforms and associated time stamps were recorded for each channel as a Plexon system format file (*.plx) using Plexon SortClient software (Version 2.6). The overhead digital FireWire camera (AVT Stingray- 640 x 480 pixel resolution at 80 Hz) tracked the red and green LED on the headstage to obtain position and heading of the mouse. Positional coordinates were recorded using the Plexon CinePlex video 36

tracking system by tracking the location of the LED lights attached to the headstage. Objects used for behavioral testing were prescreened for preference and discrimination (Hammond et al., 2004). The familiar objects (used during sample session) were two identical threaded table feet mounted on clear acrylic base (6x6 cm) and a purple toy gorilla approximately the same size used as the novel object (Figure 6). 3.2 Place Cell Criteria The putative CA1 pyramidal neurons were classified using the following criteria: 1) low baseline firing rate (<15 Hz) and irregular firing pattern; 2) dominant short interspike interval (3-10 ms) as demonstrated by interspike interval histograms showing a characteristic peak at 3-5 ms followed by a rapid exponential decay; 3) wide waveform (>300 μsec). Pyramidal neuronal activity is further defined by bursts of complex spike activity (see Figure 10). These complex spike action potentials are characterized by multiple closely spaced bursts, with the spikes within a complex spike burst exhibiting progressively decreasing amplitude (Zheng & Khanna, 2008). The putative interneurons were classified accordingly as having relatively narrow waveforms (<250 µsec) and high firing rates (> 5 Hz), and the interspike interval histograms exhibited a later peak and a much slower decay compared to putative pyramidal neurons. Interneurons were excluded from further analysis (Figure 7). To visualize location-specific firing properties of CA1 pyramidal neurons, color-coded firing rate x place maps were constructed for each neuron recorded. The place field maps represent the average firing rate in each pixel of a 32 x 32 matrix of pixels representing and 37.5 x 37.5 cm 2 area of the arena floor. The firing rate in each pixel was determined by dividing the total times the mouse s head (or LEDs) was detected in that given pixel, by the total number of spikes detected in that 37

pixel. Color-coded firing rate maps were then constructed to plot the positional firing relationship. In the color code, white pixels represent locations of the arena never visited and blue for pixels in which the firing rate was zero for the entire recording session. Firing rates greater than zero are represented in increasing order, light blue, green, yellow, orange, and red, with red representing the highest average positional firing rate. Cells were included in the analysis only if inspections of the firing rate map for the cylinder sessions revealed a distinct place field, characteristic of place cells. 3.2.1 Cue Card Rotations Prior to any behavioral testing, cue card rotations were performed to verify appropriate place cells response to a visual cue. This rotation involves placing the mouse into a cylinder arena containing a polarizing cue for a 10 minute recording session, removing the mouse and rotating the cue 90 in either direction (counterclockwise in this case) for a second 10 min session. It is expected that the place field of each CA1 place cell will rotate its position in the arena by amount approximating the rotation of the cue card (Muller & Kubie, 1987). The third and last session is identical to the first one, with the cue card facing the initial direction. It is expected that all place fields will rotate their position back to the initial location within the arena, approximating the rotation of the cue card to the initial location. Cue card rotations are typical when determining whether a pyramidal cell has a stable place field by observing if the single salient cue exerts stimulus control over the position of the cell s firing field (Knierim et al., 1995; Taube et al., 1990b; Bostock et al., 1991). If a pyramidal neuron meets the criteria stated above, as well as its place field rotates along with the dark cue card it may be considered a place cell. 38

3.2.2 Stability Measures In order to determine the stability of a place field over multiple sessions quantification is necessary. Pearson s correlation is calculated by overlapping firing rate matrices (32 x 32 pixels) from consecutive sessions and determining the relationship between the two gives a reliable measure of firing field stability. Previous studies suggest that a place field correlation close to 0.4 between sessions from the same arena can be considered to be stable although place cell stability in mice during non-spatial tasks has been debated. An experiment comparing place cell stability in mice while performing one of four possible tasks (No Task, Foraging, Novel Environment, Spatial Task) overall stability (r = 0.42) was only observed when the mouse paid specific attention to the context or were presented with a novel environment (r = 0.27) (Kentros et al., 2004). Jeffery et al. (2003) compared place cell stability over time in two distinct contexts while rats completed a hippocampal dependent task. Stability criterion for each cell was determined to be >0.35 on average over 4 sessions. A second study compared remapping of place cells during fear conditioning of rats. Results indicated that aversive stimuli could influence remapping of place cells where the context remained unaltered, and to diminish the possibility of detecting remapping when fields were stable; criteria was set at r = 0.33. Furthermore, recordings from cells that contained stable fields throughout testing had a stability correlation average of 0.6 (Moita et al., 2004). Some of these stability measures may seem low considering that aversive stimuli may affect the place memory so more strict criteria will be applied here. For the purpose of these experiments, all pyramidal neurons that do not reach the stability criteria of r = 0.4 between all three 39

sessions in the cylinder rotations as well as between habituation 1 and 2 in the square arena will be excluded from further analysis. 3.3 Data Analysis The collected spikes were analyzed through a combination of automatic and manual methods using Plexon OfflineSorter software (Version 3.2.1). Since the neuronal activity detected by the tetrodes is the collective change in voltage in the extracellular space (i.e., multi-unit activity), sorting action potentials or spikes from individual neurons must be carefully performed. Sorting the spikes that belong to one neuron from spikes that belong to other neurons and to diminish electrical noise detected by the wires is a process referred to as cluster cutting. Cluster cutting was carried out to sort the spikes using manual and automatic processes. Valley seeking is an automatic sorting method used to separate spikes from different neurons, where plots are constructed of the characteristic response properties of a set of spikes (e.g., spike amplitude) as detected on each of two tetrode channels (e.g., channel 1 vs. 2, 1 vs. 3, 1 vs. 4, 2 vs.3, 2 vs.4 and 3 vs.4). Generally, one can observe that clusters of spikes can be resolved from these plots. This automatic clustering was used to initially guide spike sorting prior to manual sorting but was never used alone to separate spikes. Although the automatic valley seeking is fast and convenient, it rarely provides the most accurate results based on the statistical properties of the waveforms (Kretzberg et al., 2009). More advanced automatic techniques for cluster cutting can be time consuming and are often used when data is analyzed by multiple researchers but these data were all analyzed by the same person, reducing the possibility of error. Manual cluster cutting refers to the laborious process of reviewing the waveforms of the spikes within the clusters to confirm similar waveform 40

characteristics. Manual cluster cutting was directed using multiple sorting parameters; including waveform, 2 and 3 dimensional clustering, autocorrelograms, and spike count (Figure 8). Sorted pyramidal neuronal spikes were combined with the coordinates with Plexon CinePlex Editor and analyzed with NeuroExplorer (Version 3.266, Nex Technologies, Littleton, MA). Place by firing rate map images were exported to MATLAB R2011b (Version 7.13.0.564), smoothed and correlation scores obtained between all 32x32 pixel matrices. Correlation stability scores were exported to Microsoft Excel 2010 grand mean standardized, organized and further analyzed using parametric statistics in SigmaPlot 11.0 (Systat Software, Inc. SigmaPlot for Windows). Behavioral measures were manually scored from a video file using XNote Timer (Version 1.11) by an experienced researcher and coded into the Excel file. Times spent actively exploring both identical objects during the 10 min sample session and the novel and familiar objects during the 10 min test session were matched to previously determined criteria (30 sec with both objects or 38 sec with either object during sample and minimum 30 sec total exploration during test session). Exploration of the objects was measured as the time the mouse spent facing the object with the nose less than 2.0 cm away. All other behavior such as grooming close to the object was not considered active exploration. All animals that did not meet both exploration criteria were excluded from further analysis. Discrimination ratios were calculated for each mouse as the difference in time spent exploring the novel object minus the time spent exploring the familiar object divided by the total time (sec) spent exploring objects (T Novel object T Familiar object / T Novel object + T Familiar object ). Discrimination ratios were grand mean standardized and exported to 41

SigmaPlot 11.0 (Systat Software, Inc. SigmaPlot for Windows) for regression analysis. Mean values were calculated and a student s t-test conducted to determine if performance on the task was different from chance (i.e., a discrimination ratio of 0), and to examine effects of protocol and delay on the discrimination ratio. 42

PART IV: RESULTS Histological verification revealed that tetrodes passed through the medial dorsal CA1 region of the hippocampus (Figure 9). The pyramidal neurons recorded from the dorsal CA1 region of the hippocampus in mice running the novel object recognition task demonstrated complex spike activity (Figure 10), comparable to CA1 complex spike activity reported in other studies (Zheng & Khanna, 2008). Of the total of 163 hippocampal neurons recorded from the 20 mice during the cylinder sessions, 109 of those units were categorized pyramidal neurons. Further, 172 hippocampal neurons from the same 20 mice recorded within the square arena, 112 were analyzed as pyramidal neurons and 73 met place cell criteria between both arenas, firing in a stable location specific manner in both recording arenas. Only place cell cells that remained stable during the cue card rotations and reached stability criteria (r > 0.4) between habituation 1 and 2 during testing in mice that were not excluded due to lack of object exploration were included in all subsequent analyses of the influence of the novel object recognition task performance on hippocampal place cell activity. 4.1 Place Cell Recording Results Recordings from place cells in the dorsal CA1 region of the hippocampus of mice performing variations of the novel object recognition task demonstrated overall high stability during multiple sessions over time. Overall average stability of place fields 43

obtained between Habituation 1 & 2, Habituation 2 & Sample as well as Sample & Test sessions are compared during the analysis. Protocol 1: Cue Card NOR. Recordings from mice (n=5) performing the Cue Card novel object recognition task, where a polarizing visual cue is provided, yielded 11 place cells that contained stable place fields within both arenas. The Cue Card protocol yielded high average stability (r = 0.771) between all sessions and delay intervals with very low variations (Figure 11 A). Furthermore, the overall average stability was r = 0.79 for cells in mice performing the task with < 5 min delay interval and r = 0.73 when there were 20 min between all sessions. Bigger differences were observed when the correlation between sample & test sessions were examined dependent on delay. When the mice received the < 5 min delay, the correlation between object sessions was r = 0.84, and r = 0.74 when the 20 min delay was imposed. Protocol 2: Conventional NOR. The Conventional protocol yielded 25 stable place sells within both arenas, recorded from 6 mice. An overall average stability measure of r = 0.699 (Figure 11 B), was obtained independent of delay. Cells from mice performing the Conventional protocol with a delay < 5 min between sessions yielded stability r = 0.688 and r = 0.696 when the delay was 20 min. Comparing only the sample & test stabilities, dependent on delay a correlations of 0.752 for the < 5 min and 0.683 for the 20 min were observed. Protocol 3: Drop-in NOR. The Drop-in protocol yielded 11 stable place cells recorded from 5 mice. Overall average stability obtained from place cells yielded in a stability correlation of 0.704 (Figure 11 C). Stability correlation measure obtained from 44

mice performing the protocol with a < 5 min delay was 0.712 and r = 0.61 was obtained when the delay was 20 min. The overall stability measures dependent on delay were r = 0.69 for the 20 min (n = 9) delay intervals and r = 0.82 for the <5 min (n = 7) delay between sessions, independent on protocols. A three factor (session: Habituation 1 & 2, Habituation 2 & Sample, Sample & Test; Delay: <5 min, 20 min; NOR Protocol: Conventional, Cue Card, Drop-in) ANOVA was performed on the mean stability scores for all 54 dorsal CA1 cells recorded within all sessions and delay between the three protocols. The ANOVA yielded a significant main effect of protocol (F 2, 179 = 4.762, P = 0.01). Post-Hoc (Holm-Sidak multiple comparisons) test indicated that the mean stability scores of place cells in the Cue Card protocol were significantly higher than the Conventional and Drop-in NOR protocols. The overall ANOVA also yielded a significant main effect of session (F 2, 179 = 4.174, P = 0.009) on place cell stability measures, and the corresponding post-hoc tests indicated that stability was significantly lower between the first two measures (Habituation 1 & 2, Habituation 2 & Sample) compared to the Sample & Test session but no difference was observed between Habituation 1 & 2 and Habituation 2 & S. The overall ANOVA yielded a non-significant effect of delay (F 1, 179 = 3.881, n.s.) and of the Session x Delay interaction (F 2, 179 = 1.558, n.s.). A two factor (protocol and delay) ANOVA was performed on the stability measures of place cells specifically between the Sample & Test sessions to assess the effect of objects placed into the arena, independent of prior sessions within the empty arena. The ANOVA failed to yield significant main effect of protocol (F 2, 59 = 0.332, 45

n.s.); however, a significant main effect of delay emerged (F 1, 59 = 9.566, P = 0.003) with higher stability scores during the < 5 min delay compared to 20 min. The interaction effect between protocol x delay did not reach significance (F 2, 59 = 1.962, n.s). In order to further determine where the main effect of protocol loses significance and delay becomes a significant main effect, additional two-factor ANOVAs (session and delay) were run for each of the three protocols. Stability scores of the Conventional NOR and the Cue Card NOR protocol were not influenced by delay, session, or the interaction between the two variables. However, a significant interaction effect was found between session x delay for the Drop-in protocol (F 2, 32 = 4.401, P = 0.022) whereas the main effects of session and delay did not reach significance (F 2, 32 = 2.566, n.s), (F 1, 32 = 0.143, n.s) respectively. Holm-Sidak Post-hoc analysis showed increased stability between the < 5 min sample & test sessions compared to the 20 min delay within the Drop-in protocol. Lastly, a regression analysis was run for the standardized average stability scores and the standardized discrimination ratios from each mouse. Best Subset Regression demonstrated that the main contributor to predict discrimination ratios was stability scores whereas the type of protocol strengthened the relationship. Removing the effects of delay from the model enhanced the relationship. Multiple linear regression was run examining the model fit (Discrimination ratio = 0.436 + (0.532 * Stability) (0.0225 * Protocol) yielding an R = 0.531 and R 2 = 0.282. Stability correlations (P = 0.045) were a significant predictor of discrimination ratios based on a linear combination of the independent variables. Separate scatter plots regression analyses were graphed for the effects of protocol and delay on the relationship between stability correlations and discrimination ratios (Figure 12). 46

4.1.1 Place Field Rotations Results Rotations of place fields were observed in sessions where visual cues were limited and objects were not present prior to the mouse entering the arena. Rotations of place fields were still determined to be stable when all cells recorded from the same session were rotated back to their expected orientation and a relatively high correlation obtained between those sessions (Figure 13). Rotations were never observed in place cells recorded from mice completing novel object recognition with the cue card present but rotations were seen in both the conventional and the Drop-in protocols. Place fields obtained from mice running the Conventional NOR only rotated in 1 case (out of 6) where both the sample and test sessions rotated 90 clockwise compared to the empty arena sessions. The place fields always remained stable between the two object sessions. Rotations of place fields were observed in 4 out of 5 mice performing the drop-in protocol with two cases demonstrating place field rotation between the sample and test sessions. 4.1.2 Object Specific Activity Results In one case, a CA1 neuron discharged at a high firing rate during specific periods as the mouse performed the sample and test session during the Cue Card protocol. Subsequent analysis revealed that this neuron s peak firing occurred when the mouse was positioned at the same location as the two objects, and the cell appeared to demonstrate object-location activity with a correlation of 0.8994 between sample and test sessions. Further analysis from other sessions (cue card rotations, habituation sessions and follow up cylinder as well as empty arena) yielded no location specific activity, with the cell essentially remaining silent during all sessions that did not include objects within the 47

arena (Figure 15). This object-specific activity was obtained from a mouse during successful performance of the Cue Card NOR with a 20 min delay intervals, demonstrating a discrimination ratio of 0.352. This mouse was however excluded from the general analysis due to insufficient exploration during the sample session; with object explorations of 16.53 and 8.57 seconds with the identical objects. Interestingly, this mouse demonstrated sufficient exploration during the test session; spending 23.26 sec with the familiar object and 48.53 sec with the novel object, indicating that the encoding, consolidation and retrieval of the sample session objects occurred without difficulties. We cannot; however, exclude the possibility that test session object discrimination is due to chance, since the mouse did not meet the minimum exploration criteria during the sample session and therefore object memory cannot be inferred. 4.1.3 Novel Object Recognition Results Of the 20 mice that underwent novel object recognition testing during place cell recordings, 4 mice (3 did not meet sample session exploration criteria and 1 failed to explore objects more than 30 sec during the test) did not reach the exploration criteria during the 10 min object sessions and were removed from further analysis. Mice performed the task successfully, they demonstrated a higher discrimination ratio than would be expected by chance (t 16 = 3.953; P = 0.001). A two factor (Protocol and Delay) ANOVA was conducted on discrimination ratio scores to determine whether behavioral performance was influenced by protocol or delay intervals. The ANOVA yielded no significant effect of protocol (F 2, 15 = 0.681, n.s.), no significant effects of delay (F 1, 15 = 3.396, n.s.), and no significant interaction between the two variables (F 2, 15 = 0.168, n.s.). Further, Student s t-test revealed no significant difference between overall total 48

exploration during the sample session and total exploration during the test session (t 16 = - 0.556, n.s.). 4.2 Place Cell Stability Discussion The stability measures obtained from these experiments can aid in determining what features affect place cell stability and comparing the effects observed by varying polarizing cues. Looking at the overall place cell stability between protocols, it is evident that place fields are most stable during the NOR task run in the presence of a polarizing cue card; that is, place cells appear to ignore the information provided by the addition of objects placed into the arena when more prominent cues are available. Overall stability was also observed during the Conventional as well as the Drop-in protocols but these were significantly lower than recordings from mice performing novel object recognition with a polarizing cue within the arena. This would indicate that the lack of cues in the empty arena do not provide adequate spatial information to obtain maximum stability, but mice are nonetheless able to create and maintain stable place fields in a sterile environment (Figure 14). These results contradict the findings from Kentros et al., (2004) where place cell stability during free exploration (no task) yielded an average correlation below 0.15. Kentros et al., (2004) reported higher place cell stability measures were observed when the mice were encouraged to pay more attention to their environment by performing a spatial task similar in demand to the Morris water maze. The authors interpreted the pattern of results as evidence that when the mouse is actively engaged in a spatial task that requires attention to distal landmark cues, hippocampal neuronal activity is more tightly locked to spatial location. It is noteworthy that in most place cell recording studies there is little demand on the rodent to attend to environmental cues 49

most recordings are conducted while the rat or mouse freely explores the environment or chases food pellets dropped into the arena. Thus, in the majority of these recording studies in which little attention is paid to whether the rodent is attending to cues, place cells are remarkably stable. Kentros et al., (2004) suggested that this might be a species difference in the spatial correlate of hippocampal neuronal activity. Regardless, the requirement of attention to support stable location by firing rate maps of hippocampal activity is an interesting argument that could be applied to the current results. That is, it may be that mice running any of the three novel object recognition protocols are paying specific attention to details of their environment, possibly since it is novel to them, and that is why the hippocampal place cell stability measures are high. The place cell criteria for the stability scores that were imposed between the cue card rotations and between habituation 1 & 2 is very strict (must reach r = 0.4), essentially excluding all non-stable cell activity, however, only 3 place cells were excluded due to low stability between habituation 1 & 2, where they had met the stability criteria in the cylinder. It is possible that the stability measures from these recordings are so high for all protocols because of this restriction of range but the mean differences in stability measures between the three protocols still reaches significance, indicating a true protocol effect due to the polarizing cue. 4.2.1 Use of Objects as Landmark Cues An interesting increase in stability was observed between Sample & Test sessions, compared to the other measures in the Conventional and Drop-in protocols. This could be interpreted as an increased stability due to the addition of cues into their environment. Compared to the Cue Card protocol, where stability remained relatively unchanged 50

throughout the experiment, adding the objects into the sample and test sessions for the other two protocols gave the mice further spatial information to anchor their place cells to. Cressant et al. recorded from the dorsal CA1 in rats with objects located near the periphery of the recording cylinder and found that the angular position of place fields were strongly influenced by the object locations. Their experiments demonstrate that rodents can use objects to anchor the reference direction when placed in a certain arrangement by acting as a navigational cue (Cressant et al., 1999). Place fields have been shown to be mainly influenced by objects in a cylinder when placed on the periphery of the arena, but seem unaffected by objects placed towards the center (Cressant et al., 1997). This suggests that placing the objects in opposite corners within the square arena during object recognition may influence the hippocampus differently than if the objects were centrally placed. Further testing will be required in order to come to a clear conclusion as to whether object placement within the square arena affects object discrimination. It is unlikely, but possible; that the increase in hippocampal place field stability was attributed to experience within the context and the more recording sessions experienced the more stable the place fields become due to enhanced ease of retrieval. This possibility is not excluded; however, previous experiments have demonstrated place cell stability over time within the same context recorded in the same day. Within rats, multiple place cells recorded in consecutive sessions yielded average stability correlations between 0.52 to 0.616 in the same context, while performing a hippocampal dependent behavioral task (Jeffery et al., 2003). Furthermore, looking at how the recorded cells stability develops over time within the same context; stability measures 51

tend to decrease non-significantly while overall maintaining a high stability. This indicates that the increase in stability measures across sessions is unlikely contributed to enhanced familiarity with the square arena, but more likely contributed to the improved spatial information they receive from the objects. 4.2.2 Influence of Delay between Sessions on Place Cell Stability Significant delay effects between the <5 min and 20 min intervals were only observed between sessions that contained objects. Increased place field stability between the sample and test sessions for the <5 min delay compared to the 20 min could be contributed to the differences in object memory consolidation time allotted to the mice. The lower stability during the 20 min delay is unlikely contributed to instability in the spatial memory. Kentros et al., (2004) stated that short term-stability tended to be higher than long term when recorded with delays of 30 min and 6 hours; however, for the purpose of this experiment, 20 min delay interval is still considered relatively short when examining spatial memory. Further, stability of environmental cues has been shown to influence how humans and rodents navigate. The importance of landmark stability has been shown to determine whether the cue/object can be used as an anchoring cue for navigation (Chan et al., 2012). The stability of objects as environmental cues has been shown to influence whether place fields can use them to anchor a reference point to them. Knierim et al., (1995) demonstrated that place field stability is based on the animals previous experience and how reliably stable cues are within the environment are perceived to be. It is probable that during the <5 min delay, the object identity information had not yet been consolidated by the hippocampus, however; the object 52

location information remains in the spatial working memory where it contributes to the increased stability of the place fields. When the protocols are further examined separately, an unpredicted session x protocol interaction is observed within the Drop-in protocol. Whereas the Conventional and the Cue Card protocols demonstrated overall consistent stability between sessions and no significant differences between delay intervals, the Drop-in protocol revealed a significantly higher stability measures between Sample & Test session when a <5 min delay interval was imposed between sessions, compared to the 20 min delay intervals. This finding could reflect a variety of influences, but more evidence needs to be collected to make a strong conclusion. One likely explanation is that the hippocampus is rapidly forced to perform two distinct tasks (spatial mapping and object exploration) when the objects are placed into the arena during the sample and test sessions, causing a decrease in stability during the 20 minute delay and an increase in place field stability for the <5 minute delay interval. Previous studies have suggested that short-term memory for objects may be hippocampal independent whereas place cell responses are relatively immediate in a familiar environment. It is possible that during the 20-minute delay interval, the hippocampus is actively consolidating the object memory when the mouse is then placed into the empty context during the Drop-in protocol, mismatching the expectation of seeing the objects. The mouse may then attempt to retrieve the memory of the empty arena, only to find that objects appear again. This may cause a mismatch in the expected and actual place cell representation of the environment, which results in lower stability measures between the two object sessions. This interruption in maximal place cell stability may also reflect a different or divergent allocation of resources within the 53

hippocampus, dedicated to object memory consolidation. For the <5 minute delay, the hippocampus receives minimal object consolidation interval before the mouse is placed back into the empty context. Now, since the object memory is still weak or an unconsolidated memory (perhaps residing in the PER cortex), it does not interfere with the hippocampal-dependent retrieval of place field information and thus, all of the hippocampal resources can be devoted to obtaining maximal hippocampal place field stability, supported by the short-term memory of the object locations. When the objects appear 2 min later, the hippocampus receives those anchoring points to increase the stability of the place fields compared to the previous session. 4.2.3 Novel Object Recognition Discussion Mice that underwent the electrode implantation were individually housed (1 per home cage) to protect the electrode. These mice were also tested at relatively old age (6-10 months), compared to conventional mice tested using novel object recognition, andage related cognitive impairments have been reported in middle age (10-12 months) and old (18-20 months) C57BL/6J mice (Benice et al., 2006). These variables are likely the main contributors to the slightly lower discrimination ratio obtained from the hippocampal recording mice, compared to mice only tested behaviorally. Furthermore, during coding of the videos, neophobia or the avoidance of the mice approaching the objects was noted in a few of the mice. The main results demonstrate that electrode implanted mice were able to encode and consolidate the memory of an object seen during the sample session and later retrieve it during the test session for the three different variations of novel object recognition (Figure 16 A). This suggests that the mechanical destruction of the hippocampus and 54

overlying cortex caused by electrode placement within the dorsal CA1 is not sufficient to impair object memory processing. Furthermore, discrimination of the novel object was not affected by the protocol type, indicating that the inclusion of more spatial information in the arena, or dissociating the objects from the sterile context does not affect the object memory encoding, consolidation or retrieval. Discrimination ratios were not significantly influenced by the delay interval of <5 or 20 min, although the <5 min delay demonstrated noticeably better discrimination than mice tested after a 20 min delay (Figure 16 B). This increase in preference during shorter delay intervals could be contributed to the short term memory functions and it is possible that significance would emerge if more subjects were added. This could also provide further support for the notion that short-term/weak object memory does not require the hippocampus, or has not become hippocampal dependent yet. 4.2.4 Correlation between Place Field Stability and Object Memory There appears to be a positive relationship between place cell stability between the sample and test sessions and performance during the novel object recognition paradigm. Mice with higher place field stability are more likely to successfully encode and retrieve the memory of the familiar object and therefor prefer the novel object during the test session. The mouse hippocampus seems to contribute both to spatial navigation and object memory as seen by performances during both tasks. Consequently, this effect may be enhanced by the weak vs. strong memory, depending on whether the object memory has become hippocampal dependent after adequate consolidation seen when the delay intervals are compared. 55

PART IV: GENERAL DISCUSSION The present set of experiments was designed to examine the influence of NOR performance and the presence of 3D objects on the firing properties of hippocampal CA1 neurons in freely moving C57BL/6J mice. The goal of these studies was to determine whether performance of the hippocampal-dependent object memory task altered the location-specific firing properties of CA1 neurons (i.e., place cells), and to evaluate hippocampal neuronal activity for evidence of object-specific firing. Place cell recordings from mice successfully performing the novel object recognition paradigm demonstrate that a new spatial cognitive map is not created when objects are added into the sterile testing arena. This provides indirect support for the role the rodent hippocampus plays in object memory due to lack of novel spatial processing when the environment is altered by adding objects. Furthermore, a positive relationship between place cell stability and object discrimination between the sample and test sessions was observed; independent of protocol, suggesting functional independence but simultaneous processing of both spatial and object memory within the rodent hippocampus. 5.1 Rotation of Internal Spatial Representation A stable spatial representation over time may represent an established cognitive map of a given environment. The ensemble activity of multiple place cells recorded from one mouse within a familiar arena is thought to represent the internal cognitive map 56

created and maintained over time. In an environment containing salient cues (i.e. cue card); the spatial representation may anchor itself to the most prominent feature visible and remain stable as long as the environment remains unchanged. In an environment lacking in such cues, the arena geometry may serve as a reference for the cognitive map. When high ambiguity regarding the symmetry of the environment is present (i.e. empty square arena), unpredicted rotations of the stable map may occur. Rotations of place fields within the sterile symmetrical square arena were observed only under recording conditions in which the cue card was not present (see Figure 5 for an example). Such rotations of the place fields were not observed during recording sessions that began with the release of the mouse into the arena already containing the objects but without the cue card. The fact that all recorded cells that reached stability criteria within a given animal, rotated in the same manner within the empty arena provides further support for the cognitive map theory, stating that place cells are the building blocks of an internal spatial map (Wilson & McNaughton, 1993). This result indicates that it is the entire spatial reference frame that has rotated between sessions and not individual cells remapping. Interestingly, the 20-min delay Drop-in protocol was the only variation of NOR in which place field rotations were observed between object sessions. It is conceivable that this is due to the lack of cues at the time of entrance to the environment, and the 2-minute empty arena exploration the mice received in the Drop-in protocol before the objects are manually placed into the arena, is sufficient time for the mice to retrieve and stabilize the previously encoded and consolidated spatial map. Furthermore, the difference between the delay intervals where rotations were seen, may have contributed to the deterioration of short-term object location memory due to the 20 min delay, whereas the <5 min delay 57

is not sufficient time for the working memory to be lost. Evidence of rotations of CA1 place fields have been shown previously to be induced by altering the entry point of the rat into a symmetrical cylindrical environment containing 2 identical cue cards on opposite sides. Sharp et al. (1990) found that adding the second cue card to a familiar cylinder, place fields in rats remained largely unaffected but when they introduced the animal into the arena from the opposite side, place fields rotated by 180. These results suggest that the internal spatial representation of the rats rotated to match their previous experiences while using the symmetrical environmental cues to stabilize their cognitive maps. During the current experiment, mice were released into the arena as close to the center of the arena as possible but always from the same direction. Given the positions of the objects within opposite corners of the arena, the objects should have appeared symmetrical and thus provided little orienting feature. It is possible that the direction the mice are facing when they touch the arena s floor determines how they perceive their surroundings (i.e., align their internal spatial reference frame) and retrieve the map of the sterile square arena when no other cues are available. Behavioral map rotations have also been documented within a rectangular arena deprived of spatial cues. Rats were trained to retrieve food from one corner of a rectangular arena using geometrical and environmental cues. When the cues were removed, rats were left using geometrical cues and performance dropped from 88% down to about 50%; searching mainly in the target corner and the diagonal corner, demonstrating a 180 rotation of their cognitive map (Cheng, 1986). Furthermore, when rats were trained to find a hidden platform in the Morris water maze using 3 different intra-maze object cues; placed either in an isosceles or equilateral triangular 58

configuration, performance was significantly better when spatial ambiguity was lower. When objects were arranged in an isosceles configuration, rats demonstrated better performance on the spatial navigation task; however, when the objects were placed in an equilateral configuration or an over-symmetrical arrangement, poorer performance was observed. When the 3 objects in the isosceles triangle position swapped locations (e.g., A-B-C to C-B-A), only 8/10 rats went straight to the goal location, compared to all of them in the standard condition during the previous session. Analysis of swimming paths suggested confusion of a subset of the rats by the interchanged object identities (Benhamou & Poucet, 1998). This result indicates that navigation can be guided by the geometrical configuration of available cues, provided that the configuration provides unambiguous geometric information by which orientation is possible (e.g., and isosceles triangle as compared to an equilateral triangle), and secondly by landmark identity. Taken together, it appears that a polarizing cue within an environment blocks place field rotation by providing a stable navigational cue, whereas in an environment that is symmetrical and/or lacks any salient environmental cues, the entry point to that arena likely acts as the main egocentric navigational cue. Further experiments need to be carried out to determine the factors that enhance or inhibit these place map rotations but that is not within the scope of the current thesis. 5.2 Object Specific Activity Object location activity was detected on one occasion during the experiments. One CA1 pyramidal neuron was recorded in a mouse performing the novel object recognition task with the cue card present in the arena with 20-minute delay intervals. Interestingly, this animal did not meet the exploration criterion for the sample session, yet 59

exhibited adequate exploration of the arena for a place field to emerge at the location of both objects (Figure 15). Analysis of sessions where the arena did not contain objects (cylinder and square arena) yielded no location specific activity where the cell reached above 0.5 Hz. This specific cell was mainly active when the mouse was close to either object, both during the sample and test sessions; firing close to 6 Hz, indicating that it is not responsive to object identity per se but possibly processes location of an object within the size and shape of the ones used during the experiment. If this cell were to be considered a true place cell, it would indicate remapping between the two habituation sessions and the object sessions (sample and test). Furthermore, this cell demonstrated two distinct firing locations far away from each other, a phenomenon considered relatively rare for neurons in the CA1 region recorded within a familiar arena. However, Burke et al. (2011) reported a significant increase in place fields per CA1 neuron, and a decrease in place field size when objects were presented on a circular track, compared to that found during a recording session on the empty circular track. The Burke et al. (2011) report that place cells are affected by the presence of 3-dimensional objects is consistent with another report (Manns & Eichenbaum, 2009); however, both are for the most in contrast to what was observed in the present study. The object specific activity observed in the current experiment may provide further evidence for altered place cell activity within the hippocampus when 3- dimensional local cues are added into the environment; however, this specific cell s activity was not associated with a place within the cylinder or the empty square arena, indicating a partial remapping by one cell whereas the other 5 cells recorded from the mouse simultaneously remained stable within all sessions of the square arena and the 60

cylinder. This finding would contradict the cognitive map theory if remapping would be attributed to this cell s activity. Similar activity has been observed during dorsal CA1 recordings in rats, where fields emerged and remained close to a specific object (Deshmukh & Knierim, 2013) during repeated exposures. However, the recording location within the Ca1 region reported in Burke et al. was considerably more posterior, compared to the location landmark-vector cells recorded from in rats (Deshmukh & Knierim, 2013), as well as the recording location used within the current study. These data suggest that the hippocampus does not simply process spatial information, but integrates environmental object cues into a single representation of the environment and the information may be organized hierarchically. Further experiments are required to determine object specific or object location specific activity within the mouse hippocampus. 5.3 The Cognitive Map The cognitive map refers to a stored internal representation of encountered environments that gives us the ability to navigate within the environment as well as when we are not physically there. This special function allows us to create novel routes within our map and associate specific events to a given location. Support for the cognitive map theory is increasingly growing with results from human and animal studies. The gap between the functional symmetry between the human and rodent hippocampus for spatial processing is closing as research focuses in on specific research questions. The rotations of place fields observed during the present experiments provide further insight into how profound this internal representation can be. A mouse placed within an empty sterile box, with extremely limited cues can create an internal 61

representation of the context that remains stable during the following entrances; but without a polarizing anchoring reference point, the internal representation can rotate, anchoring the map only to the geometric cues available. If this experiment were to be repeated, using a cylinder instead of a square arena during behavioral testing, it is quite likely that the stability of place fields would not remain as high between sessions. The lack of geometric cues within a cylinder, compared to the square arena would essentially deprive the mice of most environmental cues, and the protocol containing a polarizing cue card would likely be the only protocol resulting in place fields that reach stability criteria. The remarkable ability of the mouse hippocampus to calculate current location using limited visual cues, vestibular input (Stackman et al., 2002), walking speed (Slawinska & Kasicki, 1998), and maintain it over extended periods of time using the mental representation of the environment remains a fascinating topic within behavioral neuroscience. Future research may be capable of determining which hippocampal place cells become active within different geometrical environments and what features influence the recruitment of specific neurons. Place cell activity has mainly been contributed to physical location dependent on distal and proximal cues within the environment; however, recent experiments have demonstrated that the rodent hippocampal place cells can code for multiple types of environmental stimuli and develop over time whereas the context remains the same. Distinctness, size, and stability of environmental cues seem to be the main contributors to how we create and retrieve cognitive maps. The landmark cues that guide behavioral choices also seem to specifically contribute to whether they become encoded into the map (Chan et al., 2012). When driving a familiar route, we tend to notice buildings close 62

to an intersection we plan to make a turn at more so than the buildings around intersections we simply cross. This may not always be consciously encoded, but most certainly will be retrieved when giving someone directions for that specific route. For experimental rodents, these navigational cues could be environmental cues associated with some significance, such a cue close to a reward location or a lever they must press to avoid foot shock. Place cells do not always remain stable over long periods of time when there is a task at hand (Moita et al., 2004; Komorowski et al., 2009). Learning about a context or to complete a task can alter place field location, suggesting that they become anchored to some cognitive processing distinct from location. While rats learned a conditional discrimination task, where they had to associate a novel scent with a reward in a specific context and not in another; CA1 hippocampal pyramidal neurons changed their firing location to represent the location of either match or mismatch digging locations over time (Komorowski et al., 2009). Throughout task learning, place cells begun developing item-place association firing fields seemingly independent from location alone. Comparable activity has been seen in rats swimming a circular track; similar to Morris water maze, with a hidden platform submerged in the opaque water. Hollup et al. (2001) reported increased place cell activity around the escape platform compared to the rest of the circular pool. These data indicate that hippocampal neuronal activity is not only for the cognitive spatial map, but places more importance on meaningful regions within that map. Despite seeing task dependent modulation within the dorsal CA1 of the rodent hippocampus, complete remapping may occur in a novel environment whereas the learned hippocampal dependent task remains intact. Rats were successfully trained to 63

retrieve food from a specific corner within a black arena during a tone-cue. When the rats were placed into a white arena and place cells recorded, complete remapping observed while the hippocampal dependent task was performed successfully (Jeffery et al., 2003). This suggests that the hippocampus does not depend on a specific spatial map to generalize spatial tasks. This study however did not report increased place field activity at the goal location, contradicting the previously discussed experiments. Taken together, the functional significance of hippocampal CA1 place fields have not yet been fully defined and future experiments need to be carried out to determine factors that influence the way we perceive and remember our environment and objects within it. 5.4 Conclusion The present study provides support for the cognitive map theory proposed by O Keefe & Nadel in 1978, stating that the hippocampus holds a general map of one s environment. Further support is provided for the rodent hippocampus participating in object memory processing, through invalidations of the object-in-context representation proposed to be a contributing factor for the role of the hippocampus during novel object recognition paradigm. A positive relationship was observed between place cell stability and behavioral performance during novel object recognition testing, suggesting that the hippocampus plays a role both spatial and object memory processes by integrating geometrical, distal and proximal cues within the arena to signify one single representation of the environment. The object location activity observed during the current experiments supports the view that the hippocampus acts as a cognitive map; simultaneously 64

processing both spatial and environmental cues, resulting in a single cognitive representation. 65

APPENDIX B A Figure 1 Schematic of memory organization. A) Nondeclarative memory organization, thought to depend on brain regions outside of the medial temporal lobe. B) Declarative memory dissociation within the medial temporal lobe. (Adapted from, Squire, 2004) 66

Figure 2. The hippocampal circuit within the medial temporal lobe. (Strien et al., 2009) Figure 3. The where and what pathways bringing in sensory information, converging in the hippocampus. (Adapted from Hunsaker et al., 2007) 67

Figure 4. Novel Object Recognition protocol for behavioral testing. During normal hippocampal function, the rodent should be able to encode, consolidate and retrieve the object memory; resulting in preference for novel object during Test session. When the hippocampus is inactivated after the Sample session or before the Test session, the rodent is unable to consolidate or retrieve the object memory, respectively. These impairments result in the mouse failing to exhibit novel object preference during the Test session, and instead exhibiting equal preference for both objects during test session. 68

Figure 5. Novel Object Recognition Recording task procedure.. Top figure shows the complete experimental layout. Bottom figure demonstrates a schematic showing the various recording sessions during Novel Object Recognition. A) Cue Card NOR protocol. B) Conventional NOR protocol. C) Drop-in NOR protocol Place Cell recordings. 69

Figure 6. Images of objects used for behavioral novel object recognition testing as well as for the recording experiments. Left: toy gorilla used as the novel object. Right: threaded metal foot, used as familiar object. Figure 7. Representative examples for pyramidal and interneurons recorded from dorsal CA1 region of the mouse hippocampus. Pyramidal neuron on the left in blue (waveform) with corresponding autocorrelogram below. Interneuron on right in red (waveform) with appropriate autocorrelogram below. 70

A) B) C) Figure 8. A) Example cluster cutting parameters using Offline Sorter V3 (Waveform, Autocorrelogram, and cluster). B) 2-dimensional clustering where interneuron blue, Pyramidal neuron yellow and noise signals green are demonstrated. C) 3-dimensional clustering of the same spikes. 71

Figure 9. Histological verifications confirmed tetrode location within the intermediate dorsal CA1 of hippocampus. Representative histology image of the mouse hippocampus after electrode implantation. Recording track seen above dorsal CA1 and disruption of overlying cortex. (Zheng & Khanna, 2008) Figure 10. Complex spike activity demonstrated by pyramidal neurons recorded form the CA1 region of the hippocampus of mice running the three different protocols of NOR. Top left shows a place cell recorded from the test session in the conventional NOR protocol with a <5 min delay between sessions. Middle shows place cell activity from a neuron in a mouse performing the Cue Card NOR test session with a 20 min delay between sessions. Bottom demonstrates spike activity from a mouse performing the test session in the Drop-in protocol with a 20 min delay between sessions. Right: comparable complex spike activity recorded from rat dorsal CA1. Arrow indicates time of paw formalin injection, resulting in reduced neuronal activity (Zheng & Khanna, 2008). 72

1.0 A) Cue Card NOR 0.8 Stability Correlation Stability Correlation 0.6 0.4 0.2 0.0 1.0 0.8 0.6 0.4 0.2 Hab1 & 2 5min B) Hab2 & S 5 min S & T 5 min Hab1 & 2 20 min Sessions and Delay Conventional NOR Hab2 & S 20 min S & T 20 min Figure 11. A) Mean stability of place field maps between recording sessions within the Cue Card protocol, as a function of delay. B) Mean stability of place field maps between recording sessions within the Conventional protocol, as a function of delay. C) Mean stability of place field maps between recording sessions within the Drop-in protocol, as a function of delay. Error bars SEM. 0.0 Hab 1& 2 5 min Hab 2 & S 5 min S & T 5 min Hab 1 & 2 20 min Hab 2 & S 20 min S & T 20 min Sessions and Delay 1.0 C) * D r o p - in N O R * S ta b ility C o rre la tio n 0.8 0.6 0.4 0.2 0.0 H a b 1 & 2 5 m in H a b 2 & S 5 m in S & T 5 m in H a b 1 & 2 2 0 m in H a b 2 & S 2 0 m in S & T 2 0 m in S e s s io n s a n d D e la y 73

Standardized Discrimination Ratio 2.0 1.5 1.0 0.5 0.0-0.5-1.0-1.5 A) Protocol Regression Conventional CueCard Drop-in -2.0-2.0-1.5-1.0-0.5 0.0 0.5 1.0 1.5 2.0 Standardized Stability scores Standardized Discrimination Ratio 2.0 1.5 1.0 0.5 0.0-0.5-1.0-1.5 B) Delay Regression 20 min < 5 min -2.0-2.0-1.5-1.0-0.5 0.0 0.5 1.0 1.5 2.0 Standardized Stability scores 74

Figure 13. Example of place field rotations within the empty square arena. Here, 180 rotations are seen between habituation 2 and sample, again 270 seen between test session and follow up session. Rotations between sample and test sessions were only seen in mice performing the Drop-in protocol with a 20 min delay interval. Warm and red colors represent highest firing rate location within the arena, blue and colder colors represent low firing rate or the cell is silent. 75

76 Figure 14. Example of place fields within the three different protocols with the in field firing frequency above each figure and the correlation between adjacent sessions stated below and between the images. Top represents place fields recorded from one cell within the Cue Card protocol. Middle represents place fields recorded from one cell within the Conventional protocol. Bottom represents place fields recorded from one cell within the Drop-in protocol. All examples are shown where the mice received 20 min delay between sessions.

D) Figure 15. Neuronal activity recorded during object exploration- location specific activity only at locations of familiar and novel objects during test session of Cue card protocol with 20 min delay interval. Representative examples taken at various times during the 600 sec (10 min) session A) Mouse engaging in active exploration of the familiar (foot) object in lower left corner. B) Mouse actively exploring the novel object (Gorilla) in upper right corner. C) Mouse freely moving around the recording chamber- no active exploration. D) Representative pyramidal neuron place firing from sessions during the NOR task. Cell fires only at object locations and remains silent in all other sessions, both in cylinder and square arena. F) Waveform E) Autocorrelogram and from the recorded cell. F) E) 77