THE EFFECT OF VESTIBULAR LESIONS ON THE
HIPPOCAMPAL CHOLINERGIC SYSTEM
PHILLIP NORMAN AITKEN
A thesis submitted for the degree of Doctor of Philosophy
University of Otago Dunedin New Zealand 2016 ABSTRACT Bilateral vestibular loss (BVL) produces profound behavioural changes, some of which do not appear to be directly associated with the acute loss of vestibular information, such as hyperlocomotion and spatial memory dysfunction. These behaviours appear to be due to long-term changes within the basal ganglia and limbic system. Both of these areas of the brain are associated with locomotion and memory. The hippocampus, in particular, has been extensively studied in relation to vestibular function. However, the exact mechanism which produces locomotor hyperactivity and cognitive dysfunction following BVL has yet to be discovered. Cholinergic signalling within the hippocampus is strongly associated with learning, memory, theta rhythm, and long-term potentiation (LTP). In the striatum, acetylcholine (ACh) release is correlated with procedural memory and locomotor activity. The aim of this study was to examine the effect of vestibular loss on behaviour, as well as examining how vestibular loss alters cholinergic signalling within the theta-generating pathway, hippocampus, and striatum. The behaviour of animals that underwent BVL or sham lesions was assessed in the open field to determine and distinguish the specific behavioural profile produced by BVL caused by intratympanic sodium arsanilate injections. BVL rats demonstrated reduced thigmotaxis, increased locomotor activity, an increase in transitions between each zone of the open field, and increased whole body rotations. The measured behavioural effects of BVL could correctly predict whether animals had received a BVL with a high degree of accuracy at both day 3 and day 23 post-BVL (83% and 100%, respectively).
Further experiments aimed to determine whether BVL produces changes to hippocampal and striatal cholinergic signalling. Initially, the distribution and density of muscarinic M1 and M2 receptors in the striatum and hippocampus were examined 7 and 30 days post-BVL. The density and distribution of M1 receptors was measured with beta-imaging autoradiography. The number of neuronal and non-neuronal cells expressing M2 receptors was measured with flow cytometry.
Thirty days following BVL in both the hippocampus and striatum, the density of M1 receptors decreased (P ≤ 0.0001) and the number of neurons expressing M2 receptors increased (P ≤ 0.05).
ii However, at the earlier 7 day time point, there was no change in muscarinic receptors in either area
(P > 0.05). Another experiment recorded local field potentials in the hippocampus using electrophysiology. Ninety days post-BVL or -unilateral vestibular loss (UVL), rats were anaesthetised with urethane and the theta rhythm generated from a tail pinch was recorded, analysed, and compared to sham-lesioned animals. The generation of type 2 theta was potentiated following BVL (P ≤ 0.05). The cholinergic neurons in the pedunculopontine tegmental nucleus
(PPT) of these rats were counted using stereology. Choline acetyltransferase (ChAT) immunohistochemistry selectively stained the cholinergic cells in the PPT. PPT cholinergic neurons increased in number contralateral to the lesion in UVL rats (P ≤ 0.05), with BVL rats exhibiting an increase in ChAT-positive neurons on both sides (P ≤ 0.05). Finally, the release of
ACh within the hippocampus following BVL was measured using microdialysis and high- performance liquid chromatography with electrochemical detection (HPLC-ECD). Microdialysis samples were collected under urethane anaesthesia. Baseline ACh release was unchanged between
BVL and sham animals (P > 0.05). Neither a tail pinch stimulus nor a rotational vestibular stimulus produced detectable changes in ACh release (P > 0.05). The results suggest that the hippocampal and striatal cholinergic systems are significantly altered following BVL. The potentiation of type
2 theta rhythm induced by somatosensory stimulation and the increase in cholinergic PPT neurons indicate potential changes to memory consolidation. Whether these changes are the cause of, or an adaptation to, the behavioural and cognitive symptoms which occur following BVL remains unknown.
iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my extremely supportive and dedicated supervisors, Prof. Paul Smith and Dr. Yiwen Zheng. Their guidance through each of my experiments and throughout the writing process has been crucial to each of my successes.
I want to thank my Doctoral Committee. Assoc. Prof. Cynthia Darlington, Dr. Catherine
Gliddon and Dr. John Ashton. I am extremely grateful for your constant enthusiasm and support, while remaining critical of my mistakes and providing solutions to them.
I want to thank Dr. Stéphane Besnard, my supervisor while I was in Caen. Allowing me to work in your lab and learn new techniques while visiting the far side of the globe was a truly wonderful experience. I also need to thank you for your fantastic translation abilities, without which I would have been truly lost.
I also want to thank the rest of the French lab group, for your help and patience while I learnt new techniques. Especially Dr. Alice Benoit, thank you for teaching me all I know about flow cytometry, and assisting with the tritiated ligand labelling for beta-imaging, I still shiver thinking about that walk-in refrigerator. Also, Bruno Philoxéne, thank you for teaching me how to perform sodium arsanilate intratympanic injections, and performing them yourself in many of the animals used in my beta-imaging and flow cytometry experiments.
Back in New Zealand, I want to thank each of the members of the Smith/Darlington/Zheng lab over the past 5 years, whether prior to or during my Doctoral studies. Primarily I want to thank
Lucy Stiles, for taking this PhD journey in parallel to me. Life was much easier, whether we were setting up the electrophysiology room, travelling to conferences, or simply getting through a bad day with your support.
I want to thank all the staff in the Department of Pharmacology and Toxicology. The morning/afternoon teas, lunches, Christmas functions, and more kept me well fed over the years.
I will certainly miss the daily ODT quiz. The helpful, friendly, and welcoming attitude throughout the entire department
iv I also want to thank John Reynolds for his help with setting up our electrophysiology room.
I want to thank Mandy Fisher, Janine Neill and other staff in the Histology Unit for decalcifying the temporal bones and allowing me to use your equipment to cut and stain them. I also want to thank Dave Grattan for the use of the HPLC-ECD equipment, and Ruth Napper and Andrew
McNaughton for use of the stereological microscope.
I want to thank Shayma Ali for reading my introduction and providing valuable feedback.
I would like to thank all the other post-grads in the Department. Our cosy room may be loud from time to time, but I could always count on someone being there to listen to me.
Without funding, this thesis would not have been completed. For funding projects and living costs while I studied I would like to thank Helen Rosa Thacker and the BHRC, the George
Duncan Memorial Scholarship, the Hope Selwyn Scholarship, the OSMS Dean’s Bequest, and the
Marie Curie SVETA grant. For funding travel to scientific conferences, allowing my research to be shared and reviewed by the best in the field, I want to thank the BHRC, Brain Research NZ, the Neurological Foundation of NZ, the Division of Health Sciences, and the Maurice and Phyllis
Paykel Trust.
Finally, I want to thank my friends and family for supporting me throughout this endeavour. Jess, you get me out of bed in the morning, both figuratively and literally. You keep me steady when I start to spiral. Thank you for being so supportive throughout this work. Finally,
I want to thank my parents. Their support and guidance shaped me into who I am today.
v TABLE OF CONTENTS
Abstract ______ii Acknowledgements ______iv Table of Contents ______vi Index of Abbreviations ______ix Index of Figures ______xii Index of Tables ______xv 1 ______1 Introduction ______1 Vestibular Nuclei ______5 Superior Vestibular Nucleus ______6 Medial Vestibular Nucleus ______6 Lateral Vestibular Nucleus ______6 Descending Vestibular Nucleus ______7 Vestibulo-Hippocampal Connections ______7 Thalamo-Cortical Pathway ______9 Head Direction Pathway ______10 Cerebello-Cortical Pathway ______11 Theta-Generating Pathway______11 Spatial Encoding Cells ______14 Head Direction Cells ______14 Grid Cells ______17 Place Cells ______18 Spatial Memory, Cognition, and the Vestibular System ______21 Effect of Vestibular Damage on the Hippocampus ______31 Hippocampal Theta Rhythm and the Vestibular System ______33 Vestibular Damage, Cholinergic System and Neurodegenerative Disease ______37 Muscarinic Acetylcholine Receptors______39 Limitations of Existing Studies ______41 Conclusion ______43 Aims and Objectives ______45 2 ______46 Chemical Labyrinthectomy ______46 Methods ______48 Subjects ______48 Intratympanic Injection ______48 Assessment of Vestibular Syndrome ______49 Temporal Bone Histology ______51 3 ______52 Open Field Behaviour of BVL Rats ______52 Introduction ______52 Methods ______57 Statistical Analysis ______60 Results ______61 Discussion ______69 4 ______72
M1 Receptor Density Following BVL ______72 Introduction ______72 Methods ______75 Quantitative M1-Receptor Autoradiography ______75
vi Equations ______76 Statistical Analyses ______77 Results ______78 Vestibular Deficiency ______78 M1 Receptor Autoradiography ______78 Discussion ______80 5 ______82
M2 Receptor Expression Following BVL ______82 Introduction ______82 Methods ______84 Subjects ______84 Tissue Dissociation and Myelin Removal ______84 Antibody Markers ______86 Statistical Analysis ______89 Results ______90 Vestibular Deficiency ______90 M2 Receptor Expression ______90 Discussion ______92 6 ______96 Hippocampal Theta Rhythm is Potentiated Following BVL______96 Introduction ______96 Methods ______98 Hippocampal Local Field Potential Recording ______98 Histology ______99 Statistical Analysis ______99 Results ______101 Vestibular Deficiency ______101 Electrode Placement ______101 Hippocampal Electrophysiology ______103 Discussion ______106 7 ______108 Cholinergic Cells in the PPT Following BVL ______108 Introduction ______108 Methods ______112 Statistical Analysis ______113 Results ______115 Discussion ______117 8 ______120 Acetylcholine Release in the Hippocampus is Not Altered Following Bilateral Vestibular Lesions ______120 Introduction ______120 Methods ______123 Hippocampal Recording and Microdialysis ______123 Histology ______125 HPLC-ECD ______125 Probe Recovery ______126 Statistical Analysis ______127 Results ______128 Vestibular Deficiency ______128 Electrode Placement ______128 Hippocampal Electrophysiology ______130 HPLC-ECD Analysis ______131 Discussion ______135
vii 9 ______137 General Discussion ______137 Limitations of Experiments ______141 Future Directions ______143 Conclusion ______145 10 ______147 References ______147 11 ______189 Appendices ______189 A) Ethovision Statistical Analysis ______189 B) Published Research______196 C) Conference Proceedings ______202 D) Copyright Permissions ______203
viii INDEX OF ABBREVIATIONS ACh Acetylcholine AChE Acetylcholinesterase aCSF Artificial cerebrospinal fluid AD Alzheimer’s disease ADHD Attention deficit / hyperactivity disorder ADN Anterior dorsal nucleus AIC Akaike’s information criterion ANOVA Analysis of variance BSA Bovine serum albumin BVL Bilateral vestibular loss CA1 Area cornu ammonis 1 CA2/3 Area cornu ammonis 2 and 3 CCD Charge coupled device Ch5 Cholinergic cell group 5 ChAT Choline acetyltransferase cVEMP Cervical vestibular evoked myogenic potentials DAB 3,3′-Diaminobenzidine DD Depersonalisation/derealisation DG Dentate gyrus of the hippocampus DTN Dorsal tegmental nucleus EC Entorhinal cortex ECD Electrochemical detection EEG Electroencephalogram FN Fastigial nuclei GABA γ-aminobutyric acid GFAP Glial fibrillary acidic protein GLMM Generalized linear mixed model GPCR G-protein coupled receptor GVS Galvanic vestibular stimulation HD Head direction cells
ix HPLC High-performance liquid chromatography h Hour(s) HRP Horseradish peroxidase Hz Hertz Int Interpositus nuclei KO Knockout LDA Linear discriminant analysis LMM Linear mixed model LMN Lateral mammillary nucleus LOO Leave-one-out LTD Long term depression LTP Long term potentiation LTS Low threshold spikes LVN Lateral vestibular nucleus mAChR Muscarinic acetylcholine receptor min Minute(s) MLR Multiple linear regression MRI Magnetic resonance imaging MS Medial septum MVN Medial vestibular nucleus NADPH-d Dihydronicotinamide adenine dinucleotide phosphate diaphorase NMDA N-methyl-D-aspartate NSB Non-specific binding PBS Phosphate buffered saline PD Parkinson’s disease PH Posterior hypothalamus PPT Pedunculopontine tegmental nucleus PrH Prepositus hypoglossi PS Postsubiculum PSP Progressive supranuclear palsy QICC Corrected quasi likelihood criterion RPO Reticularis pontis oralis
x s Second(s) SCC Semicircular canals SUM Supramammillary bodies SVN Superior vestibular nucleus Theta Theta rhythm TTX Tetrodotoxin UVL Unilateral vestibular loss UVLC Unilateral vestibular loss contralateral side UVLI Unilateral vestibular loss ipsilateral side VLN Ventral lateral nucleus of the thalamus VNC Vestibular nuclei complex VORs Vestibulo-ocular reflexes VSRs Vestibulo-spinal reflexes Xylo Xylocaine
xi INDEX OF FIGURES
Figure 1.1: Anatomy of the inner ear...... 1
Figure 1.2: Vestibular nuclei complex...... 5
Figure 1.3: Proposed pathways from the peripheral vestibular organs to the hippocampus ...... 8
Figure 1.4: Head direction cell firing...... 15
Figure 1.5: Place cell and grid cell firing patterns...... 17
Figure 1.6: Place cell firing maps following temporary vestibular inactivation...... 23
Figure 1.7: Conceptual model proposing a mechanism of cognitive dysfunction due to vestibular dysfunction...... 28
Figure 1.8: Examples of galvanic vestibular stimulation waveforms...... 30
Figure 2.1: Time course of histological lesions induced by arsanilate in the sensory epithelia of one semi-circular canal in a rat ...... 47
Figure 2.2: Contact inhibition test...... 50
Figure 2.3: Head tilt and body twisting in a UVL rat...... 50
Figure 2.4: Histology of horizontal vestibular ampulla ...... 51
Figure 3.1: Rat in the open field...... 57
Figure 3.2: Movement tracing of BVL rats in an open field...... 61
Figure 3.3: Zone duration in the open field...... 62
Figure 3.4: BVL rat activity in an open field...... 63
Figure 3.5: Zone transitions of BVL rats in the open field...... 63
Figure 3.6: Rotation frequency of BVL animals in the open field...... 64
Figure 3.7: Cluster analysis of behavioural data...... 66
Figure 4.1: General operating principle of a beta-imager...... 74
Figure 4.2: M1 acetylcholine receptor expression following bilateral vestibular lesions ...... 77
Figure 4.3: M1 mAChR density 7 and 30 days following BVL ...... 79
Figure 5.1: Tissue dissociation protocol...... 85
xii Figure 5.2: MACS magnetic separation protocol...... 86
Figure 5.3: FITC (green) and Alexa Fluor750 (purple) excitation and emission spectra...... 87
Figure 5.4: Laser calibration of cytometer samples...... 88
Figure 5.5: Myelin gating protocol...... 88
Figure 5.6: Cells counted with flow cytometry ...... 89
Figure 5.7: M2 acetylcholine receptor expression following BVL using flow cytometry ...... 91
Figure 5.8: Western blot of the anti-M2 antibody (ab2805)...... 94
Figure 6.1: Electrode tip locations...... 102
Figure 6.2: Hippocampal response to a tail pinch during urethane anaesthesia...... 103
Figure 6.3: Changes of hippocampal power distribution within frequency bands...... 104
Figure 6.4: Hippocampal frequency at the maximum power within frequency bands (Fmax). . 104
Figure 6.5: PPT power and frequency...... 105
Figure 7.1: Summary of the topographical distribution of the connectivity in the PPT...... 109
Figure 7.2: Stereological counting frame over cells in the PPT...... 114
Figure 7.3: Size difference between PPT cholinergic neurons and astrocytes...... 114
Figure 7.4: ChAT and methyl green staining in the PPT...... 115
Figure 7.5: The number of cholinergic PPT cells following vestibular lesions...... 116
Figure 8.1: Diagram of microdialysis probe, cannula and electrode...... 123
Figure 8.2: Timeline of microdialysis sampling...... 125
Figure 8.3: Schematic of HPLC-ECD...... 126
Figure 8.4: Probe and cannula tip locations...... 129
Figure 8.5: Hippocampal response to tail pinch during urethane anaesthesia...... 130
Figure 8.6: Hippocampal power distribution within frequency bands...... 131
Figure 8.7: HPLC-ECD output...... 132
Figure 8.8: Standard curve of known concentrations of ACh chloride...... 133
Figure 8.9: Relative recovery of microdialysis probes...... 133
xiii Figure 8.10: Acetylcholine release in BVL and sham rats...... 134
Figure 11.1: EthoVision analysis, non-significant results...... 189
xiv
INDEX OF TABLES
Table 1.1: Location of muscarinic receptors throughout the brain...... 39
Table 3.1: Variables measured with EthoVision XT 6™...... 59
Table 3.2: Multiple linear regression where time not moving was the dependant variable (day 3).
...... 67
Table 3.3: Multiple linear regression where relative angular velocity was the dependant variable
(day 3)...... 67
Table 4.1: Behavioural scores of animals prior to beta-imaging autoradiography ...... 78
Table 5.1: Behavioural scores of animals prior to flow cytometry analysis...... 90
Table 6.1: Behavioural scores of animals prior to electrophysiological recording ...... 101
Table 8.1: Behavioural scores of animals prior to electrophysiological recording ...... 128
Table 11.1: Multiple linear regression where distance moved (centre point) was the dependant variable (day 3)...... 190
Table 11.2: Multiple linear regression where time contracted was the dependant variable (day 3).
...... 192
Table 11.3: Multiple linear regression where time stretched was the dependant variable (day 3).
...... 192
Table 11.4: Multiple linear regression where time in the outer zone (nose point) was the dependant variable (day 3)...... 193
Table 11.5: Multiple linear regression where time in the centre zone (nose point) was the dependant variable (day 3)...... 193
Table 11.6: Multiple linear regression where time in the centre zone (centre point) was the dependant variable (day 3)...... 193
Table 11.7: Multiple linear regression where meander was the dependant variable (day 3). .... 194
Table 11.8: Multiple linear regression where time mobile was the dependant variable (day 3). 194
xv Table 11.9: Multiple linear regression where time not moving was the dependant variable (day
3)...... 194
Table 11.10: Multiple linear regression where clockwise rotation was the dependant variable
(day 3)...... 195
Table 11.11: Multiple linear regression where frequency of outer-intermediate zone transitions was the dependant variable (day 3)...... 195
Table 11.12: Multiple linear regression where total rotations was the dependant variable (day 3).
...... 195
xvi
If the human brain were so simple That we could understand it, We would be so simple That we couldn’t.
Emerson M. Pugh
xvii 1 INTRODUCTION The vestibular organs in the inner ear provide the ability to sense balance and acceleration.
The vestibular apparatus is one of the oldest evolutionary sensory organs, evolving ~500 million years ago with variations of the general structure found in a diverse range of animals (Fritzsch,
1998; Spoor et al., 2002). The vestibular sensory organs also develop early during embryogenesis, allowing for the detection of gravity and self-motion prior to birth (Baker, 1998; Ronca et al.,
2008). The vestibular organ is made of a bony labyrinth, inside of which is the membranous labyrinth which consists of tubular ducts filled with fluid called endolymph (Guild, 1927). The labyrinth structures are mirror symmetric within the inner ear on each side of the head, each containing five vestibular sensory organs, that is, three semi-circular canals (SCCs) and the two otolith organs (Figure 1.1).
Figure 1.1: Anatomy of the inner ear. Figure from Khan and Chang (2013). Copyright IOS press, used with permission.
Vestibular sensory information is transduced within the SCCs: horizontal, anterior and posterior canals, which detect rotational acceleration, and the otolith organs (the utricle and
1 saccule), which detect linear acceleration. The utricle detects horizontal acceleration while the saccule detects vertical acceleration, including gravity. At the end of the semicircular ducts is the ampulla, an enlarged region containing sensory hair cells embedded in the gelatinous cupula.
During acceleration, the endolymphatic fluid within the SCCs is displaced relative to the ducts/canals; this produces a fluid current which bends the cupula and the sensory hair cells within it. The hair cells are depolarized by movement in one direction and hyperpolarized by movement in the opposite direction (Khan and Chang, 2013). This changes the rate of firing in the afferent axons, increased during depolarization and decreased during hyperpolarization. Signals are sent through the vestibulo-cochlear nerve (cranial nerve VIII) to the vestibular nucleus located in the brainstem (Carleton and Carpenter, 1984). The otolithic sensory organs act in a similar fashion to detect linear acceleration, however rather than relying on the inertia of endolymph to stimulate the hair cells, the utricle and saccule contain otoconia. Otoconia are calcium carbonate crystals which are embedded into the surface of a gelatinous membrane within which are sensory hair cell projections. Acceleration causes the otoconia to move, causing an inertial force on the membrane which causes a deflection of the hair cells that are fixed to the underlying epithelium. Following damage to the vestibular system a vestibular syndrome occurs, including oculomotor, postural and sensory symptoms, such as spontaneous nystagmus, head tilting and unidirectional spinning following unilateral vestibular loss (UVL) (Darlington and Smith, 1989; Smith and Curthoys,
1989), and ataxia, hyperactivity, head dorsiflexion, backward locomotion, bidirectional circling and oscillopsia following bilateral vestibular loss (BVL) (Goddard et al., 2008; Ossenkopp et al.,
1992; Ossenkopp et al., 1990; Rinne et al., 1998). Vestibular compensation provides a partial restoration of normal behaviour. The compensation process can take 60 days for dynamic symptoms (those that occur during movement) to reach a steady state, while static symptoms
(those that occur while the animal is stationary) can be compensated for within 2-3 days, depending on the species (Mantokoudis et al., 2014; Smith and Curthoys, 1989).
2 Ocular and postural reflexes are dependent on the vestibular system. The vestibulo-ocular reflexes (VORs) allow for smooth eye-tracking movements during rotational and translational movement of the head. The VOR occurs so rapidly that it can have a response latency of as little as 5 ms and allow for responses to acceleration up to 15000o/s2 (Huterer and Cullen, 2002; Minor et al., 1999). The vestibulo-spinal reflexes (VSRs) maintain balance and postural control during changes in head velocity. Studies in the late 20th century suggested that the vestibular system also plays an important role in spatial navigation, memory and other cognitive functions (Grimm et al.,
1989; Smith, 1997; Smith et al., 2010a). Astronauts and cosmonauts are known to suffer from attention and memory difficulties while in space and microgravity environments (Newberg, 1994;
Watt, 1997). Numerous behavioural studies have also found that damage to the vestibular system impairs hippocampal-related learning and memory tasks (Baek et al., 2010; Besnard et al., 2012;
Brandt et al., 2005; Chapuis et al., 1992; Dallal et al., 2015; Guidetti et al., 2008; Horn et al.,
1981; Hufner et al., 2007; Machado et al., 2012a; Matthews et al., 1989; Neo et al., 2012;
Ossenkopp and Hargreaves, 1993; Russell et al., 2003a; Russell et al., 2003b; Schaeppi et al.,
1991; Schautzer et al., 2003; Smith et al., 2013; Stackman and Herbert, 2002; Wallace et al., 2002;
Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2004; Zheng et al., 2006; Zheng et al., 2007;
Zheng et al., 2009b). As the hippocampus is considered largely responsible for spatial navigation and memory (Best et al., 2001; Moser et al., 2008; O'Keefe, 1993), this suggests that the vestibular system might activate the hippocampus in some way. Electrical stimulation of the vestibular system has been found to activate the hippocampus during magnetic resonance imaging (MRI), as well as produce responses in Cornu Ammonis area 1 (CA1) and modulate the firing patterns of hippocampal place cells (Cuthbert et al., 2000; de Waele et al., 2001; Horii et al., 2004; Sharp et al., 1995; Vitte et al., 1996), while vestibular lesions disrupt place cell firing (Russell et al., 2003b;
Stackman et al., 2002). Hippocampal theta activity is also modulated by vestibular stimulation
(Cuthbert et al., 2000; Gavrilov et al., 1995; Gavrilov et al., 1996; Shin, 2010; Shin et al., 2005;
3 Tai and Leung, 2012; Tai et al., 2012) and disrupted by vestibular lesions (Neo et al., 2012; Russell et al., 2006; Stackman et al., 2002; Tai et al., 2012).
Hippocampal theta rhythm (theta) is an oscillating electrical signal within the 3-12 Hz frequency range in rats. Theta is modulated by inputs from the medial septum and hypothalamus
(Bocian and Konopacki, 2001; Smythe et al., 1991; Woodnorth et al., 2003). Early studies suggested that theta is required for learning and memory, with lesions of the medial septum disrupting theta and producing deficits in spatial memory tasks (Winson, 1978). The disruption of theta affects both spatial (Givens, 1995; Leutgeb and Mizumori, 1999; O'Keefe, 1993) and non- spatial tasks (Mizumori et al., 1990). The synchronous firing of cells that occurs during theta activity produces long-term potentiation (LTP) within the hippocampus (Greenstein et al., 1988;
Larson et al., 1986; Pavlides et al., 1988; Vertes, 2005). LTP strengthens synaptic responses following repetitive stimulation of the neurons involved. LTP has become generally accepted as the synaptic process that underlies learning and memory in the hippocampus (Hebb, 1949; Muller et al., 2002; Vertes, 2005). The disruption of hippocampal theta is thought to inhibit learning and memory as the magnitude of hippocampal LTP is reduced (Orr et al., 2001). While it has been shown that the ability to generate LTP in the hippocampus remains intact in vestibular-lesioned animals (Zheng et al., 2010), the lack of a vestibular system and accompanying theta rhythm generation likely prevents, or at least increases, the threshold for LTP as seen in aged rats (Orr et al., 2001). As theta occurs during a large range of activities, including many that involve movement of the head and therefore salient vestibular cues (Gavrilov et al., 1995; Shin, 2010), it is likely that connections between the vestibular system and hippocampus substantially influence the production of theta and spatial memory processing. This would explain why vestibular inactivation produces deficits in spatial learning and a reduction in theta activity (Neo et al., 2012;
Russell et al., 2006).
4 VESTIBULAR NUCLEI The vestibular nuclei complex (VNC) consists of the main cell groups which process vestibular information at the terminals of the VIIIth nerve (Figure 1.2). Vestibular information is sent via the VIIIth nerve to the ipsilateral VNC and cerebellum (Carleton and Carpenter, 1984).
The VNC consist of four separate bilaterally located nuclei: the superior, medial, lateral and descending vestibular nuclei, as well as smaller vestibular nuclei: Y-group, parasolitary nucleus, nucleus intercalatus and prepositus hypoglossi (PrH) (Barmack, 2003; Highstein and Holstein,
2006).
Figure 1.2: Vestibular nuclei complex. The VIIIth nerve projects to the VNC from the peripheral vestibular organs. Coronal view, 11 mm posterior to bregma. PrH, prepositus hypoglossi; M, medial vestibular nucleus; S, superior vestibular nucleus; L, lateral vestibular nucleus; D, descending vestibular nucleus; Y, Y-group. Cresyl violet section modified from Paxinos and Watson (1998). Copyright Elsevier, used with permission.
The nuclei are all located within the medulla and pons of the brainstem. There are connections between all of the major vestibular nuclei (Ito et al., 1985). The VNC not only receives vestibular information, but also receives other sensory projections, such as visual, proprioceptive, and in some species, magnetic input (Allum et al., 1976; Cazin et al., 1982; Gdowski and McCrea,
1999; Gdowski and McCrea, 2000; Henn et al., 1974; Robinson, 1977; Semm et al., 1984). The
VNC integrates these multiple sensory signals and relays them to other brain regions (Gdowski and McCrea, 1999; Robinson, 1977).
5 Superior Vestibular Nucleus
The superior vestibular nucleus (SVN) receives projections from the SCCs. The posterior and horizontal canals innervate the medial SVN while the anterior canal innervates a more lateral area (Gacek, 1969; Highstein and Holstein, 2006). The SVN connects to other VNC nuclei, with reciprocal connections occurring between the descending and medial vestibular nuclei (Epema et al., 1988). The SVN projects to the trochlear nucleus, oculomotor nuclear complex and cerebellum
(Carpenter and Cowie, 1985). Cells which are involved in producing the VORs are located in the central SVN while non-VOR neurons make up the peripheral cells (Mitsacos et al., 1983a;
Mitsacos et al., 1983b).
Medial Vestibular Nucleus
The medial vestibular nucleus (MVN) is the largest of the vestibular nuclei, both in cell number and total volume. The majority of SCC afferents terminate in the MVN, with all three canals projecting to the MVN (Gacek, 1969). These project to the magnocellular area in the rostrolateral MVN which contains efferent projections to the extraocular motor nuclei related to the VOR response (Buttner-Ennever, 1992; Epema et al., 1988). The otoliths, spinal cord and parts of the cerebellum project to the parvocellular region of the caudomedial MVN (Carleton and
Carpenter, 1984; Highstein and Holstein, 2006). The MVN has projections to the cerebellum, spinal cord and thalamus (Brodal, 1984; Carleton and Carpenter, 1984; Epema et al., 1988;
Highstein and Holstein, 2006; Scudder and Fuchs, 1992).
Lateral Vestibular Nucleus
The lateral vestibular nucleus (LVN) is divided into two parts, the dorsal LVN and the ventral LVN. The ventral LVN receives input from the SCCs. The dorsal LVN is the major termination site of saccular afferent fibres which innervate much of the dorsal LVN (Imagawa et al., 1998; Newlands and Perachio, 2003; Newlands et al., 2003). The LVN is involved in coordinating eye movements during movement of the head and producing the VSRs which
6 produce compensatory limb movements during body movement (Chan et al., 1977; Xerri et al.,
1988).
Descending Vestibular Nucleus
The descending vestibular nucleus is innervated by primary afferents from both the otolith organs and SCCs (Buttner-Ennever, 1992). It contains a large proportion of fibre bundles which give it the appearance of being less densely populated by neurons than other vestibular nuclei. The nucleus is unevenly innervated with the dorsal descending vestibular nucleus receiving a larger proportion of VIIIth nerve innervation than the ventral descending vestibular nucleus (Carleton and
Carpenter, 1984). Like the LVN, the descending vestibular nucleus is mainly involved in the VSRs
(Xerri et al., 1988).
VESTIBULO-HIPPOCAMPAL CONNECTIONS The vestibular system and the hippocampus are hypothesised to be linked by several polysynaptic pathways (Figure 1.3; Smith, 1997; Smith et al., 2005a). It is currently unknown as to which of the following pathways might provide signals which are associated with hippocampal spatial learning. There is also uncertainty about whether some of these pathways exist functionally.
Locating the pathway(s) involved in spatial learning could reveal treatment options such as drug therapy, electrical stimulation, or transcranial magnetic stimulation not only in vestibular-deficient patients, but also in other patients who suffer from spatial memory deficits.
7
Figure 1.3: Proposed pathways from the peripheral vestibular organs to the hippocampus on a diagram of a horizontally sliced rat brain. Thalamo-cortical pathway (brown). Theta-generating pathway (green). Cerebello-cortical pathway (red). Head direction pathway (yellow). Abbreviations: ADN – anterior dorsal nucleus; DTN – dorsal tegmental nucleus; EC – entorhinal cortex; FN – fastigial nuclei; HCF – hippocampal formation; Int – interpositus nuclei; LMN – lateral mammillary nucleus; MS- medial septum; Parietal – parietal cortex; PH – posterior hypothalamus; PrH – prepositus hypoglossi; PPT – pedunculopontine tegmental nucleus; PS – postsubiculum; RPO – reticularis pontis oralis; SUM – supramammillary bodies; Temporal – temporal cortex; VLN – ventral lateral nucleus of the thalamus; VNC – vestibular nuclei.
8 Thalamo-Cortical Pathway
Vestibular stimulation activates cortical areas. The parietal, visual and frontal cortices all respond to vestibular stimulation (Guldin et al., 1992; Guldin and Grüsser, 1998). Cortical neurons are connected to the vestibular sensory organs through trisynaptic pathways. Although there are monosynaptic projections from the cortex to the vestibular system, the projections from the vestibular system to the cortex are all polysynaptic (Guldin et al., 1992). Cortical neurons that respond to vestibular information receive a convergence of vestibular, visual and somatosensory inputs suggesting that the majority of these cells are involved in postural and gaze control (Berthoz,
1996; de Waele et al., 2001; Fukushima, 1997). Disynaptic connections between the VNC and the parietal cortex through the thalamus have been suggested to be part of a vestibulo-hippocampal pathway (de Waele et al., 2001; Hicks, 2005; Smith et al., 2005a). Four sites in the thalamus receive vestibular information from the vestibular nucleus and project it to the cortex: the ventrolateral geniculate nucleus, lateral posterior nucleus, ventral posterior nucleus and the medial geniculate nucleus (Abraham et al., 1977; Buttner and Lang, 1979; Faugier-Grimaud and Ventre,
1989; Magnin and Kennedy, 1979; Shiroyama et al., 1999; Smith et al., 2005a). Other areas in the thalamus when lesioned unilaterally produce shifts in subjective visual vertical; these include the posterior nuclei, nuclei dorsomedialis, intralamellaris, centrales thalami, ventrooralis internus, ventrointermedii, ventrocaudales, nuclei parafascicularis thalami and nuclei endymalis thalami
(Baier et al., 2016; Dieterich and Brandt, 2015).
In primates, projections from the thalamic nuclei ascend to the parietoinsular vestibular cortex (Berthoz, 1996; de Waele et al., 2001; Dieterich and Brandt, 2015; Faugier-Grimaud and
Ventre, 1989; Kirsch et al., 2016). Five pathways have been identified: two ipsilateral, two which cross in the pons or midbrain, and one pathway which bypasses the thalamus, projecting directly to the insular cortex ipsilaterally (Kirsch et al., 2016). In rats the region of the cortex involved in this pathway is the associative parietal cortex (Save et al., 2005; Save and Poucet, 2000). This region is considered analogous to the primate parietoinsular vestibular cortex (Bucci et al., 1999;
9 Kolb and Walkey, 1987; Save and Poucet, 2000). These regions of the parietal cortex have afferent connections to the hippocampus via the entorhinal and perirhinal cortices (McNaughton et al.,
1994; Save and Poucet, 2009; Whitlock et al., 2008). It is therefore likely that some vestibular responses reach the hippocampus via this vestibulo-thalamocortical pathway. In rats, lesions of the associative parietal cortex alter hippocampal place cell activity, suggesting a role in spatial processing (Save et al., 2005). As this area of the cortex receives visual, vestibular and somatosensory signals, integrated in the thalamic nuclei discussed previously (Wijesinghe et al.,
2015), it is unknown if spatial processing in this region is modulated by vestibular or other inputs.
It has been suggested that more direct connections from the vestibular sensory organs to the hippocampus are possible via the head direction and theta-generating pathways (Smith, 1997;
Smith et al., 2005a; Taube et al., 1996).
Head Direction Pathway
The pathway involved in the activation of head direction cells may also transmit vestibular information to the hippocampus (Taube, 2007). Head direction cells are located throughout the limbic system and fire in response to the direction of the head in the horizontal plane. The response is generated from vestibular stimulation and modulated by visual, motor and proprioceptive input
(Taube, 2007). Functional horizontal canals are essential for the head direction signal; animals with bilateral lesions, canal occlusion or mice with a genetic horizontal canal malformation lack the directional signal completely (Muir et al., 2009; Stackman and Taube, 1997; Taube, 2007;
Valerio and Taube, 2016). The vestibular information relating to the direction of the head is thought to project to the hippocampus via another long polysynaptic pathway. The VNC projects to the supragenual nucleus and PrH. These then project to the dorsal tegmental nucleus, which connects to the lateral mammillary nucleus. The lateral mammillary nucleus projects to the anterior dorsal nucleus of the thalamus, and on to the postsubiculum, entorhinal cortex and hippocampus
(Biazoli et al., 2006; Brown et al., 2005; Clark et al., 2012; Liu et al., 1984; Shibata, 1987; Shinder and Taube, 2010; Smith et al., 2005a; Taube, 2007; Taube et al., 1996). The head direction cell
10 information is integrated with place and grid cell information within the hippocampus, providing the animal with a sense of location in space (Taube, 2007). This makes the head direction pathway a likely source of vestibular information in the hippocampus. It may work in concert with one of the other vestibulo-hippocampal pathways to maintain normal hippocampal activity.
Cerebello-Cortical Pathway
The cerebello-cortical pathway is mainly involved in the production of smooth pursuit eye movements (Fukushima, 2003) and the pathway has not yet been fully mapped. The vestibular nerve projects directly to the cerebellum, however there are also projections to the flocculus and fastigial nucleus from the VNC (Hitier et al., 2014). The cerebellum process self-motion information, integrating vestibular and body motion cues, before sending it on to the hippocampus to build spatial representations in place cells. It then uses the spatial representation to plan the best trajectory for spatial navigation (see Rochefort et al., 2013 for review). VNC projections into the cerebellum are thought to then project from cerebellar neurons to the thalamus via the dentate and posterior interpositus nuclei (Fukushima, 2003; Hufner et al., 2007; Tanaka, 2005). From the thalamus, vestibular information is thought to reach the hippocampus through either the frontal, temporal or parietal cortices (Hufner et al., 2007). Direct connections from the cerebellum to the hippocampus have also been proposed, but whether these transmit vestibular information is unknown (see Hitier et al., 2014 for review). More research is required to examine this pathway and its possible involvement with the hippocampus and cortical areas.
Theta-Generating Pathway
Theta is a large, rhythmic, sinusoidal signal that can be recorded extracellularly in most mammals (Vertes, 2005). Theta is present during locomotor activity and rapid eye movement
(REM) sleep (Bland, 1986; Boyce et al., 2016; Winson, 1978). Tactile, olfactory, visual, auditory and vestibular stimulation all produce theta (Gavrilov et al., 1995; Nowacka et al., 2002; Senba and Iwahara, 1974; Shin, 2010; Vanderwolf, 1992). The brainstem contains nuclei which are known to both produce theta rhythm and receive projections from the VNC (Bland and Oddie,
11 1998; Vertes and Kocsis, 1997). These include the pedunculopontine tegmental nucleus (PPT) and reticularis pontis oralis (RPO) (Horowitz et al., 2005; Semba and Fibiger, 1992). The PPT is a major acetylcholine (ACh)-containing cell group in the brainstem, cholinergic cell group 5 (Ch5)
(Jones and Beaudet, 1987). The RPO projects to the PPT, supramammillary nucleus (SUM), posterior hypothalamic nucleus (PH) and intra-thalamic nuclei (Bland and Oddie, 1998; Vertes and Kocsis, 1997; Vertes and Martin, 1988). The PPT projects to the RPO, SUM, the septum, substantia nigra, lateral hypothalamus, median raphe nucleus, medial thalamic and intralaminar thalamic nuclei (Bland and Oddie, 1998; Pahapill and Lozano, 2000; Pignatelli et al., 2012; Semba and Fibiger, 1992; Woolf and Butcher, 1989). Both the PPT and RPO respond to vestibular stimulation (Aravamuthan and Angelaki, 2012; Bland and Oddie, 1998; Seemungal et al., 2010;
Vertes and Kocsis, 1997). When either the RPO or PPT is stimulated, hippocampal theta is elicited, with increased stimulation producing a linear increase in theta frequency (Bland and Oddie, 1998;
Kinney et al., 1998; Klemm, 1972; McNaughton and Sedgwick, 1978; Oddie et al., 1994; Vertes and Kocsis, 1997). RPO activity is also increased during theta-generating activity, such as a tail pinch. A tail pinch caused 85% of RPO neurons to change their firing, with 64% increasing and
21% decreasing the frequency of firing (n=72) and theta also being elicited (Nunez et al., 1991).
Injections of procaine suppress PPT activity, causing a reduction in theta and suggesting a critical role for the PPT in theta generation (Nowacka et al., 2002). While the RPO is thought to be the main brainstem site involved in the generation of theta (Vertes and Kocsis, 1997), the PPT modulates RPO activity with direct cholinergic projections (Semba and Fibiger, 1992; Shiromani et al., 1988; Vertes and Kocsis, 1997). The RPO is thought to trigger the onset of theta while the
PPT maintains theta (Takano and Hanada, 2009). The effect of the PPT on learning and memory has been examined in lesion and stimulation studies. PPT deep-brain stimulation in Parkinson’s disease patients has been found to improve working memory (Alessandro et al., 2010; Costa et al.,
2010). In rats, complete bilateral PPT lesions impair performance in the radial arm maze (Dellu et al., 1991; Keating and Winn, 2002). Conversely, in delayed non-matching to position tasks there
12 is no change in performance following PPT lesions (Leri and Franklin, 1998; Steckler et al., 1994).
It was suggested that the impaired performance in the radial maze task is due to disruption of attention, however the loss of theta generation following PPT lesioning indicates hippocampal spatial cell disruption. Place and grid cells require intact theta to accurately maintain their spatial fields (Brandon et al., 2011; Geisler et al., 2010; Koenig et al., 2011; Moser et al., 2008). Further studies are needed to examine the effect of PPT lesions on hippocampal spatial cell firing fields.
Within the theta-generating pathway the brainstem nuclei project to the SUM and the PH, which then project to the medial septum/diagonal band of Broca (MS) (Figure 1.3). The MS then projects to the hippocampus (Bland and Oddie, 1998; Hitier et al., 2014; Pignatelli et al., 2012;
Vertes and Kocsis, 1997). The SUM modulates the frequency of theta while the PH modulates the amplitude (Bocian and Konopacki, 2001; Kirk, 1998; Woodnorth et al., 2003). SUM cells fire synchronously with theta (Kirk and McNaughton, 1991; Kirk and McNaughton, 1993). PH neurons fire tonically during periods of theta activity (Bland et al., 1995). Projections from the PH to both the SUM and MS may produce changes in theta frequency as well as amplitude due to changes in SUM firing (Kirk, 1998). The selective inactivation of the PH and SUM by muscimol or procaine administration impairs theta, affecting both frequency and amplitude (Bocian and
Konopacki, 2001; Bocian and Konopacki, 2007; Oddie et al., 1994). Both the SUM and PH drive theta cells in the MS and hippocampus through glutamatergic afferents, with a positive correlation between SUM/PH stimulation intensity and rates of cell firing in the MS and hippocampus (Bland et al., 1990; Bland et al., 1994; Colom et al., 1987; Smythe et al., 1991). The PH and SUM also receive descending inputs from the MS that limit the neuronal firing rates during theta (Kirk et al.,
1996). The bursting SUM cells and the tonic PH cells appear to act together to modulate ascending theta activity.
The MS provides major inputs to the hippocampus and contains excitatory cholinergic and inhibitory GABAergic cells. The MS projects throughout the hippocampal formation, the hippocampus proper (dentate gyrus (DG), CA1, CA2/3), subiculum, presubiculum, and
13 parasubiculum (Crutcher et al., 1981). Vertes and Kocsis (1997) suggested that the cholinergic and GABAergic cells fire rhythmically to pace theta activity by inhibiting hippocampal interneurons and simultaneously activating hippocampal pyramidal cells. The coordinated discharge of cholinergic and GABAergic cells produces positive peaks of theta, blocking the inhibitory interneurons and producing a rhythmic firing of pyramidal cells. Negative theta peaks occur when there is a pause in the firing of MS cells, allowing hippocampal interneurons to fire, which prevents pyramidal cells from firing (Bland and Oddie, 1998; Vertes and Kocsis, 1997).
This results in the rhythmic sinusoidal wave of the theta frequency (Bland and Oddie, 1998; Vertes and Kocsis, 1997).
SPATIAL ENCODING CELLS Within the hippocampus and connecting areas, such as the entorhinal cortex, subiculum, and thalamus, are cells which fire specifically in relation to spatial information. These are place cells, grid cells and head direction cells. Place cells provide a representation of the animal’s location in space relative to landmarks (O'Keefe, 1976). Grid cells are activated at equidistant locations within an environment which is independent of visual stimulation, however vision does modulate grid cell firing (Hafting et al., 2005). Head direction cells fire in response to head orientation in relation to cues in the environment (Taube et al., 1990a). Each of these cell types responds to vestibular stimulation and is either involved in the generation of, or is regulated by, theta. These spatial encoding cells form the spatial representation system (Blair and Sharp, 1996;
Brandon et al., 2011; Burgess et al., 2007; Knierim et al., 1995; Moser et al., 2008; O'Keefe and
Burgess, 2005; O'Keefe and Recce, 1993; Stackman et al., 2002; Stackman and Taube, 1997;
Taube, 2007; Wiener et al., 1995).
Head Direction Cells
The aptly named head direction cells (HD cells) fire in relation to the animal’s head direction within the environment (Taube et al., 1990a). An optimum direction exists for each HD cell (Figure 1.4). The firing rate of HD cells increases linearly as the animal turns its head to the
14 optimum direction, with the opposite direction producing no or very little firing. Unlike grid and place cells, head direction cells can be found in several areas throughout the limbic system and are not necessarily confined to the hippocampal formation. These include the post-subiculum (Taube et al., 1990a; Taube et al., 1990b), retrosplenial cortex (Chen et al., 1994; Cho and Sharp, 2001), thalamic nuclei (Mizumori and Williams, 1993; Taube, 1995; Taube and Muller, 1998), lateral mammillary bodies (Stackman and Taube, 1998), entorhinal cortex (Sargolini et al., 2006), dorsal striatum (Wiener, 1993), medial prestriate cortex (Chen et al., 1994), CA1 of the hippocampus
(Leutgeb et al., 2000), and dorsal tegmental nucleus (Sharp et al., 2001). Head direction cells function like an intrinsic compass, with individual cells firing consistently at the same directional heading independent of the animal’s location. The specificity of each cell is modulated by multiple sensory cues, although unlike a compass there is no evidence for magnetic modulation of HD cells in mammals, with visual cues being able to rotate the firing field away from compass readings
(Taube et al., 1990b). However, this has not been directly tested using magnetic fields.
Figure 1.4: Head direction cell firing. The cell fires only when the head of the animal is turned towards a certain direction. Figure from Taube (2007) with permission. Copyright Annual Review of Neuroscience.
Vestibular sensory information is the primary detector of head acceleration and therefore movement and is necessary for the initiation of HD cell firing, with vestibular lesions abolishing the directional firing of HD cells (Stackman and Taube, 1997). Visual landmarks and proprioceptive information, while not as critical as vestibular information, are necessary in order
15 to modulate the placement of the cell firing field (Stackman and Taube, 1998; Taube, 2007).
Changes in vestibular stimulation have been studied by allowing animals to navigate in various dimensional planes. Both the SCCs and otolith organs have been found to be necessary for HD cell specificity. Bilateral occlusion of the SCCs produce a complete loss of HD cell specificity.
Although the cells still fire when the animal turns, they only distinguish the direction of the turn and not the position of the head in space (Muir et al., 2009). While vertical navigation does not alter HD firing (Stackman et al., 2000), rats navigating upside down lose the directional firing of
HD cells (Calton and Taube, 2005) and otolith-deficient mice lose directional specificity (Yoder and Taube, 2009). This suggests that each of the SCCs, which provide information necessary for horizontal and vertical head movement detection, as well as the natural stimulation of the otolith organs, particularly the saccule which detects changes in gravitational stimulation, is necessary to provide sensory information to HD cells. In particular, the horizontal SCC is required for HD signalling, with mutant mice devoid of the horizontal SCC having an almost complete lack of HD cell specificity (Valerio and Taube, 2016). As with SCC occlusion (Muir et al., 2009), HD cells retained normal firing patterns, but lacked directional specificity in these mutant mice. Whether the vestibular sensory information is projected solely through the previously described head direction pathway or incorporates other vestibulo-hippocampal routes, is unknown. A population of HD cells in the anteroventral thalamic nucleus (32%) produce rhythmic theta bursting, which is locked to the directionality of the cell (Tsanov et al., 2011). Also, lesions of the anterior thalamic nuclei disrupt the firing of grid and HD cells in the entorhinal cortex, but leave theta unaffected
(Winter et al., 2015). This suggests that there is an integration of the HD and theta activity, although it is likely that theta activity in the anteroventral thalamic nucleus is paced from the
MS/hippocampus, as the anteroventral nucleus is not part of the classical theta-generating pathway.
16 Grid Cells
Grid cells, which discharge at multiple sites throughout an environment, are located in the entorhinal cortex, pre- and post-subiculum (Boccara et al., 2010; Fyhn et al., 2004). The resulting map of firing sites forms a grid of equilateral triangles across the environment (Figure 1.5), suggesting an intrinsic measurement system involved in spatial navigation (Hafting et al., 2005;
Moser et al., 2008). Specifically, neighbouring cells maintain comparable grid spacing and orientation, however the grid phase, the specific locations at which the cells fire, shifts in an apparently random fashion (Hafting et al., 2005). The grid spacing is larger in ventral cells compared to dorsal cells and it is possible that they also have different grid orientations (Hafting et al., 2005). Grid cell firing persists in the absence of visual cues although there is a small decrease in the specificity of the firing fields.
Figure 1.5: Place cell and grid cell firing patterns. Black is the animal’s path travelling through an open field. Red shows positions where the recorded cell fired an action potential: A) Place cells in the hippocampus fire at a single location in the environment; B) Grid cells in the entorhinal cortex fire in a triangular pattern throughout the open field. Figure from Moser et al. (2008) with permission. Copyright Annual Review of Neuroscience.
Grid cells require intact MS theta generation in order to retain their spatial selectivity, suggesting a phasic regulation of grid cells by the theta frequency (Brandon et al., 2011; Koenig
17 et al., 2011). The cholinergic activity of the MS, which is involved in the production and pacing of theta, has been linked to grid cell firing (Barry et al., 2012b). Novel environments produce increased cholinergic activity (Giovannini et al., 2001), expanded grid cell firing patterns (Barry et al., 2012a), and reduced theta frequency (Jeewajee et al., 2008). Systemic cholinergic blockade using scopolamine has been found to disrupt grid cell firing (Newman et al., 2014). This suggests that the spatial deficits seen after MS lesions may be a result of grid cell desynchronisation following the disruption of cholinergic firing associated with theta and grid fields (Barry et al.,
2012b; Brandon et al., 2011). Due to the requirement of intact MS theta for grid cell activation, and as theta is disrupted following vestibular lesions (Neo et al., 2012; Russell et al., 2006; Tai et al., 2012) or produced following vestibular stimulation (Gavrilov et al., 1995; Gavrilov et al.,
1996; Shin, 2010), it is unsurprising that vestibular signals are involved in modulating the firing patterns in the spatial map produced by grid cells. Temporary inactivation of the vestibular system in rats using tetrodotoxin (TTX) disrupts entorhinal cortex (EC) velocity-controlled theta oscillations, which leads to a disruption of the grid cell firing field (Jacob et al., 2014).
Place Cells
Place cells produce a dynamic representation of an animal’s position in space. Place cells are hippocampal CA1 and CA3 pyramidal cells, and DG granule cells, which fire when an animal is in a certain location (Figure 1.5), each cell seemingly encoding a different site, providing a complete map or ‘place field’ of the environment within the local population of cells (Moser et al.,
2008; O'Keefe, 1976). Place cells have been found to respond to a number of different sensory cues, including somatosensory (Hill and Best, 1981; Save et al., 1998), olfactory (Save et al.,
2000), visual (Muller and Kubie, 1987; O'Keefe, 1976; O'Keefe and Conway, 1978), and vestibular cues (Gavrilov et al., 1998; Stackman et al., 2002; Wiener et al., 1995), in order to form a representation of the surrounding environment. Once the place field has been formed for a certain environment, the removal of environmental cues does not necessarily lead to a disruption in the place field (O'Keefe and Conway, 1978), suggesting a mechanism similar to navigating in a
18 familiar environment without vision, such as in the dark. Rotating the maze cues in the animal’s absence will rotate the place field when the animal is reintroduced to the maze to match the new cue positions (O'Keefe and Conway, 1978). Place cells fire even when the animal is restrained, removing any tactile, motor, or proprioceptive cues. Gavrilov et al. (1998) found that rats which had been trained to remain still while being driven by a mobile robot, exhibited place cell firing and the formation of place fields even in the dark, suggesting that vestibular cues alone are enough to maintain an accurate representation of space in the hippocampus. Supporting the importance of vestibular stimuli in the generation of place fields, it has been shown that vestibular lesions disrupt place cell firing patterns, producing a degradation of spatial specificity in the place field (Russell et al., 2003a; Stackman et al., 2002). However, a study of place cell activity in space flight found no difference between place field representations in microgravity compared to earth gravity, although some animals required a secondary exposure to the maze environment before their place fields developed, suggesting use of an allocentric learning strategy where the animal developed its place field from external cues (Knierim et al., 2000). This is opposed to egocentric learning where animals learn to navigate the environment in relation to their body (Christie et al., 2005).
Therefore, under microgravity place fields can still be generated, although it may take longer than on earth, with compensation needed for the low gravity environment. The contradictions between this and the lesion experiments suggest that even in microgravity there is enough vestibular stimulation produced from the animals’ other linear and rotational movement to regulate place field firing, although external maze cues are likely needed for place cell coherence.
Place cells appear to integrate information from grid cells, with grid cell firing patterns being able to predict changes in place cell responses (Fyhn et al., 2007). Place cells can either change their firing rate while retaining a stable place field or undergo a complete shift in both firing and field activity in response to an altered or novel environment. The grid cell field will often shift when place fields shift, allowing for a prediction of place field activity and suggesting direct connections between the two spatial mapping fields (Fyhn et al., 2007). Lesioning grid cells
19 in the entorhinal cortex produces a disruption of place firing, reducing discharge rate and altering field size (Brun et al., 2008; Van Cauter et al., 2008). A recent optogenetic and electrophysiological tracing study has shown that grid cells are the main cell type involved in producing the place cell signal, however several other cell types are also involved in generating place cell firing, such as HD cells, border cells and other non-spatial cells (Zhang et al., 2013a).
These other cells may explain why place cells develop before grid cells in infant rats (Langston et al., 2010; Wills et al., 2010). HD cells are necessary for normal place cell firing (Zhang et al.,
2013a), and develop earlier than either place or grid cells (Langston et al., 2010; Wills et al., 2010).
Calton et al. (2003) performed bilateral lesions of the anterodorsal thalamic nuclei and postsubiculum, areas which contain the highest ratio of HD cells. Place cells in lesioned animals showed an increased outfield firing rate, as well as decreased infield specificity, spatial coherence and spatial information. The size of the place field and the infield firing rate were unaffected by
HD cell lesions. Place cells from lesioned animals responded more to the directional heading of the animal, apparently due to the visual stimuli overcompensating for the loss of HD signals. An intact HD cell system is therefore required for the location-specific modulation of place fields. On the other hand, CA1 hippocampal lesioning, which destroys place cells, does not alter HD cell responses (Golob and Taube, 1997). Overall, these results suggest that the main function of place cells may be to integrate HD and grid cell information, with grid cells modulating field size and firing rate and HD cells modulating outfield firing rate, spatial coherence and information content.
Theta activity and place cell firing are linked, although place cells fire at a faster frequency than theta (Geisler et al., 2010; Moser et al., 2008). O'Keefe and Recce (1993) found that hippocampal place cells fire in phase with theta when the animal initially enters the field location, but as the animal navigates through the environment, the place cell firing shifts forward on each theta cycle. This ‘phase precession’ allows for additional spatial information to be carried by the phase of spike discharge within the theta cycle (Dragoi and Buzsaki, 2006; Huxter et al., 2008;
O'Keefe and Recce, 1993; Skaggs et al., 1996). Disruption of theta activity using cholinergic
20 blockade of the MS impairs spatial learning (Elvander et al., 2004; Rogers and Kesner, 2003) and disrupts the spatial association of place cell firing (Brazhnik et al., 2003). Vestibular inactivation over a short time period, (24-48 h) produces a disruption of rhythmic cell firing in a subpopulation of theta cells and disruptions to both place and HD cell firing patterns (Stackman et al., 2002).
SPATIAL MEMORY, COGNITION, AND THE VESTIBULAR SYSTEM Spatial memory is modulated by the firing of place, grid and head direction cells in the hippocampus, which respond to the animal’s current position and direction in space (Dudchenko and Taube, 1997; Moser et al., 2008). Stackman et al. (2002) examined the responses of place cells and head direction cells following temporary BVLs in rats. Even with the addition of visual cues, cell firing was disrupted. Both place and HD cell activity was disrupted during BVL but recovered at a rate similar to the recovery of vestibular function (Figure 1.6). This suggests that vestibular activation is necessary to maintain functioning place and HD cells and may explain the spatial memory deficits exhibited in vestibular-lesioned animals. However, there was no change in theta in the electroencephalogram (EEG) during the BVL period, although this was not analysed statistically. There appeared to be disruption to the firing activity of a subpopulation of theta cells, however without statistical analysis the difference could not be determined. The changes to place cell firing are supported by Russell et al. (2003b), who found permanent place cell disruption 60 days following surgical BVL. Contrary to these results, a recent study which used a virtual reality projection in order to reduce vestibular activity during movement, found that the generation of place cell firing is not dependant on vestibular cues (Ravassard et al., 2013). Animals were either tested on a real-world track or an identical virtual reality track. The track was linear with distinct visual cues and the animals turned once they reached the end of the track. By projecting a scene around the animal, which was fixed in space with a harness and running on a rotating ball, this experiment attempted to eliminate as much vestibular stimulation as possible. However, as the animals were running, they would have been subject to a large amount of high acceleration, multi- directional vestibular stimulation. Even if the amplitude of head movement is very small, the 21 acceleration is what stimulates the vestibular system, therefore there could be high acceleration movement while the animal appears to be stationary. Having the animal running will increase the number of accelerations being detected by the animal. During testing, there was a decrease in place cell firing, theta frequency and a widening of place fields during the virtual reality task, overall producing a decrease in spatial selectivity compared to real world testing. This suggests that matched vestibular-visual cues are necessary for the modulation of place cell firing.
22
Figure 1.6: Place cell firing maps following temporary vestibular inactivation. Each row (a-j) is an individual cell. Increasing rates of discharge are coded from yellow, orange, red, green, blue, and purple, with yellow pixels depicting locations visited where no spikes were fired. Pixels that were never visited during the recording session are coded white. Place field coherence is lost following BVL and recovers when vestibular function is restored. Figure from Stackman et al. (2002). Copyright John Wiley and Sons, used with permission.
Animals which undergo vestibular lesions have been found to have an impaired performance in spatial memory tests, such as the radial arm maze and foraging task (Baek et al.,
2010; Besnard et al., 2012; Russell et al., 2003a; Stackman and Herbert, 2002; Wallace et al., 23 2002; Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2006; Zheng et al., 2007). This is shown to be the case in both UVL and BVL animals, even after accounting for motor deficits which occur following these lesions (Zheng et al., 2006; Zheng et al., 2009b). However, the spatial memory deficits appear to resolve in UVL rats (Zheng et al., 2006) whereas in BVL rats they do not (Baek et al., 2010). Early behavioural studies found significant impairment in Morris water maze and six arm radial maze performance following either BVL or vestibular stimulation immediately prior to testing (Matthews et al., 1989; Semenov and Bures, 1989). This led to a later study which examined the ability of BVL animals to perform a foraging task relying solely on self- motion cues (Wallace et al., 2002). Initially, the training of the rats was performed in the light, allowing the animals to familiarize and learn the task. Rats were trained to find a randomly allocated food reward and return the food to a cued home base. Following training, testing for vestibular influence on the performance was performed in the dark, forcing the rats to rely only on self-motion cues. This led to no change in the behaviour of control rats, however vestibular- lesioned animals were unable to accurately return to the home base once they managed to find a food pellet. Since this study, experiments on the vestibular influence on spatial learning and memory have become more elaborate, with electrophysiological, genetic knockout (KO) and long term behavioural studies all confirming the importance of the vestibular system in navigation and learning (Baek et al., 2010; Machado et al., 2012a; Russell et al., 2003a; Russell et al., 2003b;
Russell et al., 2006; Stackman et al., 2002; Zheng et al., 2009a; Zheng et al., 2006; Zheng et al.,
2009b).
Machado et al. (2012a) tested a genetic KO mouse model which was devoid of otoconia, the calcium carbonate stones which activate the sensory hairs within the utricle and saccule. These mice, named het/het mice, provide a model to test the lack of gravisensors while the semi-circular canals remain functional. Deficiency in the otoconia leaves the mice with severe behavioural deficits similar to those seen in BVL animals – hyperactivity, head bobbing, circling and head tilting. Het/het mice were found to have a decrease in spatial memory performance in a Y-maze,
24 however there was no change in performance on an object recognition memory task. A second study by Yoder and Kirby (2014) found that otoconia-deficient tilted mice were unaffected in a
Barnes maze task, but suffered from spatial memory deficits in a radial arm maze. These results suggest that the otoliths are necessary for some, but not all, aspects of spatial memory.
A study of the long term effects of surgical BVL found that after 14 months post-surgery, the performance of rats was impaired in a foraging task (Baek et al., 2010). The severity of spatial deficits appeared to increase in comparison to an earlier study by the same group (Zheng et al.,
2009b). The earlier study examined performance in a foraging task 5-7 months following BVL surgery. Following BVL, it was found that animals were no longer able to use allocentric cues for navigation and exhibited a significant decrease in spatial performance in the dark. A second finding by Baek et al. (2010) was that the cannabinoid receptor agonist WIN,212-2, improved BVL animals’ performance during trials in the dark; however there was no effect on sham animals. The increase in performance was abolished by pre-treatment with the cannabinoid CB1 receptor inverse agonist AM251. This suggests that BVL may alter the endocannabinoid system which is involved in hippocampal spatial learning and memory.
Hypergravity produces deficits in spatial memory similar to those of BVL animals. Mitani et al. (2004) examined the spatial memory effects of 2g exposure in rats. Following two weeks of hypergravity induced by centrifugation, rats underwent spatial testing in a radial arm maze.
Hypergravity animals had reduced accuracy in the test compared to controls during the first five days of testing but there was no significant difference following these initial days. This suggests that exposure to hypergravity, and the subsequent removal from that environment, disrupts spatial working learning and memory for up to five days following exposure. This result is supported by other experiments examining spatial performance following centrifugation (Feng et al., 2010; Hao et al., 2004; Mandillo et al., 2003; Sun et al., 2009). Spatial memory impairment appears to be due to increased cell death in the hippocampus following changes in gravitational stimulation (Feng et al., 2010; Sun et al., 2009). Hippocampal cell death is linked to increased stress (Mizoguchi et al.,
25 1992); however, as neither study examined markers for stress, such as corticosterone levels, it is impossible to determine if the cell death was purely due to vestibular stimulation or was stress- induced as stress also produces a decrease in spatial memory performance (Conrad et al., 1996; de
Quervain et al., 1998; Luine et al., 1994). Anxiety disorders are more prevalent in vestibular- deficient humans than the normal population (Jacob and Furman, 2001) and anxiety levels are known to alter hippocampal theta rhythm (Gray and MacNaughton, 2000). Evidence of stress, examining corticosterone and cortisol levels, has not been found to be a cause of spatial impairment in vestibular-lesioned animals (Gliddon et al., 2003; Russell et al., 2006; Zheng et al.,
2008). These studies did not differentiate between free and bound corticosterone, which is important in determining stress-induced corticosterone release (Breuner and Orchinik, 2002).
However, corticosterone levels were increased both during and following hypergravity in rats
(Petrak et al., 2008). Hypergravity exposure and BVL have very similar profiles in regards to spatial memory, with animals even becoming hyperactive following both procedures. Studies examining spatial memory during hypergravity have not been performed due to the complexities of the centrifuge apparatus.
The human vestibular organs are very fragile, with vestibular loss caused by gentamicin toxicity at “safe” doses (Black et al., 2004) and mild head trauma, even in cases with no whiplash, such as concussion or head-strike (Grimm et al., 1989). Gentamicin toxicity may only be present following hospital discharge, 1-3 weeks following therapy (Black et al., 2004). Vestibular dysfunction increases the difficulty of normal daily living activities, with reduced performance in cognitive tasks and an increased prevalence of falls (Bigelow and Agrawal, 2015; Bigelow et al.,
2015; Harun et al., 2015; Semenov et al., 2016). Human patients with total BVL have spatial memory disruption and reduced hippocampal volume (Bigelow et al., 2015; Brandt et al., 2005;
Seo et al., 2016; Smith et al., 2005b). Cognitive measures of visuospatial ability, working memory and attention are consistently impaired following vestibular loss (Bigelow et al., 2015; Semenov et al., 2016). The atrophy of vestibular cortical regions, postural and gaze instability, and affective
26 disorders following vestibular dysfunction are thought to lead to cognitive impairment (Figure 1.7)
(Bigelow and Agrawal, 2015; Gurvich et al., 2013). Partial BVL has also been found to lead to cognitive dysfunction and atrophy in the hippocampus (Kremmyda et al., 2016). Unilateral vestibular dysfunction is more complicated. Patients with right vestibular failure have spatial memory and navigation deficits while the volume of the hippocampus is unchanged (Hufner et al.,
2007). Patients with left vestibular failure test normally in the spatial memory tasks (Hufner et al.,
2007). It is thought that this difference is due to there being a dominance of the right labyrinth and the right vestibular cortex in right-handed patients (Bense et al., 2004; Dieterich et al., 2003).
Unilateral vestibular loss (UVL) increases attentional demand during reaction time tasks even when the patient is sitting (Redfern et al., 2004; Talkowski et al., 2005). Patients with superior semi-circular canal dehiscence or otic capsule defects have an opening in the bone overlaying the canal/otolith. These patients suffer from vestibular vertigo and oscillopsia in response to pressure or sound which causes the exposed endolymphatic fluid to shift within the membranous labyrinth
(Minor et al., 1998). These patients have cognitive dysfunction, however this can be reversed by repairing the dehiscence (Wackym et al., 2016).
27
Figure 1.7: Conceptual model proposing a mechanism of cognitive dysfunction due to vestibular dysfunction. Figure from Bigelow and Agrawal (2015). Copyright IOS press, used with permission.
Vestibular disorders have also been linked to several psychiatric disorders (see Gurvich et al., 2013 for review). Depression, anxiety, mania, attention-deficit/hyperactivity disorder (ADHD) and psychosis have all been linked to vestibular dysfunction. Each of the listed psychiatric disorders are known to affect regions in the brain that are also involved in vestibular processing, such as the amygdala, hippocampus, thalamus, putamen, prefrontal cortex, parietal lobe, raphe nuclei and locus coeruleus (Gurvich et al., 2013). Patients with vestibular vertigo have a three- fold increase in the prevalence of affective disorders such as depression, anxiety and panic disorders and report difficulty concentrating or remembering (Bigelow et al., 2016). Patients with vestibular disease have an increased rate of depersonalisation/derealisation (DD) symptoms such as déjà vu, difficulty concentrating, blurred thoughts, sensation of shifting ground, and feeling
'spaced out' (Jauregui-Renaud et al., 2008a; Jauregui-Renaud et al., 2008b; Sang et al., 2006).
These DD symptoms can be elicited in healthy individuals using caloric vestibular stimulation
(Sang et al., 2006). DD symptoms are highly correlated with unipolar depression and panic
28 disorders (Hunter et al., 2004), therefore these symptoms may indicate the existence of other psychiatric disorders in patients with vestibular dysfunction.
Bachtold et al. (2001) suggest that stimulating the vestibular system improves both verbal and spatial memory. Participants were given unilateral cold water caloric stimulation, which induces convection currents in the vestibular inner ear, producing movement of the endolymph and stimulation of the hair cells. During the stimulation subjects were asked to memorise either the location of objects (spatial memory test) or a list of words (verbal memory test). Following this, participants were asked to recall their respective lists. The spatial memory test demonstrated a significant improvement in recall speed in those subjects who underwent caloric stimulation of the left ear compared to controls. There was no difference between the right ear stimulation and control subjects in this task. The verbal memory test showed the opposite result, with improvement occurring in the right ear stimulation group and no difference between the left ear stimulation and controls. As the object recall task has been found to activate the right parahippocampal gyrus
(Milner et al., 1997) and the word recall task activates the left temporal lobe (Basso et al., 1982;
Pulvermuller, 1999), it was suggested that unilateral caloric stimulation produces an increase in cognitive processes mediated by structures contralateral to caloric vestibular stimulation.
Supporting this finding, a study using galvanic vestibular stimulation (GVS) found an improvement in visual memory recall in humans (Wilkinson et al., 2008). GVS activates the vestibular nerves with electrodes applied to the scalp overlying the mastoid process bilaterally, and a small current is then passed through the head. Sensory perception and processing of GVS can be improved by overlaying a stochastic resonance noise signal (noisy GVS; Figure 1.8) in the stimulation protocol (Forbes et al., 2014; Moss et al., 2004; Smith et al., 2010b). The experimental task consisted of the memorisation of unfamiliar faces and their respective names. Following memorisation, the participants were asked specific questions comparing features of each face while undergoing GVS. Participants who underwent GVS with the anode on the left side and cathode on the right showed a significant decrease in recall time compared to controls and those
29 with the anode and cathode reversed. This was only true for subjects who underwent noisy GVS stimulation, as constant current GVS produced no change in recall time. As visual memory recall is associated with the left hemisphere (Barton and Cherkasova, 2003) and GVS activates the cortex contralateral to the anode (Dieterich et al., 2003; Fink et al., 2003), these data support the Bachtold et al. (2001) unilateral stimulation study. However, when suprathreshold GVS is applied a significant decrease in performance in match-to-sample and perspective-taking tasks can occur
(Dilda et al., 2012). Overall it appears that there may possibly be cognitive benefits to subthreshold noisy GVS, however suprathreshold GVS can produce negative effects, therefore the thresholds must be carefully determined.
A B
C D
Figure 1.8: Examples of galvanic vestibular stimulation waveforms. A) Square wave stimulation. B) Sinewave stimulation. C) Constant current stimulation. D) Noisy stochastic stimulation. Figure D modified from Forbes et al. (2014). Copyright Forbes et al. (2014), open access.
Evidence of a negative vestibular influence on spatial tasks which involve other sensory processing has also been found in humans (Furman et al., 2003; Furman et al., 2012). Furman et al. (2012) showed an increase in reaction time during a spatial choice right-left lateralization task which involved listening for a sound while receiving rotational vestibular stimulation. A non- spatial auditory frequency discrimination task was also tested. The combination of visual and vestibular processing during auditory tasks increased reaction times significantly. Performance in spatial tasks was affected to a greater degree than non-spatial tasks. This suggests that visual and
30 vestibular signals interfere with auditory cognitive processing, leading to a reduction in reaction time during concurrent stimulation.
EFFECT OF VESTIBULAR DAMAGE ON THE HIPPOCAMPUS Changes in vestibular activity have been found to alter hippocampal volume in a similar manner to extensive navigational training, as seen in taxi drivers (Maguire et al., 2000). A study examining professional dancers and slackliners reported an increase in posterior hippocampal volume and decrease in anterior hippocampal volume, with a correlation between the experience of the athletes and the relative hippocampal changes (Hufner et al., 2011). Blind individuals have increased anterior hippocampal volume and superior navigational skills compared to age-matched controls (Fortin et al., 2008). There is also evidence of posterior hippocampal reduction (Chebat et al., 2007; Lepore et al., 2009). This is thought to be due to increased reliance on vestibular and proprioceptive cues, although increased use of verbal memory techniques is also likely (Fortin et al., 2008). Zheng et al. (2012a) measured rat hippocampal volume 16 months post-BVL and found no change in volume, as well as no change in neuronal number. Using MRI, Besnard et al. (2012) measured total rat brain volume and total hippocampal volume. There was no significant difference between lesioned animals and sham animals. Besnard et al. (2012) showed that following bilateral sodium arsanilate transtympanic injections, with one side injected 3 weeks following the other, performance in the spatial memory tasks of the Y-maze and radial arm maze was decreased. No change was found during the non-spatial object recognition task. Although hippocampal volume remains unchanged following BVL in rats, there are changes occurring at the dendritic level. BVL rats have significantly fewer basal dendritic intersections in area CA1, which may decrease LTP in the hippocampus (Balabhadrapatruni et al., 2016). In human subjects there are conflicting results, with some studies finding no changes in hippocampal volume in patients with either BVL or UVL (Cutfield et al., 2014; Hufner et al., 2007) and others which find a significant reduction in hippocampal volume (Brandt et al., 2005; Kremmyda et al., 2016; Seo et al., 2016; Van Cruijsen et al., 2007; zu Eulenburg et al., 2010). Resolution of imaging, time since onset of the vestibular
31 loss and the extent of the lesion may contribute to the wide variation in human studies. A study examining 27 BVL patients found no difference in overall hippocampal volume compared to controls but a significant reduction bilaterally in hippocampal region CA3 (Gottlich et al., 2016).
The lack of atrophy in animal studies may be due to the different time frame between lesion and testing (10 weeks vs. 5-10 years in human studies). It is possible that more time is needed following
BVL for the hippocampus to atrophy. The different kinds of lesions (BVL / UVL / Meniere’s disease / vestibular neuritis) are also likely to produce different effects in the hippocampus.
N-methyl-D-aspartate (NMDA) receptors have been studied following BVL and UVL due to their association with hippocampal LTP (Bliss and Collingridge, 1993). Using autoradiography it was found that NMDA receptor levels in the hippocampus were increased but the affinity of
NMDA receptors was decreased (Besnard et al., 2012). This may suggest a compensatory glutamatergic mechanism following vestibular lesions as NMDA receptors are required for hippocampal memory function (Alvarez et al., 2007; Gao et al., 2010). Zheng et al. (2013) examined NMDA receptor subunits within hippocampal subregions, CA1, CA2/3 and the DG, at multiple time-points following BVL using western blotting. They found no change in any NMDA receptor subunit within the individual regions of the hippocampus. Liu et al. (2003) found a decrease in the NR1 and NR2A NMDA subunits in the CA2/3 region following surgical UVL, using western blotting. The difference in the lesion procedure, with Besnard et al. (2012) performing sodium arsanilate injections unilaterally over time to achieve BVL, and Zheng et al.
(2013) performing simultaneous surgical BVL, is the most likely explanation as to why these experiments produced conflicting results. The sensitivity of the western blot technique of single regions compared to receptor autoradiography of the entire hippocampal formation may also explain the different findings.
A study examining the electrophysiological effects of UVL on field potentials in hippocampal brain slices reported long term changes in CA1 neuronal activity (Zheng et al., 2003).
Brain slices were taken from animals which underwent UVL and field potentials in the CA1 were
32 recorded in response to electrical stimulation of the Schaffer collaterals. UVL animals were found to have a decreased population spike amplitude and decreased field excitatory post-synaptic potential slope. This effect increased with time following UVL and occurred bilaterally. Changes in paired-pulse inhibition and facilitation were also observed. These results suggest that CA1 neurons suffer decreased excitability following UVL, possibly due to long-term compensatory changes in hippocampal plasticity. In vivo studies have found that electrical stimulation of the medial vestibular nucleus produced increased firing rates of CA1 place cells (Horii et al., 2004), while BVL abolished CA1 location-related place field activity (Russell et al., 2003b), with LTP and baseline field potentials remaining unchanged (Zheng et al., 2010). These results show a difference between UVL and BVL hippocampal compensation, with UVL but not BVL producing changes in field potentials. This may also be due to differences between in vivo and in vitro recording.
HIPPOCAMPAL THETA RHYTHM AND THE VESTIBULAR SYSTEM The importance of theta in the production of hippocampal function and spatial memory has long been known. Disrupting theta produces severe deficits in both spatial and non-spatial memory tasks (Buzsáki, 2005; Leutgeb and Mizumori, 1999; Mizumori et al., 1990; O'Keefe, 1993; Vertes,
2005; Winson, 1978). Theta disruption produces a reduction in LTP, leading to inhibition of learning and memory (Orr et al., 2001; Vertes, 2005). Hippocampal theta activity in response to vestibular stimulation was first examined by Gavrilov et al. (1995) and Gavrilov et al. (1996).
During their experiments, rats were rotated by a robotic platform and hippocampal EEG was recorded. Theta power in the 7-8.5 Hz band was found to be increased during rotation. There was no difference in theta activity between clockwise and counter-clockwise rotation or between rotation in the light and dark. This suggests that a high frequency theta is produced during vestibular stimulation independent of visual stimuli in awake, unanaesthetised animals. It was initially thought that the high frequency theta activity following vestibular stimulation was type 1 atropine-resistant theta (Gavrilov et al., 1995; Gavrilov et al., 1996). However, more recent studies
33 illustrate that rotational acceleration produces an atropine-sensitive type 2 theta activity (Shin,
2010; Tai et al., 2012). Although the majority of high frequency theta activity is type 1, there are cases where atropine-sensitive type 2 theta rhythms can be within the high frequency range (Bland and Oddie, 2001).
An experiment examining the effects of pre-treatment with atropine, a non-selective muscarinic cholinergic antagonist, in awake mice during passive whole body rotation, reported that vestibular-induced theta activity was abolished following atropine administration (Shin,
2010). It was concluded that theta activity associated with vestibular stimulation is atropine- sensitive and therefore can be classed as type 2 theta rhythm. Atropine also produced a loss of the orientation homeostasis response, the behavioural response in which an animal circles in the opposite direction of a rotating treadmill in order to maintain a stable position in space. An earlier study, which supports the finding that vestibular-induced theta is cholinergically dependant, showed that there is an increased release of ACh in the hippocampus following vestibular stimulation (Horii et al., 1994). This ACh increase was then prevented by injecting a glutamatergic antagonist into the medial vestibular nucleus. Together, these results suggest a close link between cholinergic theta activity, vestibular stimulation, and an animal’s ability to determine its position in space.
Tai et al. (2012) recorded theta activity in rats using implanted electrodes within the CA1 region of the hippocampus while animals underwent passive whole-body rotation. Theta frequency was found to be relative to the speed of angular rotation, with higher frequencies occurring during fast rotations and lower frequencies being recorded during slower rotations. There was no alteration in the power of hippocampal theta, which contrasts with an earlier study that reported power increases with faster rotations (Gavrilov et al., 1996). Animals which underwent lesions of either the vestibular receptors or cholinergic neurons in the medial septum displayed a reduction in rotation-induced theta power. Theta was also greatly attenuated following the administration of the muscarinic ACh receptor (mAChR) antagonist, atropine sulphate, however activation of a
34 small level of type 1 atropine-resistant theta still occurred. In contrast to previous studies, theta frequency was found to be in the lower range of <7 Hz, compared to 7-8.5 Hz in rats (Gavrilov et al., 1995; Gavrilov et al., 1996) and 7-10 Hz in mice (Shin, 2010). The authors suggested that the difference in the length of vestibular stimulation, with previous studies using short phasic (2.5 - 4 s) stimulation rather than tonic stimulation (30 s - 5 min), may have produced the alteration in the theta frequency range. The decision to use an intratympanic chemical injection to lesion the vestibular system may be the reason that theta activity was attenuated, rather than abolished in lesioned animals. Chemical lesions have been shown to be imperfect for complete vestibular lesioning, as their effects are dependent on the absorption of the drug through the round window, which can be highly variable (Jensen, 1983; Saxon et al., 2001; Smith et al., 2005a). Although a behavioural test was performed to confirm the effectiveness of the lesion, vestibular nerve activity and VOR responses may still have been active (Saxon et al., 2001). These injections do not damage surrounding tissues (Vignaux et al., 2012) but it is necessary to perform histology to determine the effectiveness of intratympanic chemical lesions. Another problem with the design of the experiment was the failure to address excess noise and vibration during recording. An electric drill was used to rotate the chamber containing the animal, therefore increased rotation speed likely meant increased noise and vibrations from the drill motor. As both sound and vibration can produce theta activity (Pyykko and Starck, 1985; Sainsbury and Montoya, 1984) and otolith stimulation (Curthoys et al., 2009), this confounds the rotational experimental results.
Lesion experiments, examining the effects of the loss of the vestibular system on hippocampal theta activity, have also been performed. Russell et al. (2006) examined the long- term effects of BVL on hippocampal EEG activity and single unit firing in rats. Sixty days after bilateral surgical labyrinthectomy, animals underwent an open field foraging task and movement was tracked using an infrared camera. Movement recording was synchronised to EEG and single unit recording in order to determine the direction and acceleration of movements which elicited responses. Hippocampal EEG results demonstrated a disruption in the theta range at 6-9 Hz and
35 10-12 Hz. Single unit spontaneous firing rates (i.e. not in response to places in the environment) of place cells and theta cells increased significantly in BVL animals. This is contradictory to the findings by Stackman et al. (2002), who found no change in the spontaneous firing rate of either cell type. As Stackman et al. (2002) examined BVL following intratympanic TTX administration, which produced a rapid onset but temporary disruption of vestibular activity, it is likely that the difference in spontaneous cell firing rates between the two studies is due to the different lesion techniques and recovery time leading to vestibular compensation. As TTX lesions only disrupt vestibular function for 48 h, there was not enough time for vestibular compensation to occur. The compensation process partially restores resting activity in the VNC (Ris and Godaux, 1998), therefore it is likely that downstream regions of the vestibular pathways are altered during and after compensation, which may have produced the changes in hippocampal spontaneous cell firing rates.
Attempting to repair the cognitive and emotional disorders caused by BVL using MS stimulation produced a restoration in hippocampal theta activity but no change in behaviour
(Carter, 2010; Neo et al., 2012). Following BVL, the animals displayed emotional and cognitive deficits in the open field, elevated T-maze and spatial nonmatching to sample tests and had a reduced theta power. Stimulation of the medial septum was given throughout behavioural tests and successfully restored hippocampal theta activity of 7.5-8 Hz, but did not improve emotional or spatial learning behaviours. The inability of MS stimulation to restore normal behavioural responses in BVL animals may have been due to stimulation not being synchronised with movement, which would have more accurately replicated vestibular stimuli. MS stimulation may have also produced unwanted responses due to potential antidromic activation of hippocampal pathways. As vestibular stimulation reaches the hippocampus through a number of polysynaptic pathways, it is possible that regulation of anxiety and spatial cognition occurs in a location upstream from the MS, or even via a separate pathway. With the necessity to have normal place, grid and HD cell firing for spatial navigation, it is probable that restoration of theta activity without
36 the synchronous activation of these cells, particularly in response to vestibular stimulation, would not lead to an improvement in spatial cognitive performance.
VESTIBULAR DAMAGE, CHOLINERGIC SYSTEM AND NEURODEGENERATIVE DISEASE There is a concurrence of vestibular disorders and neurodegenerative disorders, such as
Parkinson’s disease (PD), Alzheimer’s disease (AD), and progressive supranuclear palsy (PSP)
(Jellinger, 1988; Lee, 2011; Reichert et al., 1982; Zingler et al., 2007). While in some cases the neurodegeneration produced by the disease will cause degradation of the VNC, as can be seen in
AD and PSP patients who develop neurofibrillary tangles within the VNC (Cruz-Sanchez et al.,
1992; Powell et al., 1974; Ransmayr et al., 1994), there is evidence to suggest that vestibular loss may lead to, or at the least increase, the progression of the disease (Previc, 2013). Vestibular loss can lead to atrophy of the hippocampus (Brandt et al., 2005), which is one of the earliest sites of neurodegeneration in AD patients (Fox et al., 1996). This suggests that vestibular loss may accelerate the progression of early stage AD as hippocampal atrophy has already begun to occur.
Hippocampal degeneration produces disturbances in spatial memory in both vestibular patients
(Brandt et al., 2005; Harun et al., 2016) and AD patients (Deweer et al., 1995). As AD is treated using acetylcholinesterase inhibitors, which slow the progression of the disease, it is thought that degradation of cholinergic transmission and areas of the brain which contain large amounts of cholinergic neurons are related to the neurodegeneration seen in AD (Francis et al., 1999; Terry and Buccafusco, 2003).
The PPT is a major cholinergic nucleus in the brainstem (Ch5) and is involved in the theta- generating pathway, receiving stimulation from the VNC and projecting via the SUM/PH and MS to the hippocampus (Figure 1.3). AD patients suffer neurofibrillary tangles in the PPT (Mufson et al., 1988) as well as ~30% neuronal loss (Jellinger, 1988). PD and PSP patients suffer an even greater loss of neurons in the PPT as well as neurofibrillary tangles (Hirsch et al., 1987; Jellinger,
1988; Zweig et al., 1989; Zweig et al., 1987). The PPT is reciprocally connected to the substantia nigra, the dopaminergic nucleus that degenerates and produces Parkinsonian symptoms. Loss of 37 PPT neurons in PD patients correlates to the severity of Parkinsonian symptoms, being associated with the frequency of falls, as well as gait and postural symptoms (Bloem et al., 2004; Bohnen et al., 2009; Ferraye et al., 2010; Hirsch et al., 1987; Kuo et al., 2008). Experimental treatments have shown that deep brain electrical stimulation of the PPT improves PD motor symptoms, with reductions in falls and freezing, as well as improvements in postural control (Ferraye et al., 2010;
Khan et al., 2011; Moro et al., 2010; Pereira et al., 2008; Plaha and Gill, 2005; Stefani et al., 2007;
Thevathasan et al., 2011). It appears that vestibular stimulation can also improve PD symptoms, with stimulation producing immediate improvements in postural control and gait (Pal et al., 2009;
Tamlin et al., 1993; Yamamoto et al., 2005). Vestibular stimulation, independently of visual stimuli, activates the majority (72%) of macaque PPT cells (Aravamuthan and Angelaki, 2012).
Cholinergic agents, including nicotine exposure from smoking, have been found to improve cognition in PD (Aarsland et al., 2002; Kelton et al., 2000; Nielsen et al., 2013; Searles Nielsen et al., 2012). As the PPT is a major cholinergic nucleus, it is possible that these cholinergic agents are acting to increase or restore PPT activity, suggesting a role for the PPT in cognition as well as motor control. A study supporting this theory examined the effects of deep brain stimulation of the PPT in PD patients (Alessandro et al., 2010). A significant improvement in a number of cognitive processes was found, with deep brain stimulation improving working memory, preventing executive dysfunction and restoring normal sleep quality.
The current research suggests that vestibular damage is associated with neurodegenerative disorders. Cholinergic neurons in the PPT receive input from the VNC and are associated with both cognitive and motor functions. AD and PD cognitive dysfunction may be partly related to, or be exaggerated by, vestibular damage and disruption to the cholinergic neurons in the theta- generating pathway. This indicates that vestibular / PPT dysfunction is likely to be a common association between neurodegenerative diseases and the severity of the symptoms (Harun et al.,
2016; Previc, 2013).
38 MUSCARINIC ACETYLCHOLINE RECEPTORS
There are five types of mAChR, M1-M5 (Bonner et al., 1987; Bonner et al., 1988). They are G-protein coupled receptors (GPCRs) and can all be found in the brain (Table 1.1). M1, M3 and M5 are primarily coupled to Gq GPCRs and upregulate phospholipase C and inositol triphosphate, while M2 and M4 are coupled to Gi GPCRs and inhibit adenylyl cyclase and cAMP
(Felder, 1995). M1, M2 and M4 are the most abundant receptors in the brain (Levey, 1993). Each of the five receptors are in the hippocampus. Due to the prominent role the hippocampus has in memory formation, mAChRs have been implicated in memory-related disorders (Bruno et al.,
1986; Conn et al., 2009; Wess et al., 2007) and targeted for nootropic drug development (Kitamura et al., 1990; Petkov and Popova, 1987).
Table 1.1: Location of muscarinic receptors throughout the brain.
Brain region M1 M2 M3 M4 M5
Striatum +++ ++ - +++ -
Hippocampus +++ + ++ ++ +
PPT - +++ - - -
Thalamus + +++ ++ + +
Neocortex +++ ++ ++ + -
Substantia nigra - + - + ++
Amygdala +++ + + + +
Basal forebrain - +++ + + - Relative densities determined by in situ hybridisation Densities are: – undetectable, + low, ++ medium, +++ high. Modified from Levey et al. (1991). Copyright J. Neuroscience, used with permission.
A study which examined the effects of specific M1 and M2, and non-specific muscarinic antagonists, in the hippocampus found that non-specific blockade of mAChRs in the hippocampus disrupted learning and memory in the Morris water maze. They also found that M1 receptor
39 blockade disrupted long term memory, however memory formation remained intact. Antagonism of the M2 receptor had no effect on water maze performance when injected directly into the hippocampus (Herrera‐Morales et al., 2007). Romanelli Ferreira et al. (2003) found that an M1 selective agonist improved memory consolidation while an antagonist caused amnesia in an inhibitory avoidance task. Earlier studies also examined mAChRs’ effects on memory in the hippocampus. Jerusalinsky et al. (1998) found that a selective M4 antagonist produced retrograde amnesia when injected into the hippocampus. Another study which examined the effects of both mAChR and nicotinic receptors on short-term spatial memory in a delayed matching to position task found complementary results (Andrews et al., 1994). In this study the drugs were injected into the lateral ventricle, rather than directly into the hippocampus. Nicotinic antagonists produced only a small effect on performance and only at the highest doses tested. M1 and M1/M3 antagonists as well as non-specific mAChR antagonists disrupted performance dose-dependently. An M2 antagonist produced no change in performance. Overall, these studies illustrate the hippocampal
M1, M3 and M4 receptors’ essential roles in memory and learning, while blockade of the hippocampal M2 receptor and nicotinic receptors does not cause significant disruptions to spatial memory.
In aged animals ACh release is reduced in the cortex and hippocampus. This correlates with impaired cognitive abilities in object recognition and passive avoidance tasks (Vannucchi et al., 1997). The M2/M4 antagonist AFDX 384, injected intraperitoneally, increased ACh release and restored cognitive performance in aged rats. Another study has examined the function of these receptors in KO mice models (Tzavara et al., 2003). Using M2 and M4 single receptor knockouts as well as an M2/M4 double knockout, it was found that basal ACh levels were increased in the hippocampus in all KO animals. Learning and memory was impaired during a passive avoidance test in the M2 and M2/M4 KO animals but not the M4 KO. Together, these studies have found that the M2 receptor does play a role in learning and memory in aged animals, and removing the functionality of the M2 receptor, either by antagonist or genetic KO, increases ACh in the
40 hippocampus and cortex. The conflicting behavioural data, with M2/4 KO animals having impaired learning and memory while M2/4 antagonists restoring cognitive function, is likely due to the different methods utilised. The brains of KO animals have been substantially altered by the complete removal of M2 and M4 receptors and compensatory mechanisms have likely taken place in order for the brain to function somewhat normally. Antagonists are a short-term blockade of the receptor and therefore have an acute affect. While either removal or blockade of the M2 receptors produces an increase in hippocampal ACh release, the cognitive performance that follows is dependent on whether the loss of M2 or M4 function is acute or chronic. Further support for the role of the M2 receptor in learning and memory is found in studies which used a variety of M2 selective antagonists administered systemically (subcutaneous, oral or intra-peritoneal). These studies all found that M2 receptor antagonists improve learning and memory (Carey et al., 2001;
Packard et al., 1990; Quirion et al., 1995; Rowe et al., 2003; Vannucchi et al., 1997). As direct administration of M2 antagonists to the hippocampus does not alter learning and memory, whereas systemic administration produces an improvement, it is likely that there is an extra-hippocampal brain region in which the memory-related M2 receptors are located. M2 receptors are densely located in the hindbrain, particularly within the PPT and LDT (Mash and Potter, 1986). As the
PPT is implicated in spatial working memory disruption, theta generation and has links to the vestibular system and hippocampus, it is possible that this is the site of M2 receptor disruption which leads to the learning and memory deficits seen following systemic M2 receptor antagonist administration.
LIMITATIONS OF EXISTING STUDIES Studies of the vestibular system use a range of techniques to disrupt or stimulate the vestibular end organs. While surgical UVL and BVL completely destroy the vestibular labyrinth and remove all downstream vestibular activity, intratympanic injections of sodium arsanilate damage the hair cells dose-dependently, not necessarily producing the complete vestibular ablation seen with surgical techniques (Vignaux et al., 2012). However, intratympanic injections are
41 considered more similar to clinical vestibular disorders, as often only partial vestibular dysfunction occurs clinically. It is therefore important to choose the lesion technique which is best suited to the study, whether examining the anatomical and physiological influences of the vestibular system or replicating and attempting to develop treatments for clinical conditions. Another concern with these lesion techniques is the destruction of the cochlea, leading to hearing loss in the animal in addition to vestibular disruption. Although studies examining surgical BVL animals compared to sham animals with their tympanic membrane removed, in order to produce partial hearing loss, have found that hearing loss does not appear to affect cognitive ability compared to BVL animals
(Schaeppi et al., 1991; Zheng et al., 2006; Zheng et al., 2007; Zheng et al., 2008; Zheng et al.,
2009b), it is still important to consider the potential effects of losing two sensory systems simultaneously rather than just one. Although surgical UVL is largely specific to the vestibular sensory system, there is still some damage to the cochlea in guinea pigs, and profound hearing damage in human patients (Brown et al., 2016). Currently a lesion model which leaves the cochlea completely undamaged does not exist, therefore it is necessary to control for hearing loss as best as possible.
The methods used to stimulate vestibular end organs are diverse, with caloric stimulation,
GVS, whole body rotation, direct electrical stimulation and even electrical stimulation of the vestibular nucleus all being employed to test vestibular function and activity. GVS is particularly problematic as there is a large variety of stimulation protocols which are used. Each protocol has a different amplitude, frequency, length, train and shape of stimulation as well as different stimulation sites being used. There are also concerns as to what exactly is being stimulated by
GVS. GVS electrodes are placed bilaterally on the skin above the mastoid process and the electrical current must travel from the anode to the cathode across the brain. While studies suggest that GVS stimulation is localised to the vestibular nerve leading to a downstream activation of vestibular-responsive cortical regions (Lobel et al., 1999; Stephan et al., 2005), more needs to be done to determine how the current crosses the brain and which other areas may be affected. As
42 well as concerns about cortical activation during GVS there is disagreement as to whether GVS predominantly stimulates the otoliths or the semi-circular canals (Cohen et al., 2012; Curthoys and
Macdougall, 2012; Reynolds and Osler, 2012). These differences and, particularly in the case of
GVS, the lack of understanding in how they produce vestibular stimulation, make for a large degree of variability between experiments.
The continued uncertainty of the existence of specific vestibulo-hippocampal pathways and how they may affect spatial memory requires a comprehensive examination of each potential pathway. Currently research relies on tracing experiments, leading to multiple pathways being implicated. An electrophysiological or fMRI study examining major nuclei within each pathway simultaneously during vestibular stimulation is required. Although a study of this kind has been performed recently (Rancz et al., 2015), it did not measure latency of action potential firing in response to the vestibular stimulation, therefore the pathway which activates the hippocampus first remains unknown. They also failed to control for fMRI producing vestibular stimulation through
Lorentz forces (Antunes et al., 2012). The vestibular stimulation protocol was also extreme (3x nystagmus threshold), likely producing severe nystagmus which may activate other, non- vestibular brain regions. A follow up study may provide the data necessary for determining which pathway activates the hippocampus and which area or areas of the hippocampus (CA1, CA2/3 or
DG) are being activated. A similar study, using selective stimulation of the individual canals and otoliths while recording from a 16 channel hippocampal electrode array, has already been performed but the results are yet to be fully analysed (Zhang et al., 2013b). It is possible that each pathway activates different areas of the hippocampal formation, integrating the final signal into the place fields necessary for spatial learning and memory.
CONCLUSION Vestibular stimulation improves cognition and short term memory while vestibular inactivation produces severe deficits in spatial and non-spatial memory. The vestibular system and hippocampus are linked by potentially multiple polysynaptic pathways, although currently the
43 exact pathway or pathways through which vestibular information is sent to the hippocampus is/are unknown. Vestibular activation and inactivation alters hippocampal theta rhythm and the firing of place and head direction cells. This suggests that the theta-generating pathway is necessary for vestibular-hippocampal interactions. Disruption of place cells and theta rhythm are seen during spatial memory deficits following vestibular loss as well as neurodegenerative diseases, suggesting a link between neurodegenerative diseases and vestibular inactivation. Vestibular loss is linked to several psychiatric disorders and impaired cognition. There is evidence that vestibular stimulation can improve memory encoding. Muscarinic AChRs M1, M3 and M4 within the hippocampus are required for spatial learning and memory. M2 receptors have effects on learning and memory outside the hippocampus. The PPT is a cholinergic nucleus with dense M2 receptor distribution.
The PPT is involved in vestibular activation, theta generation and neurodegenerative diseases, suggesting it as one of the connection sites between the vestibular system and hippocampus in determining the modulatory role of the vestibular system on hippocampal function.
44 AIMS AND OBJECTIVES This thesis aimed to examine the effect of vestibular loss on behaviour and how vestibular loss alters cholinergic signalling within the theta-generating pathway, hippocampus and striatum.
Firstly, the behaviour of animals which underwent BVL or sham lesions was assessed in the open field in order to determine and distinguish the specific behavioural profile produced by BVL. The objective of the following experiments was to determine if BVL produces permanent changes to hippocampal and striatal cholinergic signalling. The first set of experiments aimed to examine the distribution and density of muscarinic M1 and M2 receptors following BVL in both the hippocampus and striatum. The next experiment aimed to determine whether the generation of type 2 theta was possible following BVL and UVL. Following this, the number of cholinergic cells in the PPT was counted using stereology with the aim to explain why theta is altered following
BVL and UVL. Finally, the release of ACh within the hippocampus following BVL and during vestibular stimulation was assessed with the aim to determine whether changes in hippocampal
ACh release were producing the changes to behaviour, receptor distribution, and theta generation following BVL.
45 2 CHEMICAL LABYRINTHECTOMY Destruction of vestibular function through injection of sodium arsanilate into the middle ear is a common and well established method for producing BVL and UVL syndromes in rats
(Besnard et al., 2012; Hunt et al., 1987; Ossenkopp et al., 1992; Ossenkopp and Hargreaves, 1993;
Vignaux et al., 2012; Wallace et al., 2002). Sodium arsanilate is an arsenic derivative which interferes with ATP generation (Aposhian and Aposhian, 1989), inhibits enzymes and active ion transports (Anniko and Wersall, 1977; Singh and Rana, 2007). Chemical labyrinthectomies more accurately model vestibular syndromes which occur following chemical toxicity, e.g. aminoglycoside antibiotic treatment compared to surgical ablation of the vestibular organs or
VIIIth nerve. An in-depth analysis of sodium arsanilate lesions was recently performed (Vignaux et al., 2012). The transtympanic injection of sodium arsanilate caused irreversible loss of vestibular hair cells within 3 days, with the behavioural syndrome of vestibular loss appearing within 24 h of injection, although complete loss of function did not occur until 3-7 days post-injection
(Vignaux et al., 2012). These lesions cause damage specific to the inner ear, with the VIIIth nerve largely unaffected up to 3 months afterwards. The vestibular sensory epithelia become restructured over 90 days, with large vacuoles present in the epithelium 3 and 7 days post-injection, and a monolayer of epithelial supporting-type cells forming by the 90 day time point (Figure 2.1). Only a small number of hair cells remained. This monolayer formation is also found in human patients with vestibular disorders (McCall et al., 2009; Rauch, 2010).
46
Figure 2.1: Time course of histological lesions induced by arsanilate in the sensory epithelia of one semi-circular canal in a rat. A) At low magnification, compared to transtympanic injection of control saline solution (Cont.), lesions were induced by both doses of arsanilate (5 mg and 30 mg) and could be seen at day 3, 7, 30, and 90 after the transtympanic injection. Arrows indicate large swellings obtained in the sensory epithelium. B) High magnification shows that the large swellings observed at 7 days post-injection disappeared at day 90. At this time, the semi-circular canal epithelium had been restructured, presenting a monolayer of columnar-shaped epithelial cells made up of epithelial supporting-type cells (SC). Very few hair cells (HC) were still present. . Copyright Elsevier, used with permission.
47 METHODS
Subjects
Male Wistar rats were selected as experimental subjects as this strain has been used consistently by our group and as such is supported by a large amount of vestibular experimental data (Baek et al., 2010; Balabhadrapatruni et al., 2016; Russell et al., 2003a; Russell et al., 2006;
Smith et al., 2005a; Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2006; Zheng et al.,
2007; Zheng et al., 2008; Zheng et al., 2009b; Zheng et al., 2010; Zheng et al., 2013). Animals were sourced from the University of Otago animal breeding facility. Animals were housed in groups of two to three under constant temperature (21°C ± 1°C), humidity (55% ± 5%), and brightness conditions (lower than 110 Lux). Rats had a 12:12 h normal light:dark cycle with food and water available ad libitum and were handled daily prior to and following intratympanic injection. Animals weighed between 200-250 g at the time of sodium arsanilate injection. All procedures were approved by the either the University of Otago Committee on Ethics in the Care and Use of Laboratory Animals (Ethics numbers 21/14 and 82/14; Chapters 3, 6, 7 and 8) or in accordance with the European Communities Council Directive (86/6609/EEC) as well as French legislation (Chapters 4 and 5).
Intratympanic Injection
Animals were placed in an induction chamber containing gaseous isoflurane (Attane,
Bayer) anaesthesia (5% in oxygen; flow rate: 2 L/min). Once anaesthetized so that the pedal reflex was no longer present, the animal was transferred from the induction chamber and placed in a nose holder with a nose cone to continue isoflurane administration (2% in oxygen; flow rate: 1 L/min).
Sodium arsanilate (Sigma) was mixed into a paste with saline (300 mg/ml). The tympanic membrane was identified under a surgical microscope (Zeiss) while a speculum was inserted into the ear canal. A 21 gauge needle was inserted into the middle ear cavity through the anterior part of the tympanum and 0.1 ml of sodium arsanilate (300 mg/ml) was injected. Sham control animals received saline injections and UVL animals received saline injections on the intact side.
48 Intraperitoneal (i.p.) injections of paracetamol (20 mg/animal) and amoxicillin (40 mg/animal) were given to reduce pain and prevent infectious otitis, respectively. These injections were repeated twice a day for 3 days following intratympanic injection.
Assessment of Vestibular Syndrome
The clinical scoring system described by Boadas-Vaello et al. (2005) and Besnard et al.
(2012) was used to assess the bilateral vestibular syndrome. This system of measurement examines six static and dynamic locomotor signs: head bobbing, circling, reversing, the tail-hanging reflex, contact inhibition (Figure 2.2), and air-righting reflex, each rated from 0 (no observable deficit) to
4 (severe deficit), rated subjectively by the experimenter. Rats were tested at days 1, 3, 7 and 30.
A brief description of each sign follows. Bobbing is a spontaneous and intermittent extreme backward extension of the neck leading to abnormal head movements. Circling is defined as stereotyped and spontaneous circling locomotor activity. Reversing is defined as a spontaneous retropulsion movement (the animal walking backwards). The tail-hanging reflex consists of a forelimb extension when the animal is lifted off the table; BVL rats bent themselves ventrally, sometimes "crawling" up toward their tails. Contact inhibition, related to the righting reflex, reflects the ability and speed to go from a supine to a prone position on a table. The rat is turned over into a supine position, the limbs hanging onto a core grid and the back is touching the surface of the table. Sham rats turn over to the physiological prone position while vestibular-lesioned rats stay in a supine position walking on the core grid. For the air-righting reflex, animals are held supine and dropped from a height of 15 cm onto a foam cushion. Sham rats are successful in righting themselves in the air, while vestibular-lesioned rats land on their backs.
49
Figure 2.2: Contact inhibition test. BVL rats are placed on a wire grid with their back against a solid surface. BVL rats will not right themselves unlike sham and UVL rats. This behaviour can also be seen in the animal’s home cage.
For the experiment which used UVL animals, the unilateral vestibular syndrome was assessed using a similar behavioural paradigm (Besnard et al., 2012; Saxon and White, 2006). The animals were scored between 0-4 on the severity of their head tilt, body twisting, and walking deviation. They were also assessed on whether forelimb extension was present during the tail- hanging reflex and the severity of rotation when undergoing the tail hang test. All UVL rats exhibited severe head tilt, barrel rolling (sustained, repetitive longitudinal twisting/flipping), and rotation when lifted by the tail within 3 days following the intratympanic arsanilate injection. Sham control animals all scored 0 on both BVL and UVL tests as no vestibular syndrome was present.
Figure 2.3: Head tilt and body twisting in a UVL rat. UVL rats display severe head and body twisting toward the lesioned side between 3 and 7 days post-lesion. By day 30 the degree of head tilt is much less severe.
50 Temporal Bone Histology
Temporal bones from rats in Chapter 6 were collected and fixed in formalin (10%) for four weeks. The bones were decalcified by being placed in EDTA (8.5%) and microwaved for 48 h to accelerate the decalcification process. Temporal bones were embedded in paraffin wax and sections were cut (5µm) using a microtome. The sections were then stained with haematoxylin and eosin, cleared in xylene and cover-slipped.
Histological analysis of the temporal bone under a microscope revealed a visible shift in the cell layers of the horizontal vestibular ampulla, with hair cells being replaced by supporting cells (Figure 2.4). The central nerve of the horizontal ampulla was also damaged, with increased striations in the crista connective tissue and a reduction in the density of nerve fibres. The loss of hair cells and damage to the nerve suggested complete loss of vestibular function in all lesioned ears.
Sham Lesioned
Figure 2.4: Histology of horizontal vestibular ampulla from a sham rat and the lesioned side of a UVL rat at 30 days after saline and sodium arsanilate intratympanic injections, respectively. Hair cells are in a surface layer with cilia projecting outwards (black arrowhead). Supporting cells are in a second layer underneath (white arrowhead). On the lesioned side, vestibular hair cells have withdrawn from the membrane or have been replaced with a single layer of supporting cells (white arrowhead). The central nerve has also been damaged (green arrowhead). Scale bars = 10 µm.
51 3 OPEN FIELD BEHAVIOUR OF BVL RATS
INTRODUCTION Following UVL in rodents, a vestibular syndrome occurs which includes the initial oculomotor, postural and sensory symptoms of vestibular loss, such as spontaneous nystagmus, head tilting and unidirectional spinning (Darlington and Smith, 1989; Smith and Curthoys, 1989).
Following BVL, ataxia, head dorsiflexion, backward locomotion, hyperactivity, bidirectional circling, and oscillopsia are present (Besnard et al., 2012; Goddard et al., 2008; Rinne et al., 1998).
Several days following vestibular lesioning, compensation begins to provide a partial decrease in the severity of most symptoms. In both UVL and BVL animals the compensation process can take
2-3 days for static symptoms, while dynamic symptoms can take more than 60 days to compensate
(McCall and Yates, 2011; Smith and Curthoys, 1989). The symptoms of vestibular loss are different between species; one that is currently poorly understood is locomotor hyperactivity in rodents following BVL. Humans who suffer from vestibular loss usually reduce their locomotor speed and activity (Mamoto et al., 2002) while rodents increase their locomotor activity (Baek et al., 2010; Goddard et al., 2008; Ossenkopp et al., 1992; Porter et al., 1990; Russell et al., 2003b;
Stiles et al., 2012). A study which closely examined the behaviour of rats following BVL in an open field found a significant increase in both movement velocity and time spent moving when compared to sham animals (Goddard et al., 2008). The increase in movement was seen immediately post-surgery and was consistent at 3 weeks, 3 months and 5 months following lesion surgery. These behaviours have also been seen in animals up to 14 months post-lesion (Baek et al., 2010). It is therefore likely that locomotor hyperactivity following bilateral vestibular loss in rats is permanent. Despite their increased movement speed and activity, the BVL rats spent more time immobile and stopped more often than sham animals (Basile et al., 1999; Goddard et al.,
2008). There was also a reduction in rearing behaviour, and the rearing that did occur was significantly different from normal behaviour. BVL animals tended to rear much less for
52 exploratory purposes and usually reared only when they came in contact with the wall of the testing environment (Goddard et al., 2008). This is consistent with the behaviour displayed in other hyperactive rats, for example animals that have had their hippocampus removed, suggesting that reduced rearing is due to the hyper-locomotion rather than balance disorders caused by the loss of vestibular stimulation (Goddard et al., 2008; Hannigan et al., 1984; Yang et al., 2006). Genetic
KO animals which lack vestibular function also exhibit symptoms of hyperactivity (Kaiser et al.,
2001; Khan et al., 2004; Löscher et al., 1996; Rabbath et al., 2001; Vidal et al., 2004).
One theory for hyperactivity following BVL is that increased levels of striatal dopamine following vestibular damage produce changes in the indirect loop of the basal ganglia motor circuit
(Brock and Ashby, 1996; Schirmer et al., 2007a; see Stiles and Smith, 2015 for review). Activation of the striatal motor pathway inhibits locomotor activity, while inhibition of the pathway increases locomotion (Di Chiara et al., 1994). Unilateral lesioning of striatal cholinergic interneurons induces contralateral circling which is consistent with behaviour seen following vestibular loss
(Goddard et al., 2008; Kaneko et al., 2000). A study examining the effects of dopaminergic treatments following UVL found that the specific D2 agonist bromocriptine accelerated the rate of vestibular compensation. D2 antagonists and non-specific D2 agonists decreased the rate of compensation (Petrosini and Dell'Anna, 1993). In vestibular-deficient KO rats, D2 antagonists and haloperidol, a non-specific inverse agonist at D2 receptors, were found to decrease locomotor activity, abnormal head movement, and circling behaviours (Brock and Ashby, 1996; Schirmer et al., 2007b). A study examining changes in striatal D2 receptors following surgical BVL found no changes in D2 receptor levels or locomotor activity following the administration of a D2 antagonist
(Stiles et al., 2012). Neonatal rats suffering from aminoglycoside vestibulotoxicity exhibited an increase in striatal D2 receptors (Seth et al., 1982). Giardino et al. (1996) found age-dependant changes in D2 receptor levels in the striatum following surgical BVL. D2 receptors decreased in young rats (3 months old) but both D1 and D2 receptors increased in older rats (24 months old).
Their results illustrate that the age of the animal has an influence on the modulation of striatal
53 dopamine receptors following vestibular loss. These results suggest that the mechanism of vestibular loss (genetic, lesion, unilateral or bilateral) and age of the animals are factors in the expression of striatal dopamine receptors. Even when the mechanism of vestibular loss remains the same, the response of the dopaminergic receptors is not necessarily identical between individual animals.
It is possible that changes to the striatal dopaminergic receptors are mediated by ACh. mAChRs are closely related to the aging process and are also present on the striatal neurons (Joyce,
1993; Nordberg et al., 1992; Rinne, 1987). KO mice that lack the M1 muscarinic cholinergic receptor have increased locomotor hyperactivity, suggesting that the loss of stimulatory M1 receptors in the striatum produces a reduction in activity in this inhibitory motor pathway, leading to hyperactivity (Miyakawa et al., 2001). Another M1 receptor KO mice study found an increase in striatal dopaminergic transmission in mice lacking the M1 receptor (Gerber et al., 2001).
Cholinergic activity in the striatum, hippocampus and cortex is positively correlated with increases in locomotor activity (Day et al., 1991). It is possible that the striatum is not the predominant location of hyperactivity associated with M1 receptors. The hippocampus, although largely associated with spatial memory and learning, may be the source of changes in the striatal cholinergic response. Following BVL, rats switch from hippocampal learning strategies to striatal strategies (Machado et al., 2014). The symptoms of hyperactivity and spatial memory loss following vestibular damage are similar to those observed following hippocampal lesioning (Baek et al., 2010; Goddard et al., 2008; Gray and McNaughton, 1983; Kuroiwa et al., 1991; Maren et al., 1997; Maren and Fanselow, 1997; Ossenkopp and Hargreaves, 1993; Porter et al., 1990; Shull and Holloway, 1985; van Praag et al., 1994). Although hyperactivity is not present following vestibular damage in humans, studies of attention deficit/hyperactivity disorder (ADHD) have found an association between loss of vestibular function and hyperactivity in children (Arnold et al., 1985; Shum and Pang, 2009). Attention is decreased in rats (Zheng et al., 2009a) and occasionally in human patients following vestibular damage (Brandt et al., 2005; Redfern et al.,
54 2004). The cholinergic system is likely to be involved in ADHD, with patients self-medicating with nicotine to improve their symptoms (Potter et al., 2006). The physical size of the hippocampus in ADHD children is altered, with an enlargement of the anterior hippocampus
(Plessen et al., 2006). The larger the anterior hippocampus, the fewer ADHD symptoms are expressed, suggesting a compensatory response. By adulthood, this difference in hippocampal volume is no longer detectable (Perlov et al., 2008). Whether attention is directly linked to hyperactivity following vestibular loss is unknown, although it is possible that the hyperactivity seen in rodents following vestibular loss manifests as ADHD in human patients.
The PPT is associated with locomotor activation and inhibition (Jones and Beaudet, 1987;
Kuo et al., 2008; Mathur et al., 1997). The cholinergic receptor inhibition of PPT cells with scopolamine increases locomotion dose-dependently by 2-15 times whereas cholinergic activation with carbachol reduces locomotion initially but then produces an increased response in activity
(Mathur et al., 1997). Increases in striatal dopamine levels occur following direct injections of scopolamine into the PPT through the excitation of the substantia nigra (Chapman et al., 1996).
The PPT also has direct connections from the striatum and is a target for deep brain stimulation in
Parkinson’s disease (Inglis and Winn, 1995; Mazzone et al., 2009; Pereira et al., 2008; Plaha and
Gill, 2005; Winn et al., 1997). The increases in striatal dopamine and locomotion following PPT disinhibition with scopolamine appear to be consistent with changes found following vestibular loss. This suggests that changes to the cholinergic activity of the PPT may occur following vestibular loss, which leads to hyperactivity in rodents.
Because so many of the behavioural effects of BVL in rats, which appear to be not directly linked to the loss of vestibular reflex function, remain unexplained, the aim of the current experiment was to assess rat locomotor activity in an open field using modern animal tracking software in the sodium arsanilate model of BVL. The animals that underwent microdialysis in
Chapter 8 were used for behavioural testing during their recovery period. In addition to univariate analyses of individual locomotor behaviours, we also sought to determine if vestibular loss could
55 be predicted from multiple behaviours not directly related to the loss of vestibular reflex function, using multivariate statistical analyses (Smith et al., 2013). While the striatal theory of hyperactivity is currently the preferred model, there are inconsistencies in the current data which suggest that there may be another mechanism. This experiment allowed for a discussion of rodent BVL hyperactivity in the context of cholinergic signalling in the hippocampus as microdialysis samples were collected and ACh release measured 7 days following testing in the open field (Chapter 8).
56 METHODS Rats underwent either BVL (n=7) or sham (n=8) intratympanic sodium arsanilate injections as detailed in Chapter 2. Three and 23 days following intratympanic injection, animals were placed in an open field (60 x 60 x 30 cm). Animals were placed individually in the centre of the open field
(Figure 3.1) and their behaviour was recorded for 30 min for later analysis with EthoVision XT
6.1 software™ (Noldus). Recordings were performed in black and white for increased contrast and detection optimization for EthoVision. Recordings were made at the highest resolution available in EthoVision, 768x576 (PAL), using a PICOLO DILIGENT (Euresys) frame grabber board and
N287 camera. The surfaces of the chamber were cleaned thoroughly with ethanol between animals.
Figure 3.1: Rat in the open field. Each animal was allowed to explore the open field for 30 min. The image is a screenshot of the video recording used in the analysis. The open field was divided into three concentric zones for analysis: outer, intermediate, and central.
The dimensions of the chamber were calibrated in EthoVision by inputting a known length.
Tracking was limited to within the confines of the chamber. The centre, intermediate and outer zones of the chamber were delineated using the 100x100 mm square lines on the surface of the chamber (Figure 3.1). Detection thresholds were set with the grey scale nose-tail advanced model- based detection (range 160-255, 25 fps). Detection was monitored during each recording for
57 adherence accuracy. EthoVision automatically detected the nose, middle point, and tail base of each animal throughout the recordings. Recordings were monitored for adherence to detection protocol and any errors were manually corrected. Twenty-seven variables were selected for analysis (Table 3.1). Some variables require definition. Meander was calculated from relative turn angle of the centre point divided by distance moved. Angular velocity (either relative or absolute) was calculated as turn angle (either relative or absolute) divided by time difference between samples. Elongation thresholds were set at stretched > 70% of body length and contracted < 30% of body length. Moving and Not Moving were detected from the centre point with thresholds of
0.5 and 0.2 cm/s, respectively. Mobility was detected from the percentage of pixels of the subject displaced between frames and had thresholds of Highly Mobile > 20% and Immobile < 1%.
58 Table 3.1: Variables measured with EthoVision XT 6™.
VARIABLE BODY POINT MEASUREMENT UNIT
DISTANCE MOVED Nose-point Total cm
DISTANCE MOVED Centre-point Total cm
ELONGATION Contracted Duration s
ELONGATION Normal Duration s
ELONGATION Stretched Duration s
OUTER ZONE Nose-point Duration s
CENTRE ZONE Nose-point Duration s
INTERMED ZONE Nose-point Duration s
OUTER ZONE Centre-point Duration s
CENTRE ZONE Centre-point Duration s
INTERMEDIATE ZONE Centre-point Duration s
MEANDER Centre-point Mean º/cm
MOBILITY Whole body Duration Immobile s
MOBILITY Whole body Duration Mobile s
MOBILITY Whole body Duration Highly Mobile s
MOVEMENT Centre-point Duration Moving s
MOVEMENT Centre-point Duration Not Moving s
ROTATION (CCW) Centre-Nose Frequency
ROTATION (CW) Centre-Nose Frequency
ROTATION (TOTAL) Centre-Nose Frequency
TURN ANGLE Centre-point Mean º
VELOCITY Nose-point Mean cm/s
VELOCITY Centre-point Mean cm/s
ABSOLUTE ANGULAR Centre-point Mean º/s VELOCITY
RELATIVE ANGULAR Centre-point Mean º/s VELOCITY
OUTER/INTERMEDIATE ZONE Centre-point Frequency TRANSITION
INTERMEDIATE/CENTRE Centre-point Frequency ZONE TRANSITION
59 Statistical Analysis
Initially statistical analysis was performed using a repeated measures two-way ANOVA with Sidak’s multiple comparisons test in Prism 6 (Graphpad). This allowed for the analysis of each individual variable to assess the effects of BVL and time. Further statistical analysis was performed using linear discriminant analysis (LDA) and cluster analysis to determine whether individual variables could be predictors of BVL and multiple linear regression (MLR) was used to determine whether one variable could be predicted from the others. These analyses were performed in SPSS 22. The LDA used was stepwise and Box’s M test was used to confirm the equality of the variance/covariance matrices to ensure the LDA was valid. Significance was determined using
Wilks’ lambda. The success of the LDA was tested using cross-validation with a leave-one-out
(LOO) method (Manly, 2005). The cluster analyses were hierarchical and agglomerative and were performed using Ward’s minimal variance algorithm on the squared Euclidean distance; for these analyses the data were first standardised (i.e. z transformed) (Manly, 2005). MLR was used to determine whether each of the variables could be predicted using the remaining variables. The
MLRs were stepwise and were considered to be valid only if the Durban-Watson statistic was < 2 and/or the tolerance value was > 0.1 (Field, 2013). The adjusted R2 value was used to judge the success of the MLR and the ANOVA and t tests for individual coefficients were used to test the significance of the regression (Manly, 2005). P ≤ 0.05 was considered significant in all tests.
60 RESULTS BVL animals exhibited significantly different behaviours in the open field compared to sham animals (Figure 3.2). BVL rats showed reduced thigmotaxis, with more time spent in the central zones (Figures 3.2 and 3.3). The centre point of BVL animals was more often in the intermediate zone (Treatment: P ≤ 0.05; F (1,10) = 7.2) while the nose point was more often in the central zone (Treatment: P ≤ 0.05; F (1,10) = 8.1; Figure 3.3).
Day 3
Day 23 A
Day 3
Day 23
Day 3
B Day 23
Day 3
Day 23
Figure 3.2: Movement tracing of BVL rats in an open field. A) Centre-point traces over the 15 min analysis period. B) Nose-point traces. Blue = sham, Red = BVL. BVL animals appeared to have less thigmotaxis and increased movement compared to sham animals. Each pair of images (day 3 and 23) represent a single rat.
61
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Twenty-three days post-lesion, BVL animals showed increased activity in the open field, with the total distance moved and velocity increasing significantly for both the centre and nose point measurements (Day 23: Distance nose: P ≤ 0.0001; t(20) = 5.484; Distance centre: P ≤ 0.001; t(20) = 4.658; Velocity nose: P ≤ 0.0001; t(20) = 5.586; Velocity centre: P ≤ 0.0001; t(20) = 5.815;
Figure 3.4). The increase in activity was also reflected in the number of transitions between each zone of the field. Transitions between the outer and intermediate zone increased (Day 23: P ≤ 0.01; t(20) = 3.787) as well as transitions between the intermediate and centre zones (Day 23: P ≤ 0.001; t(20) = 4.191; Figure 3.5). The zone transition measurements detected any part of the animal crossing into a new zone and were not based on either the centre or nose points as determined in
Figures 3.2 and 3.3.
BVL animals showed increased whole body rotations following lesions (Figure 3.6). Three days following BVL there was a significant increase in clockwise rotations (Day 3: P ≤ 0.05; t(20)
= 2.497). Twenty-three days following BVL there was a significant increase in counter-clockwise rotations (Day 23: P ≤ 0.001; t(20) = 4.28). The total number of rotations, calculated by adding both clockwise and counter-clockwise rotations, showed a significant increase only 23 days post- lesion (Day 23: P ≤ 0.001; t(20) = 4.733). No other variable measured showed significant effects following analysis with a two-way ANOVA (Appendix A, Figure 11.1).
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movement of the animals. Mean ± SEM. *** = P ≤ 0.001, **** = P ≤ 0.0001.
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Figure 3.5: Zone transitions of BVL rats in the open field. BVL animals significantly increased the number of zone crossings 23 but not 3 days following lesion. Mean ± SEM. ** = P ≤ 0.01, *** = P ≤ 0.001.
63 * *** 3 0 s
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Figure 3.6: Rotation frequency of BVL animals in the open field. BVL animals had significantly more counter-clockwise rotations than sham animals 23 days following lesions and significantly more clockwise rotations 3 days following lesions. When the total number of rotations were combined, an increase in BVL rotations was observed only 23 days following the lesion. Mean ± SEM. * = P ≤ 0.05, *** = P ≤ 0.001.
LDA demonstrated that at day 3 animals could be successfully classified as either sham or
BVL using the single variable of relative angular velocity (P ≤ 0.05; χ2 = 4.9). This was cross- validated using the LOO method, which was 83% successful in classifying the animals. At day 23, using the same testing parameters, three variables were required to successfully classify animals: velocity (nose), total rotations and meander (P ≤ 0.0001; χ2 = 25.8). This was cross-validated using the LOO method, which was 100% successful in classifying the animals.
Cluster analysis illustrated some separation between sham and BVL animals (Figure 3.7).
There was some grouping of the data into the separate treatment groups, especially at day 3 (Figure
3.7, A).
MLR analysis was performed on day 3 predictors which did not have excessive multicollinearity (VIF < 10, Tolerance > 0.2, Durban-Watson > 1.0 and < 2.0). No day 23
64 predictors had acceptable criteria due to excessive correlation in the data. Treatment was a factor when Not Moving (Table 3.2) or Relative Angular Velocity was a dependant variable (Table 3.3), but was not a predictor for any other analysis (Appendix A, Tables 11.1-11.12).
65 A
B
Figure 3.7: Cluster analysis of behavioural data. A) Day 3 data. B) Day 23 data. Individual animal z scores were assessed.
66 Table 3.2: Multiple linear regression where time not moving was the dependant variable (day 3).
Unstandardized Coefficients Standardized Coefficients Model B Std. Error β P value 1 Constant 717.52 79.20 0.00 Mobile -0.81 0.14 -0.88 0.00 2 Constant 681.09 43.03 0.00 Mobile -0.84 0.07 -0.92 0.00 Treatment 110.83 21.80 0.41 0.00 Note: R2 = .77 for Model 1, ΔR2 = .17 for Model 2.
Table 3.3: Multiple linear regression where relative angular velocity was the dependant variable (day 3).
Unstandardized Standardized Coefficients Coefficients Model P value Std. B β Error 1 Constant 2.97 0.98 0.01 Clockwise Rotation -0.34 0.07 -0.83 0.00 2 Constant -0.31 1.07 0.78 Clockwise Rotation -0.32 0.05 -0.78 0.00 Counter-Clockwise Rotation 0.44 0.11 0.44 0.00 3 Constant 2.66 1.17 0.05 Clockwise Rotation -0.22 0.04 -0.53 0.00 Counter-Clockwise Rotation 0.66 0.11 0.67 0.00 Distance Moved (nose) 0.00 0.00 -0.41 0.01 4 Constant 5.54 1.05 0.00 Clockwise Rotation -0.18 0.03 -0.45 0.00 Counter-Clockwise Rotation 0.82 0.08 0.83 0.00 Distance Moved (nose) 0.00 0.00 -0.51 0.00 Stretched -0.01 0.00 -0.22 0.01 5 Constant 3.81 0.78 0.00 Clockwise Rotation -0.12 0.02 -0.30 0.00 Counter-Clockwise Rotation 0.92 0.05 0.94 0.00 Distance Moved (nose) 0.00 0.00 -0.32 0.01 Stretched -0.01 0.00 -0.25 0.00 Centre-Intermed Transitions -0.30 0.08 -0.36 0.01
67 6 Constant 3.47 0.52 0.00 Clockwise Rotation -0.12 0.02 -0.29 0.00 Counter-Clockwise Rotation 0.91 0.04 0.92 0.00 Distance Moved (nose) 0.00 0.00 -0.37 0.00 Stretched -0.01 0.00 -0.26 0.00 Centre-Intermed Transitions -0.34 0.05 -0.40 0.00 Mobile 0.00 0.00 0.12 0.03 7 Constant 3.88 0.35 0.00 Clockwise Rotation -0.14 0.01 -0.33 0.00 Counter-Clockwise Rotation 0.90 0.02 0.92 0.00 Distance Moved (nose) 0.00 0.00 -0.38 0.00 Stretched -0.01 0.00 -0.26 0.00 Centre-Intermed Transitions -0.39 0.04 -0.47 0.00 Mobile 0.00 0.00 0.10 0.02 Outer-Intermed Transitions 0.04 0.01 0.13 0.04 8 Constant 3.86 0.19 0.00 Clockwise Rotation -0.14 0.01 -0.34 0.00 Counter-Clockwise Rotation 0.91 0.01 0.93 0.00 Distance Moved (nose) 0.00 0.00 -0.40 0.00 Stretched -0.01 0.00 -0.26 0.00 Centre-Intermed Transitions -0.39 0.02 -0.47 0.00 Mobile 0.00 0.00 0.12 0.00 Outer-Intermed Transitions 0.03 0.01 0.13 0.01 Treatment 0.21 0.06 0.03 0.05 Note: R2 = .69 for Model 1, ΔR2 = .19 for Model 2, ΔR2 = .07 for Model 3, ΔR2 = .03 for Model 4, ΔR2 = .01 for Model 5, ΔR2 = .00 for Model 6, ΔR2 = .00 for Model 7, ΔR2 = .00 for Model 8.
68 DISCUSSION BVL produces significant changes to the behaviour of rats in the open field. The findings of the present experiment are consistent with previous studies, where rodents increased locomotor activity following BVL (Baek et al., 2010; Goddard et al., 2008; Ossenkopp et al., 1992; Porter et al., 1990; Russell et al., 2003b; Stiles et al., 2012). Both distance moved and velocity of BVL rats increased compared to shams at day 23 post-lesion. A similar increase was seen for both the nose and centre point, suggesting that increases in whole body movement, rather than nose/head movement, are produced following BVL. There was no difference in distance moved or velocity at day 3 post-lesion, suggesting that locomotor hyperactivity is not a direct consequence of peripheral vestibular loss, but a phenomenon which develops over time involving complex changes in the CNS. Previous experiments noted that BVL rats spend more time immobile and stopped more often than sham animals (Basile et al., 1999; Goddard et al., 2008). An increase in immobility was not seen in the present experiment with either the immobility or non-moving measurements. Another finding of previous studies was that BVL rats have altered rearing behaviours (Basile et al., 1999; Goddard et al., 2008; Zheng et al., 2012b). Rearing could not be directly measured with EthoVision, although the amount of time the animal spent contracted was measured (Appendix A, Figure 11.1) and a non-significant decrease (P = 0.06; 32.7 s (sham) vs
10.7 s (BVL)) was seen at day 3 post-BVL; however, this reversed at day 23 (P = 0.4; 13.55 s
(sham) vs 24.4 s (BVL)). As animals seen from above contract during rearing behaviour, this can be used to estimate the time animals spent rearing, although it is non-specific since other behaviours where the animal’s body contracts, such as grooming, will be detected. The non- specificity of the body elongation measurements is likely to be why there was no change seen between sham and BVL animals.
BVL animals performed whole body rotations, both clockwise and counter-clockwise, significantly more often than sham animals. The preference for the direction of rotation changed between day 3 (clockwise) and 23 (counter-clockwise). It is well known that BVL rats turn in tight
69 circles (Besnard et al., 2012; Boadas-Vaello et al., 2005; Goddard et al., 2008; Rinne et al., 1998;
Stiles et al., 2012); this measure is used as one of the measurements of lesion severity throughout this thesis. However, that BVL rats have a directional preference to their rotation, and then change direction with time, has not been previously reported. BVL animals following surgical labyrinthectomy, have no preference for rotation direction, exhibiting bi-directional circling (Stiles et al., 2012). The preferential rotations may be because of the sodium arsanilate lesions. While producing severe BVL syndromes and damage to the vestibular structures of the inner ear, sodium arsanilate may not completely destroy every vestibular sensory hair cell and remaining hair cells may continue to respond to vestibular signals, unlike surgical lesions which aspirate the canals and otoliths. Therefore, the directional circling seen in BVL animals in the current study may reflect different degrees of lesion between the right and left side due to the progressive damage caused by sodium arsanilate. UVL animals show preferential circling behaviour also, turning in the direction of the lesioned side; however an ipsilateral head tilt usually accompanies this (Smith and
Curthoys, 1989), which was not present in any of the BVL rats in the current experiment.
Thigmotaxis was decreased in BVL animals as seen by an increase in inner zone activity
(Figures 3.2 and 3.3). At day 23 there was also an increase in the number of zone transitions (Figure
3.5). This is similar to previous results (Goddard et al., 2008; Machado et al., 2012b; Zheng et al.,
2012b). Goddard et al. (2008) argued that the time spent in the outer zone by BVL animals was not necessarily representative of thigmotaxis, as BVL rats did not have consistent wall contact and would instead rear or return to the centre immediately after reaching the outer wall. It appears that
BVL rats enter the outer zone as a consequence of their hyper-locomotion, rather than due to the normal safety-seeking thigmotaxis behaviour. Contrary to these results, short-term inactivation of the vestibular system using trans-tympanic injections of TTX increases thigmotaxis, possibly due to increased anxiety immediately following loss of the vestibular sensory system (Stackman et al.,
2002).
70 Attempting to distinguish BVL animals from sham animals through multivariate statistics was successful. Cluster analysis suggested some separation of the two treatment groups. Multiple linear regression at day 3 found that treatment was a predictor of two variables: relative angular velocity and time spent not moving. This was consistent with the results of the stepwise linear discriminant analysis which found that relative angular velocity could predict BVL at day 3 (83% accurate), while velocity (nose), total rotations and meander could predict BVL at day 23 (100% accurate).
Hyperactivity in rodents following BVL is an established symptom which occurs following either surgical (Baek et al., 2010; Goddard et al., 2008; Russell et al., 2003b; Stiles et al., 2012;
Zheng et al., 2012b) or chemical BVL (Machado et al., 2012b). Mutant mice with either selective otolith (Machado et al., 2012a) or SCC dysfunction (Hadrys et al., 1998) exhibit hyperactivity.
The exact mechanisms which lead to hyperactivity remain unclear, although it is likely that receptor and synaptic changes in the striatum and/or hippocampus are involved (Brandt et al.,
2005; Gray and McNaughton, 1983; Machado et al., 2014; Stiles and Smith, 2015; Stiles et al.,
2012). There is also the possibility of changes to pontine regions early in the vestibular- hippocampal/striatal pathways which drive changes in dopamine and ACh release, such as in the
PPT and LDT (Chapman et al., 1996; Mathur et al., 1997; Mazzone et al., 2009; Potter et al.,
2006). However, the widespread changes in the brain following BVL make determining the exact mechanism of locomotor hyperactivity difficult. Humans do not display hyperactivity in the same manner as rodents following BVL, although there is some evidence to link ADHD in childhood to vestibular dysfunction (Arnold et al., 1985; Shum and Pang, 2009). Further studies are needed to examine the effects of BVL on human locomotor activity and attention.
71 4
M1 RECEPTOR DENSITY FOLLOWING BVL
INTRODUCTION
Out of the five mAChRs the M1 receptor is the most widely and densely expressed within the hippocampus (Levey, 1993; Levey et al., 1995b; Mash and Potter, 1986). M1 receptors are predominantly located post-synaptically and these G-protein-coupled receptors are involved in the production of both LTP (Calabresi et al., 1999; de Sevilla et al., 2008) and LTD (Kamsler et al.,
2010). Synaptic metaplasticity within the hippocampus has also been shown to be mediated through the activation of M1 receptors (Hulme et al., 2012). Behavioural studies have found that blocking the M1 receptor inhibits spatial learning and memory in a variety of tasks, while the administration of M1 selective agonists can potentially enhance spatial working and reference memory (Brandeis et al., 1990; Caccamo et al., 2006; Hagan et al., 1987). The dorsal hippocampus, rather than the ventral hippocampus, is necessary for spatial learning and memory in rats (Moser et al., 1993). The striatum also receives vestibular input, although like the hippocampus, this is through indirect pathways from the vestibular nuclei, via the cortex or parafascicular nucleus (Lai et al., 2000; Stiles and Smith, 2015). Localized pharmacological blockade of M1 receptors in the striatum has been found to disrupt reversal learning (McCool et al., 2008; Tzavos et al., 2004). BVL rats switch from declarative spatial memory strategies, utilizing the hippocampus, to procedural memory strategies, probably mediated by the striatum, during the reverse T-maze task (Machado et al., 2014). This change in spatial learning strategy following BVL is also seen following hippocampal lesions (Packard and McGaugh, 1996). This suggests that BVL rats lose hippocampal function causing them to switch to a striatal procedural memory strategy (Machado et al., 2014). Therefore, the cholinergic system in both the hippocampus and striatum, may be involved in BVL-induced spatial memory impairment.
As well as memory, the mAChRs in the striatum and hippocampus may be implicated in the hyper-locomotor activity observed following BVL (Avni et al., 2009; Goddard et al., 2008;
72 Ossenkopp et al., 1992; Ossenkopp et al., 1990; Stiles et al., 2012). Cholinergic and dopaminergic systems in the basal ganglia both positively and negatively regulate each other, depending on the receptor subtype (Di Chiara et al., 1994; Gerber et al., 2001). Mice lacking the M1 receptor have elevated levels of extracellular dopamine in the striatum, producing hyper-locomotor behaviors
(Gerber et al., 2001; Miyakawa et al., 2001). However, hyper-locomotion is not necessarily produced by basal ganglia disturbance, as selective hippocampal, MS and forebrain lesions have been found to produce increased locomotor activity (Coutureau et al., 2000; Kuroiwa et al., 1991;
Lecourtier et al., 2010; van Praag et al., 1994). The MS is the largest source of hippocampal ACh
(Lewis and Shute, 1967), therefore it is a target for lesion studies examining the effects of hippocampal ACh. Combined lesions of both GABAergic and cholinergic MS neurons produce hyper-locomotion, however individually lesioning these neurons does not produce the hyperactivity (Lecourtier et al., 2010).
Beta-imaging autoradiography produces rapid, high resolution detection of beta-particles from tritium isotopes. Autoradiography is used to detect the localization, density and affinity of ligands in tissue (Barthe et al., 2004; Gattu et al., 1997; Mash and Potter, 1986; Spencer et al.,
3 1986). Using beta-imaging autoradiography and [ H]pirenzepine, a tritiated M1 mAChR antagonist, the density of M1 mAChRs can be quantified in the rat brain. Tritium particles are individually detected by the beta-imager. A gaseous scintillation detector with a parallel plate proportional avalanche chamber converts the electrons to light which can be recorded in real time with a Charge Coupled Device (CCD) camera (Figure 4.1; Barthe et al., 2004).
73
Figure 4.1: General operating principle of a beta-imager. When an emitted β-particle or an electron interacts with the detection medium, part of its energy is converted into light. The light is recorded by a Charge Coupled Device (CCD) camera and displayed on a computer. Figure from Barthe et al. (2004). Copyright Elsevier, used with permission.
While increases in ACh release have been found in the hippocampus during electrical stimulation of the vestibular system (Horii et al., 1994), and LTP induced by rotation (i.e., natural vestibular stimulation) has been shown to be abolished by pre-treatment with the muscarinic ACh receptor antagonist, atropine (Tai and Leung, 2012), no study to date has examined the effect of complete BVL on the hippocampal or striatal cholinergic receptors. The complete loss of vestibular input is hypothesized to have a significant effect on the density of hippocampal and striatal muscarinic ACh receptors. Therefore, the objective of the current study was to determine the density of M1 receptors in the hippocampus and the striatum, in rats with BVL induced by the intratympanic injection of sodium arsanilate.
74 METHODS Animals were housed as described in Chapter 2. Experiments were carried out in accordance with the European Communities Council Directive (86/6609/EEC) as well as French legislation. Twenty-three male Wistar rats (250–300 g, Janvier, France) underwent either bilateral intratympanic injections of sodium arsanilate or saline and behavioural testing to determine lesion severity.
Quantitative M1-Receptor Autoradiography
At either 7 or 30 days post-BVL, rats (BVL: n = 6 day 7, n = 5 day 30; sham: n = 6 day 7, n = 6 day 30) were anaesthetized with isoflurane (5%) in oxygen (2 L/min). After decapitation, the brains were removed, immediately frozen in isopentane for 10 s, and stored at −80°C. Brains were cryo-cut coronally using a cryostat (Leica Biosystems) and 20 µm sections from six different levels of the brain were collected (2 mm, 1 mm, 0 mm, −2 mm, −3 mm and −4 mm from bregma;
Paxinos and Watson, 1998). Sections were preincubated in Tris-HCl buffer for 15 min, then
3 incubated for 1 h with 3 nM [ H]pirenzepine (ACh M1 receptor antagonist, Perkin-Elmer, 1 mCi) in 50 nM Tris-HCl buffer at 4°C (Flynn et al., 1997; Tayebati et al., 2004).
Nonspecific binding (NSB) of tritiated pirenzepine was determined after incubation for 1 h with 3 nM [3H]pirenzepine and 3 µM pirenzepine (Sigma-Aldrich) in 50 mM Tris-HCl buffer.
All sections were washed four times in Tris-HCl for 2 min then in deionized water for 5 s. Sections were air-dried overnight at 4° C and rendered conductive by adhering copper foil tape (3M,
Belgium) to the free side. They were then placed into the gas chamber (mixture of argon and triethylamine) of a β-imager (Biospace Lab, France). Data were collected for 72 h. The area analysed was 20 × 25 cm2 and [3H] efficiency of counting was 90% on 2π radians (yield). The limits of sensitivity and resolution were 0.07 dpm and 50 μm, respectively (Barthe et al., 2004).
Regions of interest in the right and left caudate putamen and right and left dorsal hippocampus were manually delineated for each section by merging the autoradiographic images with histological sections (Figure 4.2). Regions of interest were delineated using the rat brain atlas
75 (Paxinos and Watson, 1998) and from Berendse and Groenewegen (1990) for striatal areas which receive projections from the parafascicular nucleus. Total dorsal hippocampal and striatal areas were analysed as well as separate analyses of the CA1, CA2/3, dentate gyrus and parts of the dorsal caudate putamen which receive parafascicular projections. The bound radioligand signal corresponding to the β-particle emitted from the delineated area was expressed in counts per minute per mm2 (cpm/mm2) (M3 vision software, Biospace-Instrument, France). Cpm/mm2 was then converted into nmol/mm3 of tissue, taking into account the yield (45%) and thickness of the section to which the ligand could bind (12 µm). Specific binding was determined by subtracting the NSB from total binding in corresponding brain areas.
Equations
The activity of [3H]pirenzepine (Ci/mmol) was converted to disintegrations per minute per mole (dpm/mol).
[3H]pirenzepine activity = 78.5 Ci/mmol = 78.5 x 2.2e12 (1Ci) x 1e-3 in dpm/mol =
1.727e11 dpm/mol
Binding density (nmol/mm3) was calculated by subtracting the NSB from total binding in each brain region. Section thickness, yield, and [3H]pirenzepine activity were taken into account and the final unit was converted to nmol/mm3.
Binding density (nmol/mm3) = (cpm/mm2 - nsbcpm/mm2) x (12e-3 (section thickness) x 0.45
(yield) / 1.727e11 dpm/mol ([3H]pirenzepine activity)) x 10e9 in nmol/mm3
76
Figure 4.2: M1 acetylcholine receptor expression following bilateral vestibular lesions (BVL) using beta-imaging autoradiography. The hippocampus (total, CA1, CA2/3, dentate gyrus) and caudate putamen (total, dorsal parafascicular inputs) were manually delineated using merged autoradiographic and histological images. Figure published in Aitken et al. (2016). Copyright John Wiley and Sons, used with permission.
Statistical Analyses
The results are expressed as mean ± standard error of the mean (SEM). A generalized linear mixed model (GLMM) analysis was performed on the autoradiographic data, with the treatment and day as between-group factors and the area, side and section as repeated measures (McCulloch et al., 2008). The GLMM was used in preference to a repeated measures ANOVA due to the 3 repeated measures and the potential for correlation in them to invalidate the ANOVA assumption of sphericity (Smith, 2012). The Corrected Quasi Likelihood Criterion (QICC) was used to determine the Goodness of Fit (McCulloch et al., 2008). The behavioral scores were analyzed using Mood’s median test due to the non-parametric nature of the data (Sprent and Smeeton, 2007).
77 RESULTS
Vestibular Deficiency
All sham animals were found to have a score of zero at both day 7 and day 30. Table 4.1 shows the mean clinical score of all animals in each experimental group. BVL rats expressed a strong vestibular syndrome both at 7 (χ2=12, P ≤ 0.001) and 30 days (χ2=10, P ≤ 0.01) after transtympanic chemical lesion, compared to the sham group.
Table 4.1: Behavioural scores of animals prior to beta-imaging autoradiography
Day 7 Day 30
Sham 0 (0,0) 0 (0,0)
BVL 16 (14,20) 15 (13,21)
Vestibular loss was reflected by a strong bilateral vestibular syndrome in the BVL group at day 7 and 30 compared to the sham group. P ≤ 0.01. Data are expressed as median (min,max) with the maximum possible score being 24.
M1 Receptor Autoradiography
3 [ H]pirenzepine bound selectively to M1 receptors in the cortex, hippocampus and caudate putamen, with the caudate putamen and hippocampal CA1 having the densest labelling (Figure
4.2). Seven days post BVL the binding density of [3H]pirenzepine was unchanged between sham and BVL animals in both the dorsal hippocampus and caudate putamen (Figure 4.3). Thirty days post BVL showed a significant decrease in the binding density in BVL animals in the dorsal hippocampus and caudate putamen (significant interaction: day x treatment: Wald χ2 (1,9) = 33.30,
P ≤ 0.0001); area (Wald χ2 (5,9) = 132.61, P ≤ 0.0001); section (Wald χ2 (5,9) = 27.54, P ≤
0.0001); area x section (Wald χ2 (9,9) = 33.35, P ≤ 0.0001)). Analysis of the CA1, CA2/3 and dentate gyrus demonstrated that the decrease was across the entire dorsal hippocampus and not restricted to a single area. The dorsal caudate putamen also showed a decrease in binding density independent of the total caudate putamen.
78 D a y 7 S h am
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Figure 4.3: M1 mAChR density 7 and 30 days following BVL in the hippocampal subregions (CA1, CA2/3 and the dentate gyrus (DG)), as well as the dorsal hippocampus (D Hippocamp), whole caudate putamen (CP) and dorsal caudate putamen (D CP) using beta-imaging autoradiography. Significant Area, Section, Area*Section and Day*Treatment effects (P ≤ 0.0001). Error bars are ± SEM.
79 DISCUSSION The analysis of [3H]pirenzepine binding in the present study illustrated a significant decrease in the density of M1 muscarinic ACh receptors in both the hippocampus and striatum following vestibular loss, which may suggest a reduction in ACh neurotransmission following the loss of vestibular information. Such a reduction in M1 receptor density, or similar changes in affinity, in either the hippocampus or the striatum could contribute to spatial memory deficits.
Local injections of M1 receptor antagonists into the hippocampus have been shown to produce memory deficits while M1 agonists improve memory consolidation (Brandeis et al., 1990; Hagan et al., 1987; Romanelli Ferreira et al., 2003). Similarly, M1 antagonists injected locally into the dorsomedial striatum have been found to produce learning deficits in reversal learning tasks
(McCool et al., 2008; Tzavos et al., 2004). Taken together, these results suggest a role for hippocampal and striatal M1 receptors in the BVL-induced spatial learning deficits seen in rodents.
The fact that the decrease in M1 receptors was present at 30 days post-lesion but not at 7 days post-lesion is likely due to the lesion mechanism. Sodium arsanilate lesions have a progressive toxicity, taking between 3 and 7 days for the vestibular hair cells to lesion (Vignaux et al., 2012). As the hair cells are progressively and heterogeneously destroyed, some vestibular function may remain until 7 days post-lesion. The delay in receptor down-regulation may also be partially due to the complexity of hippocampal sensory integration. With the hippocampus receiving inputs from many other sensory systems (Tesche and Karhu, 1999), any changes to ACh receptors may be delayed as the other sensory systems attempt to compensate for the lack of vestibular input.
An earlier study by Besnard et al. (2012) reported an increase in N-methyl-D-aspartate
(NMDA) receptor density at 30 days following BVL. Using bilateral sodium arsanilate transtympanic lesions, Besnard et al. (2012) found profound spatial memory disruption in BVL animals while hippocampal volume remained unchanged. It was suggested that the increase in
NMDA receptor density was a result of synaptic reorganization in the hippocampus, and was
80 partially responsible for the spatial memory deficits seen. It is likely that the M1 receptor decrease is a part of the same synaptic reorganization mechanism in response to the loss of peripheral vestibular input. Whether the changes in receptors are the cause of spatial memory deficits or a response to it remains open to speculation at this stage. The role of vestibular dysfunction in dementia has been raised recently by Previc (2013). Bilateral loss of cervical vestibular evoked myogenic potentials (cVEMP) responses is associated with a three-fold increase in AD prevalence
(Harun et al., 2016). A down-regulation of M1 receptors in the hippocampus following BVL supports the hypothesis that vestibular degeneration negatively affects the hippocampal cholinergic system. M1 receptors are also reduced in the hippocampus of AD patients (Shiozaki et al., 2001). It is likely that the combination of BVL and AD will produce an additive loss in cholinergic transmission in the hippocampus, potentially increasing the progression of dementia.
In normal aging the number of vestibular hair cells and VOR function has been reported to be reduced (Rosenhall and Rubin, 1975; Rauch et al., 2001; Chang et al., 2012; however, see
McGarvie et al., 2015 for conflicting data and Smith (2016) for a review) . Cholinergic transmission also showed a progressive loss with normal aging (Sarter and Bruno, 2004). The combination of vestibular and cholinergic degradation in aging may also lead to the spatial cognitive deficits seen in elderly individuals that are not related to dementia (Klencklen et al.,
2012).
81 5
M2 RECEPTOR EXPRESSION FOLLOWING BVL
INTRODUCTION
The M2 mAChRs inhibit adenylyl cyclase and cAMP through Gi coupled receptors (Felder,
1995). This mechanism and the pre-synaptic localization of the M2 receptors (Rouse et al., 1998) allow for the important self-regulatory inhibitory feedback to occur at the synapse. M1, M2 and M4 are the most abundant mAChRs in the brain (Levey, 1993). M2 receptors are located on dendrites and in the soma of neurons throughout the brain (Levey et al., 1995a; Levey et al., 1995b). M1 and
M2 receptors are also located on astrocytes and further muscarinic signalling occurs on other glial cell types (Guizzetti et al., 1996; Loreti et al., 2007; Murphy et al., 1986; Van Der Zee et al., 1993;
Wessler and Kirkpatrick, 2008). M2 receptors play an important role in learning and memory. M2 receptor antagonists improve learning and memory when injected systemically (Carey et al., 2001;
Packard et al., 1990; Quirion et al., 1995; Rowe et al., 2003; Vannucchi et al., 1997). Further, M2
KO mice exhibit learning and memory disruption in passive avoidance tasks (Tzavara et al., 2003).
In the striatum and hippocampus, M2 receptors are the primary muscarinic autoreceptor subtype involved in the inhibitory feedback regulation of ACh release (Billard et al., 1995; Hersch et al.,
1994; Volpicelli and Levey, 2004). M4 receptors are also inhibitory autoreceptors and have similar functionality to the M2 receptor (Dolezal and Tucek, 1998; Hersch et al., 1994). Hence, both M2 and M4 KO mice exhibit an increase in ACh in the hippocampus (Tzavara et al., 2003). The blockade of striatal M2 receptors has been shown to improve procedural memory performance, but not spatial memory performance, by increasing ACh release in the striatum (Lazaris et al., 2003).
In human AD patients, the expression of M1, M2, and M3 receptors is decreased in the hippocampus. M2 receptors are increased in the striatum, while M4 receptors are unchanged throughout the brain (Rodriguez-Puertas et al., 1997). This suggests that the expression of M2 receptors, but not M4 receptors, in the hippocampus and striatum, is indicative of the cognitive
82 decline seen in AD. With vestibular dysfunction suggested as a possible factor accelerating the progression of AD, it is likely that BVL patients have the same mAChR profile (Previc, 2013).
As the density of M1 mAChRs has been assessed previously in Chapter 4, the present experiment examined the number of M2-expressing cells. Using flow cytometry, the individual somata of M2 labelled cells could be counted and separated into neuronal and non-neuronal cells.
Flow cytometry allows for high throughput, accurate cell counting in brain tissue (Collins et al.,
2010). Unlike autoradiography, flow cytometry measures the individual cells which contain M2 receptors and not the density of these receptors. Flow cytometry also allows for double-labelling, which in the present experiment allowed for the determination of M2 receptor localization using a neuron-selective beta-tubulin III antibody. Examining the expression of hippocampal and striatal
M2 receptors following BVL would determine whether the cognitive decline seen following BVL is associated with changes in muscarinic autoreceptors. With the decrease in M1 receptor density seen in Chapter 4, it was hypothesised that M2 receptor expression would be increased following
BVL due to its opposing function in the cholinergic system. This would produce decreased cholinergic signalling through increased inhibitory feedback, leading to cognitive and spatial memory disorders.
83 METHODS
Subjects
Twenty-four male Wistar rats (250–300 g) underwent sodium arsanilate BVL or sham injection as detailed in Chapter 2. The behavioural score was assessed at day 7. At either seven or thirty days post BVL, rats (BVL: n = 5 day 7, n = 4 day 30; sham: n = 5 day 7, n = 2 day 30) were anaesthetized with 5 ml of pentobarbital sodium (125 mg/ml, i.p.). Initially all groups were n = 6, however tissue from several animals did not double label correctly during antibody labelling and analysis could not be performed. Further to this, several animals from day 30 lost their tail markings and could no longer be confidently identified as BVL or sham and were therefore excluded from analysis. Animals underwent transcardiac perfusion with saline (300 ml). The brain was removed and the dorsal hippocampus and striatum were dissected out.
Tissue Dissociation and Myelin Removal
The brain tissue was dissociated using the standard protocol of the MACS neural tissue dissociation kit (P) (Miltenyi Biotec) with the gentleMACS™ Dissociator (Miltenyi Biotec).
Following the protocol provided by Miltenyi Biotec, β-mercaptoethanol (50 mM; 20 µl) was added to Buffer X (14980 µl). The buffer X solution (1900 µl) was added to Enzyme P (50 µl) to form
Mix 1. Enzyme A (10 µl) and Buffer A (1 ml) were combined. Solution A (10 µl) was combined with Buffer Y (20 µl) to form Mix 2. Brain tissue was added to a MACS C tube (Miltenyi Biotec) with 37°C Mix 1 (1950 µl). Neuron dissociation protocol 1 was performed with the gentleMACS™ Dissociator followed by gentle (80 RPM) orbital shaker incubation for 15 min at
37°C (Figure 5.1). The MACS C tube was returned to the gentleMACS™ Dissociator for neuron dissociation protocol 2. Mix 2 (30 µl) was then added to the sample solution prior to a 10 min incubation at 37°C. Neuron dissociation protocol 3 was performed followed by a final 10 min incubation at 37°C. Following dissociation, the tissue was filtered through a 70 µm nylon mesh
(neuron soma ø ≤ 10 µm (Meitzen et al., 2011; Yamada et al., 2002)) and centrifuged (200 g for
5 min, at room temperature). The supernatant was removed and then the pellet was fixed in
84 paraformaldehyde (5 ml; 4%) for 3 h at 4° C. The tissue was centrifuged again following fixation and the supernatant discarded.
Figure 5.1: Tissue dissociation protocol. Tissue was dissociated in MACS C tubes using a gentleMACS™ Dissociator. Mix 1 (buffer X solution (1900 µl) + Enzyme P (50 µl)) was added to the tissue before undergoing neuron dissociation protocol 1. Mix 2 (Solution A (10 µl) + Buffer Y (20 µl)) was added following neuron dissociation protocol 2. This was followed by neuron dissociation protocol 3. Tissue was incubated at 37°C between dissociation protocols on an orbital shaker.
Myelin removal beads II (Miltenyi Biotec) (20 µl) and phosphate buffered saline (PBS)
(NaCl 137 mmol/L, KCl 2.7 mmol/L, Na2HPO4 10 mmol/L, KH2PO4 1.8 mmol/L) with 5% bovine serum albumin (BSA) (180 µl) were added to the pellet and incubated at 4⁰ C for 15 min. A further
1800 µl of PBS + 5% BSA was added to the suspension. The suspension was centrifuged (300 g for 10 min) and the supernatant discarded. LS Columns (Miltenyi Biotec) were prepared by attaching them to the magnetic field of the MACS separator (Miltenyi Biotec) and rinsing with
PBS buffer (2 ml). Each sample was gravity fed through LS columns (Figure 5.2). This was followed by immediately rinsing the LS columns with 1 ml PBS + 5% BSA. Tissue not labelled with magnetic separation beads was collected as it exited the LS columns. Tissue labelled with magnetic separation beads was bound within the LS columns. Labelled tissue was flushed with 5 ml PBS + 5% BSA using a syringe plunger following collection of the unlabelled tissue by removing the LS columns from the magnetic field. The tissue suspensions collected from the LS columns (~3 ml) were then fixed in 1% paraformaldehyde (5 ml) for 12 h.
85
Figure 5.2: MACS magnetic separation protocol. Cells are labelled with magnetic separation beads. Unlabelled cells are separated from labelled cells using a magnetic field and LS columns. Labelled cells (myelin) are removed from the LS columns by flushing the column with PBS following removal from the magnetic field.
Antibody Markers
Paraformaldehyde solution was removed via centrifugation (300 g for 10 min) and PBS +
5% BSA solution (1 ml) was added to each sample. Each sample was divided into four groups with equal volume to prepare for labelling. These groups were: unlabelled, M2 antibody only, beta- tubulin III antibody only, M2 and beta-tubulin III antibodies (combination). Beta tubulin III is a microtubule element specifically expressed in neurons (Jiang and Oblinger, 1992; Roskams et al.,
1998). The M2 receptor antibody (Abcam ab2805; 1:200 final concentration) and its secondary antibody FITC (Abcam ab97239; 1:100 final concentration) were added to their respective sample groups and incubated for 30 min at room temperature in the dark. The sample was centrifuged
(300 g for 10 min), supernatant discarded and replaced with PBS + 5% BSA (250 µl), then centrifuged again (300 g for 10 min) and the supernatant discarded. The unlabelled and M2- antibody only groups had PBS + BSA 5% (500 µl) added to the pellet and were stored at 4° C until flow cytometric analysis began. The “beta-tubulin III” and “combination” group pellets were washed in Triton-X (0.5%) for 30 min to increase cell permeability. The antibody to beta-tubulin
III (Abcam ab18207; 1:2000 final concentration) and its secondary antibody Alexa Fluor750
(Abcam ab175731; 1:2000 final concentration) were incubated for 60 min at room temperature in
86 the dark. The samples were then centrifuged (300 g for 10 min), the supernatant discarded and replaced with PBS + 5% BSA (500 µl).
Figure 5.3: FITC (green) and Alexa Fluor750 (purple) excitation and emission spectra. The peak excitation wavelength is indicated by the dotted line curve, while the emission wavelength is indicated with the filled curve.
Samples were then placed in the cytometer (Gallios; Beckman Coulter) and the acquisition run. Cells were excited using two lasers, one at 488 nm and another at 638 nm to excite the fluorescent antibodies (Figure 5.3). Fluorescence could then be detected with a 500-550 nm filter for FITC and a >750 nm filter for Alexa Fluor750. Data were analysed with Flowing Software
2.5.1. Unlabelled cells were analysed to determine intrinsic fluorescence and gating set for future analysis of labelled samples (Figure 5.4). The individually labelled cells were measured to calibrate the gating of FITC and Alexa Fluor750 (Figure 5.4). Double-labelled cells were used for quantification once calibration had been set.
87 A C
B D
Figure 5.4: Laser calibration of cytometer samples. The two lasers FL1 and FL8, were calibrated using unlabelled cells to remove intrinsic fluorescence from the final analysis (A and B). Cells individually labelled with either FITC (C) or Alexa Fluor750 (D) were used to assess fluorescent activity before analysis of the double labelled samples. The inset figures show the result of the gating.
A B
Figure 5.5: Myelin gating protocol. Samples containing high concentrations of myelin from flushing the LS columns were analysed with the flow cytometer. The majority of myelin tissue was gated for removal (A) and these gates were used for the following sample analysis (B).
Myelin control samples allowed for the gating and removal of myelin from labelled samples (Figure 5.5). Cells which had a linear forward scatter (FS) peak versus area (INT) crossed the laser individually and were delineated for analysis (Figure 5.6B). Using calibration controls, 88 unlabelled cells and those labelled for M2 antibodies, beta-tubulin III and co-labelled were counted
(Figure 5.6C).
A B C
Figure 5.6: Cells counted with flow cytometry were separated from myelin (A). Cells which crossed the laser individually were delineated (B). Using calibration controls (see Figure 5.4) cells unlabelled (bottom left), and those labelled for M2 antibodies (bottom right), beta-tubulin III (top left) and cells which were co-labelled (top right) were counted (C). Blue = myelin, Green = unlabelled, Pink = M2 labelled, Grey = beta-tubulin III labelled, Orange = double labelled.
Statistical Analysis
Data were logit transformed to account for the compositional nature of the data (Smith et al., 2016). A linear mixed model (LMM) was used to analyse the results. Between group factors were day post-lesion and lesion group. Brain region (hippocampus and striatum) was considered a repeated measure. The covariance structure with the lowest Akaike’s Information Criterion
(AIC) was selected. The LMM was used in preference to a repeated measures ANOVA due to the repeated measures and the potential for correlation in them to invalidate the ANOVA assumption of sphericity (Smith, 2012). The behavioral scores were analyzed using Mood’s median test due to the non-parametric nature of the data (Sprent and Smeeton, 2007).
89 RESULTS
Vestibular Deficiency
All sham animals were found to have a score of zero. BVL rats expressed a strong vestibular syndrome (χ2=16, P ≤ 0.0001) after transtympanic chemical lesion, compared to the sham group (Table 5.1).
Table 5.1: Behavioural scores of animals prior to flow cytometry analysis
Sham 0 (0,0)
BVL 17 (14,21)
Vestibular loss was reflected by a strong bilateral vestibular syndrome in the BVL group compared to the sham group. P ≤ 0.0001. Data are expressed as median (min,max) with the maximum possible score being 24.
M2 Receptor Expression
At 7 days following BVL there was no significant change in M2 receptor-expressing neurons between BVL and sham animals in either the hippocampus or striatum (Figure 3). By day
30 there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M2 muscarinic receptors. This effect was seen in both the hippocampus and striatum (M2+ve/beta- tubulin+ve double labelled cells = Treatment: F(1-23)=6.8, P ≤ 0.05; Day*Treatment: F(1-
23)=4.7, P ≤ 0.05; Area: F(1-23)=0.2, P > 0.05). Non-neuronal M2 expressing cells were increased in the striatum but not the hippocampus at day 30, there was no change at day 7 (M2+ve/beta- tubulin-ve cells = Area: F(1-10.8)=5.6, P ≤ 0.05; Day*Area: F(1-10.8)=5.7, P ≤ 0.05; and
Day*Area*Treatment: F(1-10.8)=5.3, P ≤ 0.05). Unlabelled cells exhibited a decrease in percentage over time, reflecting the increase in M2 labelled neurons seen at day 30 (Day: F(1-
23)=9.7, P ≤ 0.05). There was no significant change in the number of M2-/beta-tubulin+ve cells (P
> 0.05). Data are presented as percentage of cells, the total number of cells analysed in each sample was 8462 ± 556.
90 1 0 0 1 0 0 H ip p o c a m p u s D a y 7 S h a m H ip p o c a m p u s D a y 3 0 S h a m
8 0 B V L 8 0 B V L
)
) %
6 0 % 6 0
(
(
s
s
l
l
l
l e
4 0 e 4 0
C C
2 0 2 0
0 0
d 2 s s d 2 s s e M n n e M n n ll o o ll o o e r r e r r b u u b u u e e e e la la n N N n N N U 2 U 2 M M
1 0 0 1 0 0 S tria tu m D a y 7 S h a m S tr ia tu m D a y 3 0 S h a m
8 0 B V L 8 0 B V L
)
) %
% 6 0 6 0
(
(
s
s
l
l
l
l e
e 4 0 4 0
C C
2 0 2 0
0 0
d 2 s s d 2 s s e M n n e M n n ll o o ll o o e r r e r r b u u b u u e e e e la la n N N n N N U 2 U 2 M M
Figure 5.7: M2 acetylcholine receptor expression following BVL using flow cytometry. M2 and beta-tubulin antibodies were used to quantify M2 receptor and total neuron numbers. Significant effects (P ≤ 0.05) were found for Treatment and Day*Treatment for cells co-labelled for M2 and beta-tubulin; Day for unlabelled cells; Area, Day*Area and Day*Area*Treatment for cells labelled only for M2.
91 DISCUSSION
An increase in neurons expressing the M2 mAChR thirty days following BVL suggests a long-term change in cholinergic signalling in the hippocampus and striatum. The lack of difference at seven days post-BVL illustrates that the acute effects of vestibular loss do not produce immediate changes to the number of M2 expressing cells, but rather that an increase is observed over time. As previously discussed, sodium arsanilate lesions have a progressive toxicity. As the hair cells are progressively and heterogeneously destroyed, some vestibular function may remain until 7 days post-lesion (Vignaux et al., 2012). It is possible that the effect seen at thirty days is not the peak of the cellular plasticity, and that further increases in M2 receptor-containing cells may be observed, as later time points were not assessed. While the number of cells that express the M2 receptor increases following BVL, the flow cytometry method does not determine whether the total number of receptors has increased, or whether the affinity of the receptors or the efficacy has changed. It would be surprising if the number of cells expressing the M2 receptor increased, while the total number of receptors did not. As seen in Chapter 4, the density of M1 receptors is decreased following BVL, suggesting that there is a reduction in excitatory cholinergic signalling through reduced M1 activity. With either an increase in the affinity and/or efficacy of M2 receptors or an increase in the number of M2 neurons, there is likely to be increased inhibition of cholinergic signalling in the hippocampus and striatum.
M2 antagonists have been found to improve learning and memory performance (Carey et al., 2001; Packard et al., 1990; Quirion et al., 1995; Rowe et al., 2003), as well as increase the performance in passive avoidance and object recognition tasks (Vannucchi et al., 1997). These antagonists have been found to increase ACh release in the hippocampus, which is a suggested mechanism for the improved cognitive performance (Carey et al., 2001; Quirion et al., 1995;
Vannucchi et al., 1997). The possible involvement of the M4 mAChR cannot be ruled out. Due to the similarity of the receptor structure, many of the antagonists tested also bind to both the M2 and
M4 receptors. One study used what is thought to be a more selective M2 antagonist (SCH 57790)
92 and found similar results to studies which used the M2/4 antagonist AFDX 384 (Carey et al., 2001).
The effect of BVL on M2 mAChRs in the brain appears to be similar to the effect of aging on the muscarinic cholinergic system, where M2 binding is increased in the cortex and hippocampus, leading to a decrease in ACh release and memory impairment (Quirion et al., 1995). With the increase in M2 receptor-expressing neurons there would likely be a decrease in ACh release in the hippocampus (see Chapter 8) and a disruption of memory and learning as well as poor performance in other cognitive tasks. Memory and learning deficits are commonly reported following BVL
(Baek et al., 2010; Besnard et al., 2012; Brandt et al., 2005; Chapuis et al., 1992; Dallal et al.,
2015; Guidetti et al., 2008; Horn et al., 1981; Hufner et al., 2007; Machado et al., 2012a; Matthews et al., 1989; Neo et al., 2012; Ossenkopp and Hargreaves, 1993; Russell et al., 2003a; Russell et al., 2003b; Schaeppi et al., 1991; Schautzer et al., 2003; Smith et al., 2013; Stackman and Herbert,
2002; Wallace et al., 2002; Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2004; Zheng et al., 2006; Zheng et al., 2007; Zheng et al., 2009b). The percentage of neurons in the hippocampus remained unchanged following BVL at both 7 and 30 days post-lesion. This supports a previous study in which hippocampal neuronal number was not changed in a rodent model of BVL (Zheng et al., 2012a), although this assumes that the number of cells in each brain analysed with flow cytometry was the same.
The percentage of M2-positive cells in the striatum is as low as 2.5% in immunohistochemical studies (Hersch et al., 1994). M4 receptors are present in 44% of striatal cells. We found that ~40% of striatal cells were positive for the M2 antibody, with an extra 20% of cells positive for the antibody following BVL. This suggests that the antibody might not be specific for the M2 receptor. The antibody was a full length native protein purified from pig heart.
Heart tissue is known to contain M4 receptors (Lazareno et al., 1990). Western blot testing of the antibody (Figure 5.8) displayed multiple unknown bands at 22 kDa and 40 kDa with M2 expression at 64 kDa (molecular weight: 52 kDa). These unknown bands were observed in mouse brain tissue but not human CNS protein.
93
Figure 5.8: Western blot of the anti-M2 antibody (ab2805). Lane 1: Human brain tissue lysate - total protein (ab29466). Lane 2: Human spinal cord tissue lysate - total protein (ab29188). Lane 3: Brain (Mouse) Tissue Lysate. Lysates/proteins at 10 µg per lane. Figure from Abcam (2010). Copyright Abcam, used with permission.
While neither of the unknown bands match the M4 mAChR (molecular weight: 53 kDA) and the antibody purification technique has been shown to be specific to M2 mAChRs previously
(Wang et al., 1995), our findings and the western blot analysis from Abcam suggest non-specific binding in rodent brains. If the non-specific binding is to the M4 mAChR, which is a likely candidate due to the similarities between the M2 and M4 receptors, both being inhibitory mAChRs and having similar molecular weights, the functional changes produced by an increase in M4- positive cells in the striatum and the hippocampus following BVL might be similar to changes in
M2-positive cells. Both M2 and M4 mAChRs act as autoreceptors, modulating ACh release in the striatum (Dolezal and Tucek, 1998; Hersch et al., 1994). By increasing the activity of inhibitory autoreceptors, ACh release would be reduced in the striatum and lead to procedural learning and memory deficits, as suggested by improvements following M2/4 antagonist administration
(Bymaster et al., 1993; Lazaris et al., 2003). M2 receptors inhibit dopamine release in the striatum
(De Klippel et al., 1993; Smolders et al., 1997; Xu et al., 1989). A reduction of dopamine release in the striatum has been linked to ADHD (Volkow et al., 2007). A reduction of striatal dopamine due to an increase in M2 receptors may be the underlying cause of locomotor hyperactivity seen in
94 rodent models of BVL (Fedrowitz et al., 2000; Goddard et al., 2008; Richter et al., 1999; Stiles et al., 2012). The observed increase in M2/4 non-neuronal cells in the striatum following BVL is most likely due to an increase in astrocyte proliferation (Murphy et al., 1986). Astrocytes are known to increase neurogenesis following damage to the striatum (Duan et al., 2015; Nato et al., 2015); these new cells may be the source of the increase in neuronal M2/4-receptor positive cells following
BVL.
In conclusion, BVL produces an increase in M2/4 mAChR-containing neurons in the hippocampus and an increase in M2/4 mAChR-containing neurons and non-neuronal cells in the striatum. This increase in M2/4-containing neurons may be similar to the increase in M2 affinity observed in memory-impaired aged animals. An increase in M2/4 distribution is likely to produce a decrease in ACh release in the hippocampus and striatum resulting in learning and memory deficits. In the striatum, an increase in M2 activity may also produce an inhibition of dopamine release, producing locomotor hyperactivity in BVL rats.
95 6 HIPPOCAMPAL THETA RHYTHM IS POTENTIATED FOLLOWING BVL
INTRODUCTION Hippocampal theta rhythm can be detected by recording local field potentials (LFP) in the hippocampus or medial septum. LFPs reflect the postsynaptic activity of cell groups which are periodically synchronised. Theta is the synchronised firing of hippocampal cells and is considered an important mechanism in learning and memory formation (Givens, 1995; Leutgeb and
Mizumori, 1999; O'Keefe, 1993; Winson, 1978). Vestibular stimulation produces increased theta in rodents (Gavrilov et al., 1995; Gavrilov et al., 1996; Shin, 2010; Tai et al., 2012). Lesions of the vestibular system disrupt theta rhythm in freely behaving animals and impair spatial learning and memory (Neo et al., 2012; Russell et al., 2006). However, restoring theta in BVL rats using
MS electrical stimulation was unsuccessful in restoring spatial memory (Neo et al., 2012). MS stimulation was given at 7.5-8 Hz throughout behavioural testing in the open field, elevated T- maze and a spatial non-matching to sample test. Stimulation restored the hippocampal theta power, but did not repair the learning and memory deficits in BVL animals. Since the stimulation was not synchronized to head acceleration and at a frequency greater than natural type 1 theta, it was speculated that the constant pulse bursts provided to generate theta may have disrupted the place cell network. In addition, a type 2 theta stimulation protocol was not developed, although there is evidence for type 2 theta to be the source of vestibular stimulation-induced theta (Horii et al.,
1994; Shin, 2010; Tai et al., 2012). The location of the stimulating electrode in the medial septum, while providing direct stimulation to the hippocampus, excluded other nuclei upstream in the theta- generating pathway, such as the PH, PPT, and RPO, all of which modulate the size and frequency of theta while integrating sensory and motor information. As type 2 theta is atropine-sensitive, the large number of cholinergic cells in the PPT and its position at the beginning of the theta- generating pathway suggest that this nucleus is the driver for type 2 theta. Indeed, LFP recordings
96 in the PPT are at theta frequencies, with an increase in frequency occurring during locomotion
(Geng et al., 2016). When the local anaesthetic procaine is injected directly into the PPT to produce temporary inactivation, type 2 theta is abolished in urethane-anaesthetised rats (Nowacka et al.,
2002).
As type 2 theta has not been previously assessed following BVL, theta was measured from the LFP recordings of the hippocampus and the PPT in urethane-anaesthetised rats. In rats maintained at light levels of urethane anaesthesia, spontaneous periods of theta can be seen. At deeper levels of anaesthesia, only large irregular waves are present in the hippocampal field activity and a tail pinch or other sensory stimulation will induce theta (Kinney et al., 1998; Orzel-
Gryglewska et al., 2006; Orzel-Gryglewska et al., 2015). Tail pinch activates ~50% of PPT neurons and elicits strong type 2 theta (Carlson et al., 2004; Carlson et al., 2005; Nowacka et al.,
2002). The present experiment aimed to compare the response of hippocampal and PPT LFPs to tail pinch-induced type 2 theta in BVL and sham rats. The non-vestibular tail pinch stimulation allowed theta to be generated in BVL rats and the effect of BVL on theta generation was measured.
It was hypothesized that type 2 theta would be disrupted in BVL rats in a similar manner to the disruption of type 1 theta seen in freely behaving BVL animals (Neo et al., 2012; Russell et al.,
2006).
97 METHODS Eighteen male Wistar rats (250–300 g) underwent sodium arsanilate BVL, UVL or sham injection as described in Chapter 2 (Sham: n = 6; BVL: n = 5; UVL: n = 7). Behavioural scores were assessed at day 1, 3, 7 and 30 post-tympanic injection.
Hippocampal Local Field Potential Recording
Animals were anaesthetized with urethane (1.5 g/kg, i.p.) 90 days following the BVL procedure and the level of anaesthesia controlled by monitoring the pedal reflex. The animal was placed in a stereotaxic frame and xylocaine (with 1:100,000 adrenaline) was injected along the scalp prior to an incision being made sagittally along the midline of the scalp. A small area (about
2 x 2 mm, starting at 6.8 mm posterior and 0.7 mm lateral to bregma) of the skull overlying the
PPT was removed and the dura opened. A recording electrode (stainless steel, Teflon coated wire;
A-M 7910) was implanted unilaterally 7.8 mm posterior to bregma, 1.7 mm lateral to the midline, and 6.8 mm below the dura. In BVL and sham animals the electrode was implanted in either the left or right hippocampus using pseudorandom alternation. In unilateral vestibular lesion (UVL) animals, the electrode was implanted contralateral to the lesioned side. A second identical recording electrode was implanted within the hippocampus at a 30⁰ angle, with the final tip placement 3.8 mm posterior to bregma, 2 mm lateral to the midline and 3 mm below the dura ipsilateral to the first electrode. A ground Ag/AgCl electrode (Warner Instruments) was placed under the skin of the neck. Simultaneous recording of field activity was made for 100 min using
Spike 2 software (CED Cambridge) and a Neurolog extracellular recording system (Digitimer).
LFP activity was digitized at 600 Hz after being amplified (1000x) and band-pass filtered (0.1–
200 Hz) using a dedicated ac-differential amplifier and head stage (Neurolog, Digitimer). A plastic clip was placed 1 cm below the base of the animal’s tail for 60 s periods to elicit theta activity in the hippocampus and PPT. Recordings were analysed by Fast Fourier Transform (Spike 2, CED) to determine the frequency and amplitude of the EEG over time.
98 Histology
Following recording the animals were given an overdose of urethane and perfused through the ascending aorta with phosphate buffer (PBS, 0.1 M, pH 7.2) followed by 4% paraformaldehyde in PBS and then 4% paraformaldehyde in PBS containing 0.6 M sucrose. The brains were removed and stored overnight in 30% sucrose at 4°C to protect the tissue from freezing artefacts. Brains were snap-frozen in liquid nitrogen-cooled hexane in Cryomatrix (Thermofisher). Fifty µm parasagittal sections were cut on a cryostat. Histological dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was used to determine the exact placement of electrodes. NADPH-d staining was performed as described by Hernandez-Chan et al. (2011).
Every second section was incubated for 3 h at 37°C in 0.1 M Tris–HCl buffer (pH 8.0), containing
0.6 mM β-nicotinamide adenine dinucleotide phosphate, reduced form (Alfa Aesar), 0.81 mM nitro blue tetrazolium (Alfa Aesar), 0.3% Triton X-100 (Sigma), and 2% ethanol. After staining sections were mounted onto gelatin coated slides and allowed to dry. Sections were dehydrated through graded alcohols (70–100%), cleared in xylene and cover-slipped. Alternate sections were used for choline acetyltransferase (ChAT) immunohistochemistry as detailed in Chapter 7.
Temporal bones were collected, fixed and stained as described in Chapter 2 to assess the extent of damage to the vestibular hair cells in response to sodium arsanilate.
Statistical Analysis
Analysis of the hippocampal EEG signal was performed off-line in Spike 2 (CED). Fast
Fourier Transformation, using overlapping Hanning windows with a resolution of 0.07 Hz, was calculated for 10 artefact free 15 s samples randomly selected from baseline activity and also from
10 theta rhythm episodes 5 s following application of the tail clip for 15 s. The mean power as a percentage of baseline was calculated for the peak amplitude at five 3 Hz frequency bands (0-3, 3-
6, 6-9, 9-12, 12-15 Hz). The mean frequency of the peak amplitude within each band was also calculated (Fmax). The results are expressed as means ± standard error of the mean (SEM). A linear mixed model (LMM) analysis was performed on the electrophysiological power and Fmax
99 data, with the treatment as a between-group factor and the frequency band as repeated measures.
The covariance structure with the lowest Akaike’s Information Criterion (AIC) was selected. The
LMM was used in preference to a repeated measures ANOVA due to the repeated measures and the potential for correlation in them to invalidate the ANOVA assumption of sphericity (Smith,
2012). The behavioural scores were analysed using Mood’s median test due to the non-parametric nature of the data (Sprent and Smeeton, 2007).
100 RESULTS
Vestibular Deficiency
All sham animals were found to have no vestibular deficiency indicated by a score of zero at all days assessed (Table 6.1). A total of 5 BVL, 7 UVL and 6 sham animals were assessed. Both
BVL (χ2=40, P ≤ 0.0001) and UVL (χ2=44.57, P ≤ 0.0001) rats expressed strong vestibular syndromes at 1, 3, 7 and 30 days following transtympanic chemical lesion compared to the sham group (Table 6.1). The behavioural effects of the lesions increased in severity over time before partially compensating at day 30 in both UVL (χ2=22.67, P ≤ 0.0001) and BVL (χ2=12.59, P ≤
0.01) animals. The behavioural effects of the lesions were most severe at days 3 and 7 in both groups of animals (Table 6.1).
Table 6.1: Behavioural scores of animals prior to electrophysiological recording
Day 1 Day 3 Day 7 Day 30
Sham 0 (0,0) 0 (0,0) 0 (0,0) 0 (0,0)
UVL 4 (0,6) 10 (5,16) 8 (7,13) 5 (5,8)
BVL 12 (7,17) 17 (13,18) 15 (7,21) 10 (4,12)
Vestibular loss was reflected by a strong bilateral vestibular syndrome in the BVL group at days 1, 3 7 and 30 compared to the sham group (P ≤ 0.0001). A strong unilateral vestibular syndrome was observed in UVL rats at 1, 3, 7 and 30 days post-injection compared to sham animals (P ≤ 0.0001). Data are expressed as median (min,max) with the maximum possible score being 24 for BVL and 20 for UVL animals.
Electrode Placement
Hippocampal electrode tips were predominantly located within either CA1 or the molecular layer of the dentate gyrus (Figure 6.1). PPT electrodes were largely misplaced and recordings from within the PPT could not be made with a suitable number of animals as a result.
101 Results from the five closest electrodes were analysed regardless, but statistical analysis could not be completed due to the small number (Sham n = 1, BVL n = 1, UVL n =3).
Figure 6.1: Electrode tip locations. A) Hippocampal electrode tip placement. Sagittal view 2 mm lateral from bregma. Blue = BVL, Red = UVL, Green = Sham. B) NADPH-d stained hippocampus showing electrode track (red line). Scale bar = 1000 µm C) PPT electrode placement. Sagittal view at 1.7 mm lateral from bregma. Purple area is the PPT. Blue = BVL, Red = UVL, Green = Sham, Black = misplaced. All electrode tips were located outside or on the superior border of the PPT. Those closest to the PPT were analysed. D) NADPH-d stained PPT (within purple line) with misplaced PPT electrode track (red line). Scale bar = 1000 µm.
102 Hippocampal Electrophysiology
In all animals, the placement of a clip on the tail under urethane anaesthesia produced a shift in the frequency of the local field potential (LFP) from the delta (0-3 Hz) to the theta (3-12
Hz) range (Figure 6.2).
Figure 6.2: Hippocampal response to a tail pinch during urethane anaesthesia. A) Spectrogram and raw electrophysiological trace. Immediately after the tail pinch stimulus (p) the frequency shifts from < 2 to 4.5 Hz. This reverses shortly after removal of the stimulus (o). B) A histogram FFT of the baseline data overlaid with data taken during the tail pinch. Peak power has shifted frequency from 1 Hz to above 4 Hz following a tail pinch stimulus.
An LMM analysis revealed a significant increase in the maximum power of the 3-6 Hz frequency band in BVL animals compared to both sham and UVL animals (Treatment: F (2,11.57)
= 4.25, P ≤ 0.05; Frequency band: F (4,18.4) = 23.12, P ≤ 0.001; Treatment*Frequency band: F
(8,18.4) = 3.83, P ≤ 0.01). There were no changes in the other frequency bands (Figure 6.3). The frequency corresponding to the maximal peak power (Fmax) was not significantly different between treatment groups (Figure 6.4; Treatment: F (2,41.95) = 0.33, P ≥ 0.05; Frequency band:
F (4,20.35) = 437.4, P ≤ 0.001; Treatment*Frequency band: F (8,20.35) = 0.21, P ≥ 0.05).
103 2000 Sham * BVL 1500 UVL
1000
500
Power (% baseline) Power(% 0 0-3 3-6 6-9 9-12 12-15 Frequency range (Hz) -500
Figure 6.3: Changes of hippocampal power distribution within frequency bands. FFT analysis determined the maximum power within five frequency bands. Power is expressed as mean percentage of baseline control. Sham n = 6, BVL n = 5, UVL n = 7. Error bars are SEM. * = P ≤ 0.05.
15 Sham BVL UVL 10
Fmax(Hz) 5
0 0-3 3-6 6-9 9-12 12-15
Frequency range (Hz)
Figure 6.4: Hippocampal frequency at the maximum power within frequency bands (Fmax). FFT analysis determined the frequency at the maximum power. Sham n = 6, BVL n = 5, UVL n = 7. Error bars are SEM. No shift in frequency was observed within any frequency range.
Recordings from the PPT were not statistically analysed due to the small number of animals with correctly placed electrodes. The data that could be gathered was consistent with the hippocampal theta data, with BVL animals showing an increased power in the 3-6 Hz frequency band compared to sham and UVL animals (Figure 6.5A). Fmax appeared unchanged at all frequency bands (Figure 6.5B).
104 A B 1 5
1 5 0 0 )
e S h a m
S h a m
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l 1 0 0 0 U V L )
e B V L 1 0 z
s B V L
H
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U V L
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5 0 0 a
%
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(
F
r 5 e
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o P
-5 0 0 0 0 -3 3 -6 6 -9 9 -1 2 1 2 -1 5 0 -3 3 -6 6 -9 9 -1 2 1 2 -1 5 F re q u e n c y ra n g e (H z ) F r e q u e n c y r a n g e (H z )
Figure 6.5: PPT power and frequency. FFT analysis determined the power (A) and the Fmax (B). Power is expressed as mean percentage of baseline control. Sham n = 1, BVL n = 1, UVL n =3. Error bars are SEM.
105 DISCUSSION Increased theta power in the 3-6 Hz frequency range in BVL rats was an unexpected result.
Under deep urethane anaesthesia, theta is only present following sensory stimulation, such as a tail pinch (Orzel-Gryglewska et al., 2015). In the present experiment animals were deeply anaesthetised, with no spontaneous theta present and a tail pinch was used to elicit theta. Previous studies in freely behaving animals have found a decrease in theta power following BVL (Neo et al., 2012; Russell et al., 2006). However, these studies measured the movement dependant type 1 theta (7-9 Hz). Rotation-induced vestibular stimulation produces type 2 theta (3-7 Hz) which is inhibited by atropine in restrained, awake animals (Shin, 2010; Tai et al., 2012). It appears that
BVL increases the sensitivity of type 2 theta to non-vestibular stimuli such as the tail pinch utilised in the present experiment. Type 2 theta is produced in awake rats during periods of hyper-arousal, such as in the presence of a predator (Sainsbury et al., 1987; Sainsbury and Montoya, 1984). The type 2 theta system is thought to be involved in sensory processing and is active during type 1 theta activity, as atropine reduces the rhythmicity of type 1 theta (Bland et al., 1984; Bland, 1986).
Type 2 theta has been found to decrease in amplitude and frequency with age (Abe and Toyosawa,
1999; Lamour et al., 1989). In normal aging the number of vestibular hair cells and VOR function has been reported to be reduced (Chang et al., 2012; Rauch et al., 2001; Rosenhall and Rubin,
1975), although recently this idea has been challenged for the VORs (McGarvie et al., 2015). The decrease in vestibular function with age may apply more to otolithic function (see Smith, 2016 for review). Cholinergic transmission also shows a progressive loss with normal aging (Sarter and
Bruno, 2004). The combination of vestibular and cholinergic degradation in aging may also lead to the spatial and cognitive deficits seen in elderly individuals that are not related to dementia
(Klencklen et al., 2012). The decrease in type 2 atropine-sensitive theta with age is likely linked to the loss of cholinergic transmission. The present experiment found that BVL in young animals produced an increase in type 2 theta amplitude when stimulated with a tail pinch. It is possible that
106 an increase in cholinergic transmission is potentiating theta generation, as hippocampal ACh release is correlated largely with theta amplitude (Monmaur et al., 1997).
While electrode placement in the PPT was not accurate in most animals, the increase in theta at 3-6 Hz was also present in the PPT of a single BVL rat (Figure 6.5). Neither the hippocampus or PPT exhibited changes in the frequency of the maximum power (Fmax) between sham and vestibular-lesioned animals. The increase in type 2 theta power following BVL may suggest a compensatory mechanism, where non-vestibular stimuli produce a larger than normal theta amplitude following the loss of the peripheral vestibular sensory system. That this increased response appears to occur within the PPT and due to the PPT’s role in sensory and motor integration (Winn, 2008), it is likely that the compensation occurs at this level of the theta- generating pathway. Increased PPT activity is consistent with the hypothesis that cholinergic transmission is increased in the type 2 theta-generating system following BVL.
In conclusion, BVL produces compensatory changes in the theta-generating pathway, leading to an increase in theta power during non-vestibular stimulation. This increase in theta is restricted to type 2 theta generated under urethane anaesthesia and therefore may be due to an increase in cholinergic transmission. Type 1 theta has previously been found to decrease in power in awake animals with BVL. This is further evidence of the substantially different roles of each type of hippocampal theta rhythm.
107 7 CHOLINERGIC CELLS IN THE PPT FOLLOWING BVL
INTRODUCTION The PPT is a group of mainly cholinergic neurons in the ventral mesencephalic tegmentum
(Armstrong et al., 1983; Mesulam et al., 1983; Rye et al., 1987; Spann and Grofova, 1992). The rostral PPT is interconnected with the basal ganglia (Figure 7.1) and is largely inhibitory, containing a large proportion of GABAergic neurons (Martinez-Gonzalez et al., 2011). Non- cholinergic projections from the rostral PPT are a major source of innervation to the substantia nigra, while cholinergic projections are thought to be involved in other pathways such as the reticular activating system (Lee et al., 1988). The caudal PPT is considered to be largely excitatory, containing mostly cholinergic and glutamatergic neurons, and is related to arousal and motor systems. Projections of both cholinergic and non-cholinergic neurons between the rostral and caudal areas are involved in regulating their respective functions (Martinez-Gonzalez et al., 2011).
108
Figure 7.1: Summary of the topographical distribution of the connectivity in the PPT. The rostral PPT, which is predominantly GABAergic, maintains interconnections with the GABAergic output of the basal ganglia. In contrast, the caudal PPT, where cholinergic and glutamatergic neurons are more abundant, receives input from the cortex and dorsal raphé and projects to the thalamocortical systems, STN and locomotor regions. EP, entopeduncular nucleus; GPi, internal segment of the globus pallidus; IC, inferior colliculus; SC, superior colliculus; SN, substantia nigra; STN, subthalamic nucleus; VTA, ventral tegmental area. Figure from Martinez-Gonzalez et al. (2011). Copyright 2011 Martinez-Gonzalez, Bolam and Mena-Segovia, open access.
Cholinergic neurons in the PPT are inhibited in vitro by ACh through M2 mAChRs
(Leonard and Llinas, 1994). PPT cholinergic neurons project to the RPO and were originally thought to tonically inhibit theta-producing neurons in the RPO (Kinney et al., 1998; Vertes et al.,
1993). Local injections of carbachol, a cholinergic agonist, were thought to hyperpolarize the PPT cholinergic neurons, inhibiting their firing and generating theta through disinhibition of the RPO.
However in the same studies, cholinergic activation of the RPO by carbachol induced, rather than inhibited, theta (Kinney et al., 1998; Vertes et al., 1993). Lesions to the PPT, both selective to cholinergic cells and non-selective, disrupt theta (Bringmann, 1995; Bringmann, 1997; Jones,
1991; Jurkowlaniec et al., 2003; Nowacka et al., 2002; Orzel-Gryglewska et al., 2006). PPT stimulation has also been shown to induce long-lasting neuronal activation in the RPO neurons
(Garcia-Rill et al., 2001). PPT stimulation activates type I RPO neurons while inhibiting type II
RPO neurons, and these effects are cholinergically-mediated through muscarinic receptors (Nunez
109 et al., 2002). The PPT has two types of cholinergic cells, small neurons with short, high frequency firing and medium-large neurons with long, low frequency firing (Good et al., 2007; Takakusaki et al., 1997). Multiple mAChRs are located on the PPT cholinergic neurons: M2, M3 and M4 have all been detected (Vilaro et al., 1994). Excitatory responses are likely mediated by the M3 receptor
(Flores et al., 1996). Nicotinic receptors are also present in PPT cholinergic neurons, producing excitatory effects in early development and inhibitory effects in adults (Good et al., 2007).
Glutamate or GABA agonists injected into the PPT suppress sensory-elicited theta. Administration of glutamate or GABA antagonists enhances theta. Therefore, both excitatory (glutamate) and inhibitory (GABA) receptors in the PPT paradoxically produce the same effect when activated or blocked (Nowacka and Trojniar, 2000). Serotonin and opioid agonists also produce enhanced theta when injected into the PPT locally, hyperpolarizing PPT cholinergic cells (Kobayashi et al., 2003;
Leonard and Llinas, 1994; Leszkowicz et al., 2007; Matulewicz et al., 2010). The level of activation of the PPT likely determines whether an excitatory or an inhibitory response is produced in the cholinergic cell types. The M2/4 mediated auto-inhibition inhibits theta in response to high levels of ACh while more moderate levels would likely produce theta (Leszkowicz et al., 2007).
The theta-generating pathway, as described in Chapter 1, although necessary for the generation and modulation of theta, can be influenced by other parallel pathways. The ventral tegmental area (VTA) is thought to play a role in the modulation of theta, providing dopaminergic projections to the MS (Fitch et al., 2006; Orzel-Gryglewska et al., 2015). The VTA receives cholinergic, GABAergic and glutamatergic input from the PPT, LDT and RPO (see Orzel-
Gryglewska et al., 2015 for review). However, the VTA does not appear to respond to vestibular stimulation (Rancz et al., 2015) although there are currently no studies which assess this in depth.
The PPT is considered important in theta generation (Orzel-Gryglewska et al., 2015) and is responsive to vestibular stimulation (Aravamuthan and Angelaki, 2012). Direct PPT stimulation improves balance in PD patients (Yousif et al., 2016). Changes which occur in the PPT following vestibular loss are not well documented. Cholinergic PPT neurons are necessary to produce theta,
110 especially type 2 theta, therefore it is of interest to determine if these cells increase or decrease in number following vestibular damage. As seen in Chapter 6, an increase in type 2 theta amplitude is observed in BVL rats, therefore it is possible that PPT cholinergic neurons have increased activity following BVL, which is driving this response.
Therefore, this experiment aimed to quantify and compare the number of cholinergic neurons in the PPT between sham, BVL and UVL rats. Cholinergic cells in the PPT can be stained using either NADPH-d histology or ChAT immunohistochemistry. NADPH-d stains nitric oxide
(NO) synthase-containing cells, while ChAT is involved in the synthesis of ACh (Lolova et al.,
1996). NO stimulates ACh release (Prast and Philippu, 1992) and NADPH-d is therefore largely colocalized with ChAT-containing cells in the PPT (Lolova et al., 1996); however there is a small proportion of PPT ChAT-positive cells which are not labelled by NADPH-d (Dun et al., 1994;
Lolova et al., 1996). Therefore, NADPH-d was used to identify the sections of each brain containing the PPT and ChAT immunohistochemistry was subsequently used to identify and count cholinergic neurons in the PPT.
111 METHODS The brain tissue from Chapter 6 was processed as detailed previously. Alternate sections were stained for either NADPH-d or ChAT immunohistochemistry. ChAT immunohistochemistry was performed on the remaining sections once the location of the PPT had been determined from the NADPH-d stained tissue. Initial optimisation tested ChAT binding without use of an antigen retrieval method, however this was unsuccessful. Therefore, sections underwent antigen retrieval using citric acid (10mM, pH 6) at 90°C for 10 min. Once cooled, normal donkey serum (5%,
Sigma) in 1% BSA/PBS solution was used for blocking. Sections were incubated in ChAT goat polyclonal antibody (1:200, Merck Millipore, AB144P) for 72 h. The antibody concentration and length of incubation were determined prior to the experiment through multiple optimisation steps, testing a range of 1:100 – 1:750 with either 48 or 72 h incubations. Following primary antibody incubation, sections were incubated in donkey anti-goat IgG-HRP (1:200, Santa Cruz, sc-2020) for 1 h. Sections were visualised using a DAB (3,3’-diaminobenzidine) Peroxidase (HRP) substrate kit (Vector Labs) for 10 min. Following washing with distilled water, methyl green
(0.5%) in 0.1M sodium acetate (pH 4.2) was added to each section for 5 min at room temperature to counterstain the nuclei. Sections were rinsed with distilled water, mounted on slides, dehydrated through graded alcohols (70–100%), cleared in xylene and cover-slipped. ChAT-positive cell counting was performed using Stereo Investigator software (version 10, MBF Bioscience) on an
Olympus BX51 light microscope. The nuclei of ChAT-positive cells were counted using an optical fractionator stereological method (Figure 7.2) (Keuker et al., 2001; West et al., 1991). The optical fractionator method estimates the total number of cells from an unbiased subpopulation of cells sampled with systematic random sampling. Each section is counted in the X, Y, and Z axes using the fractionator principle. Estimates for the total number of cells within the entire counting region can be determined by extrapolating the sampled subset of cells within the area of the tissue using the fractionator formula (Equation 7.1) (MBF-Bioscience, 2016).
112
Equation 7.1: Fractionator formula.
X represents the quantity of cells. The X with the caret is the estimate of X. The lowercase x is the value measured from the sample. f is the fraction of cells sampled.
The PPT boundary was delineated with a 10x objective. Optimisation of the stereological counting method was performed initially. All counting was performed with the 100x objective.
Initial testing used a counting frame of 25 x 25 µm, grid size of 65 x 85 µm, disector height of 15
µm, and a 20% guard zone; however, this counted an insufficient number of cells per section. A second testing paradigm used a counting frame of 20 x 20 µm, grid size of 65 x 65 µm, disector height of 15 µm, and a 20% guard zone, although once again this counted an insufficient number of cells per section. Finally, a counting frame of 50 x 50 µm, grid size of 80 x 100 µm, disector height of 15 µm, and a 20% guard zone was determined to be optimal. Counting of BVL (n = 5 rats) and sham (n = 5 rats) PPT cells was performed on both hemispheres; a single hemisphere from each animal was selected pseudo-randomly for counting, with an equal number of left (n =
3) and right (n = 2) hemispheres between groups. Both hemispheres of UVL rats (n = 4) were counted and separated into the contralateral or ipsilateral side to the lesion (Figure 7.2). While
ChAT is present in both neurons and glia, the size difference between cholinergic PPT neurons
(20 µm) and astrocytes (1 µm) is significant (Figure 7.3; Kovacs et al., 2016). Only neurons with soma diameter ≥ 15 µm were counted.
Statistical Analysis
As UVL animals had repeated measures with each side (contralateral and ipsilateral) counted separately, while sham and BVL animals did not have separate sides and therefore no repeated measures, statistical analysis was performed with two separate one way ANOVAs with a
113 Bonferroni post-hoc test. A paired Student’s t-test was used for comparison between the UVL hemispheres.
Figure 7.2: Stereological counting frame over cells in the PPT. Nuclei of ChAT-positive cells within or touching the green lines were counted. Any nuclei touching the red lines were not counted. The pictured cell satisfied the criteria for counting. The light blue line is the border of the counting field, cells outside the border were not counted. Arrowhead = Nuclei of ChAT-positive cell. Brown staining is DAB ChAT-positive. Blue staining is methyl green.
Figure 7.3: Size difference between PPT cholinergic neurons and astrocytes. Green = Astrocytes labelled with glial fibrillary acidic protein (GFAP). Blue = ChAT-stained neuron. Scale bar = 10 µm. Image from Kovacs et al. (2016). Copyright Springer, used with permission.
114 RESULTS ChAT immunostaining was specific for cholinergic neurons in the PPT (Figure 7.4).
Stained neurons had a diameter of 10-20 µm and the nuclei were stained with methyl green (Figure
7.2; Figure 7.4). ChAT staining was also present in the striatum, MS, hypothalamus, LDT, facial nucleus and motor trigeminal nucleus. ChAT stained the cell body and axon fibres, and dendrites were occasionally visible (Figure 7.2 and 7.4).
Figure 7.4: ChAT and methyl green staining in the PPT. ChAT stained cholinergic neurons with DAB (brown) while methyl green stained all nuclei (blue/green). a) A low magnification photograph showing the location of the ChAT-positive cells within the PPTg (black arrowhead) and motor trigeminal nucleus (red arrowhead). b) A high magnification photograph showing the specificity of the ChAT staining. Examples of ChAT staining in c) Sham, d) UVLI, e) UVLC and f) BVL. The number of ChAT-positive cells in the PPT of vestibular-lesioned animals differed depending on the side of the lesion (Figure 7.5). BVL animals exhibited significantly more ChAT- positive cells than sham animals (P ≤ 0.05; t(11) = 3.4). The contralateral PPT of UVL animals
(UVLC) also showed significantly more ChAT-positive cells than sham animals (P ≤ 0.05; t(11)
= 2.9). There was no difference between BVL and UVLC cell numbers (P ≥ 0.99; t(11) = 0.29).
ChAT-positive cells on the ipsilateral side of UVL animals (UVLI) were not significantly different
115 from sham (P ≥ 0.99; t(11) = 0.52) but were different from both UVLC (P ≤ 0.01; t(3) = 8.8) and
BVL animals (P ≤ 0.01; t(11) = 3.76).
2 5 0 0
s l
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Figure 7.5: The number of cholinergic PPT cells following vestibular lesions. The side ipsilateral to the lesion in UVL animals was not significantly different to sham animals. The contralateral side of UVL animals showed a significantly increased number of ChAT-positive cells. The increase in cells was also seen across BVL sections. Significantly different from sham * P ≤ 0.05. Significantly different from UVLI # P ≤ 0.05. Error bars display the SEM.
116 DISCUSSION Vestibular lesions produce a significant increase in ChAT-positive cholinergic cells in the
PPT (Figure 7.5). This increase of approximately 80% occurred contralateral to the side of the lesion, with BVL rats exhibiting an increase in cells on either side. Vestibular input to cortical and limbic brain areas is usually bilateral or contralateral (Dieterich et al., 2003; Hitier et al., 2014;
Lopez and Blanke, 2011). The increase in cholinergic cells may be due to the generation of new cholinergic neurons. Only large cells were counted and although specific labelling for neurons was not used, due to the significant size difference between neurons and glia in the PPT, it is certain that these cells are neurons (Figure 7.3). In humans, the number of PPT ChAT-positive neurons decreases with age; however, centenarians have an increased number compared to people aged 70-
91, resulting in a U-shaped curve (Ransmayr et al., 2000). Neurogenesis in adults has not been reported previously in the PPT and was not examined in the present study. As there is no evidence for or against PPT neurogenesis due to a total lack of studies, it can only be speculated that the increase in ChAT-positive cells following vestibular loss may due to neurogenesis. Future studies should therefore consider PPT neurogenesis a possibility following vestibular loss. The PPT has three identified neuronal cell types, determined through electrophysiological recordings and immunohistochemistry. Type I are non-cholinergic neurons with low threshold spikes (LTS), type
II large cholinergic neurons with a transient outward current (A current) and type III small cholinergic neurons which have the properties of both type I and II (A current + LTS) (Good et al., 2007; Kang and Kitai, 1990; Kobayashi et al., 2004; Kobayashi et al., 2003; Takakusaki and
Kitai, 1997; Takakusaki et al., 1997). Approximately 60% of type II cells are ChAT-positive, while 30% of type III cells are ChAT-positive (Takakusaki et al., 1997). Type III neurons appear to differentiate into either type I or type II neurons with age (Kobayashi et al., 2004; Kobayashi et al., 2003). It is possible that this is the same mechanism which produces an increase in ChAT- positive cells following vestibular loss. There is also the possibility that type II neurons are increasing in cholinergic activity, with previously non-cholinergic cells (40%) in this population
117 increasing ChAT activity to levels detectable with immunohistochemistry. In septal cholinergic cells, nerve growth factor (NGF) increases the number of ChAT-positive cells (Gnahn et al., 1983;
Knusel and Hefti, 1988). However, NGF does not have the same effect on PPT cells and the factor required for the development and differentiation of PPT cholinergic cells is unknown (Knusel and
Hefti, 1988). An increase in ChAT-positive neurons suggests an increased capacity for ACh production and release. Cholinergic activation of the RPO by carbachol enhances theta, with the
PPT being a major source of intrinsic ACh for the RPO (Kinney et al., 1998; Vertes et al., 1993).
An increase in ACh from the PPT to the RPO might therefore enhance type 2 theta as seen in
Chapter 6 following BVL.
The PPT has a significant role in rapid eye movement (REM) sleep and wakefulness
(Alessandro et al., 2010; Datta and Siwek, 1997; Hernandez-Chan et al., 2011; Petrovic et al.,
2013). During REM sleep, type 2 theta is produced by the PPT and LDT cholinergic neurons
(Brown et al., 2012; Datta and Siwek, 1997; Leonard and Llinas, 1994). REM sleep is considered necessary to memory consolidation and hippocampal LTP (Ackermann and Rasch, 2014; Boyce et al., 2016; Fogel et al., 2007; Ravassard et al., 2016; Walker and Stickgold, 2006). During development an increase in cholinergic PPT activity is linked to REM disturbances (Kobayashi et al., 2004). Therefore, a permanent increase in PPT cholinergic activity may also inhibit REM sleep and reduce type 2 theta activity, leading to associated memory deficits. However, this hypothesis seemingly contradicts selective PPT lesion and stimulation studies. Type 2 theta is suppressed by lesioning the PPT (Nowacka et al., 2002) while carbachol PPT microinjections elicit theta (Kinney et al., 1998; Vertes et al., 1993). Lesions of the PPT, either selective for cholinergic neurons or complete, disrupt learning and memory in spatial and emotional tasks (Dellu et al., 1991; Florio et al., 1999; Fujimoto et al., 1992; Inglis et al., 2000; Inglis et al., 2001; Keating et al., 2002;
Keating and Winn, 2002; Leri and Franklin, 1998; Satorra-Marin et al., 2005; Taylor et al., 2004).
Additionally, bilateral deep brain stimulation of the PPT has been shown to reduce reaction times in working memory and recall tasks in human patients (Alessandro et al., 2010; Costa et al., 2010;
118 Stefani et al., 2010). These studies demonstrate the involvement of the PPT in REM sleep, theta generation, and spatial learning and memory.
The VNC has also been implicated in REM sleep regulation. Bilateral lesions of the vestibular nuclei abolish REM theta activity (Perenin et al., 1972; Pompeiano and Morrison, 1965;
Seguin et al., 1973). However, there are no studies which assess REM sleep following surgical or chemical BVL.
In conclusion, vestibular lesions produce an increase in ChAT-positive cholinergic neurons in the PPT bilaterally following BVL or on the contralateral side following UVL. This increase was most likely due to a change in type II and type III PPT cell function, with ChAT increasing to detectable levels in the majority of the cell population. Type III cells may also be differentiating into type II cells as previously observed (Kobayashi et al., 2004; Kobayashi et al., 2003). Memory deficits following vestibular loss may be due to increased ChAT neurons in the PPT, although this is difficult to reconcile with PPT lesion studies. It is possible that both increased and decreased cholinergic activity in the PPT will cause learning and memory disruption through alterations to type 2 theta and REM sleep. Further studies are needed to determine the exact mechanism which leads to the increase in PPT ChAT neurons following BVL and UVLC and also what effect increased PPT cholinergic neuron activity has on REM sleep and memory consolidation.
119 8 ACETYLCHOLINE RELEASE IN THE HIPPOCAMPUS IS NOT ALTERED FOLLOWING BILATERAL VESTIBULAR LESIONS
INTRODUCTION ACh release in the hippocampus increases during sensory stimulation (visual, auditory, tactile, olfactory and vestibular) and locomotor activity (Dudar et al., 1979; Horii et al., 1994;
Inglis and Fibiger, 1995). The release of ACh in the hippocampus is positively correlated to the amplitude of theta (Monmaur et al., 1997). Theta amplitude also increases during locomotion and sensory stimulation (Bland and Oddie, 2001). Lesioning the cholinergic projections from the MS
(Yoder and Pang, 2005) or brainstem will disrupt theta (Bringmann, 1995; Bringmann, 1997;
Jones, 1991; Jurkowlaniec et al., 2003; Nowacka et al., 2002; Orzel-Gryglewska et al., 2006). This illustrates the important role ACh has in maintaining hippocampal theta rhythm. Theta enhances learning and memory by producing synchronised firing, leading to LTP (Greenstein et al., 1988;
Larson et al., 1986; Pavlides et al., 1988; Vertes, 2005). ACh has also been found to enhance LTP in hippocampal synapses independently of theta (Adams et al., 2004; Huerta and Lisman, 1995).
Disrupting the development of LTP would impair memory encoding but not retrieval, as seen when mAChRs are blocked with scopolamine (Atri et al., 2004; Rasch et al., 2006). A reduction in hippocampal ACh would result in learning and memory deficits, similar to those that occur following BVL (Baek et al., 2010; Besnard et al., 2012; Brandt et al., 2005; Chapuis et al., 1992;
Dallal et al., 2015; Guidetti et al., 2008; Horn et al., 1981; Hufner et al., 2007; Machado et al.,
2012a; Matthews et al., 1989; Neo et al., 2012; Ossenkopp and Hargreaves, 1993; Russell et al.,
2003a; Russell et al., 2003b; Schaeppi et al., 1991; Schautzer et al., 2003; Smith et al., 2013;
Stackman and Herbert, 2002; Wallace et al., 2002; Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2004; Zheng et al., 2006; Zheng et al., 2007; Zheng et al., 2009b).
120 Microdialysis is a technique that can collect analytes, such as ACh, from the extracellular space in vivo. A microdialysis probe implanted in the tissue has artificial cerebrospinal fluid
(aCSF) pumped through it. Neurotransmitters pass between the extracellular space and the probe membrane freely and can be collected for analysis. Microdialysis allows for the retrieval of ACh without the use of acetylcholinesterase inhibitors. This allows for a precise measurement of ACh release during the collection window. ACh is rapidly broken down in the synaptic cleft by acetylcholinesterase, therefore neostigmine or physostigmine is often utilised in the microdialysis perfusate to increase the concentration of ACh collected. The addition of acetylcholinesterase inhibitors can produce large changes to the cholinergic system, altering drug effects and pathological states (de Boer et al., 1990; Ichikawa et al., 2000; Vinson and Justice, 1997).
Detection of ACh in the collected sample needs to be sensitive enough to detect the low levels present without the use of acetylcholinesterase inhibitors. High-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) utilises a solid phase reactor (SPR) to convert ACh into H2O2 allowing for detection levels as low as 5 fmol (EiCOM,
2016; ESA, 2015; Ichikawa et al., 2000).
The present experiment aimed to collect ACh-containing microdialysis samples from the hippocampus of urethane-anaesthetized BVL and sham rats while recording local field potentials.
ACh release was measured in response to tactile (tail pinch) and vestibular stimulation (horizontal rotations). It was hypothesised that ACh release would be increased during tail pinch stimulation in BVL rats compared to sham animals, consistent with the increased theta amplitude seen in
Chapter 6. ACh release was expected to decrease during vestibular stimulation compared to sham animals, as BVL animals lack vestibular sensory signals. It was also hypothesised that baseline
ACh release would be decreased in BVL animals due to increased M2/4 hippocampal autoreceptors as seen in Chapter 5 and previous evidence of spatial learning disruption. Temporary lesioning of the ipsilateral PPT was performed to examine the role of the PPT in hippocampal ACh release. It
121 was hypothesised that PPT lesions would significantly reduce ACh release during both tactile and vestibular stimulation in both groups of animals.
122 METHODS Thirteen male Wistar rats (250–300 g) underwent sodium arsanilate BVL or sham injections as described in Chapter 2 (Sham: n = 6; BVL: n = 7). Behavioural scores were assessed at day 1, 3, 7 and 30 post-tympanic injection.
Hippocampal Recording and Microdialysis
Thirty days post-BVL, animals were anaesthetised with 1.5 mg/kg urethane and placed in a stereotaxic frame. Xylocaine (with 1:100,000 adrenaline) was injected along the scalp and an incision made. A probe cannula and recording electrode (stainless steel, insulated apart from the flat cut tip) were implanted within the hippocampus at 4 mm posterior to bregma, 2 mm lateral to the midline and 2.5 mm below the dura and cemented into place. The electrode was twisted around the cannula with the electrode tip projecting 1 mm below the base of the cannula (Figure 8.1). An injection cannula was inserted into the PPT for xylocaine administration, 8 mm posterior to bregma, 2 mm lateral to the midline, and 7.5 mm below the dura.
Figure 8.1: Diagram of microdialysis probe, cannula and electrode. An insulated electrode (yellow line) was twisted around a microdialysis cannula (grey/beige). The electrode projected 1 mm below the base of the cannula. Once the cannula was implanted within the hippocampus, the microdialysis probe (blue) was inserted into the cannula and the membrane extended 2 mm from the cannula tip. Artificial cerebrospinal fluid was perfused through the probe (arrows) and the resulting perfusate was collected for later analysis.
123 A Ag/AgCl ground electrode (Warner Instruments) was placed under the skin of the animal’s neck. LFP activity was digitized at 600 Hz after being amplified (1000x) and band-pass filtered (0.1–200 Hz) using a dedicated ac-differential amplifier and head stage (Neurolog,
Digitimer). The microdialysis probe (CMA12, 2 mm membrane) was perfused with aCSF (125 mM NaCl, 3 mM KCl, 0.75 mM MgSO4 and 1.2 mM CaCl2, pH 7.4) and inserted into the probe cannula. A flow rate of 1 µl/min was used as it was the lowest rate which could be managed within the recording timeframe while collecting a sufficient sample size for analysis. Flow rate has an inverse relationship to dialysate concentration, therefore low flow rates are optimal (Chefer et al.,
2009). Sample collection began following equilibration, 1.5-2 h after probe insertion. Samples were taken over 20 min periods (Figure 8.2). During tail pinch periods a plastic clip was placed on the base of the animal’s tail. During vestibular stimulation periods, the animal was rotated on an orbital shaker at 70 RPM. Both tail pinch and shaker periods were repeated following xylocaine administration to the PPT. At the beginning of collection three baseline samples were collected.
Further baseline samples were collected between each experimental period. Following the first shaker stimulation, xylocaine (0.5 µl) was injected into the PPT through a cannula implanted earlier at a flow rate of 0.1 µl/min for 5 min. Xylocaine administration was repeated every 20 min until the washout period. Two samples were taken following recovery from the xylocaine injections followed by a final tail pinch collection sample. Electrophysiological recordings were analysed by Fast Fourier Transform (Spike 2, CED) to determine the frequency and amplitude of theta rhythm over time. Microdialysis samples were frozen at -80° C and later analysed by HPLC-
ECD.
124
Figure 8.2: Timeline of microdialysis sampling. The microdialysis probe was inserted into the hippocampus of a urethane-anaesthetised rat. The timeline is shown in min following probe insertion. Artificial cerebrospinal fluid was perfused through the probe for 120 min to equilibrate. Samples were taken every 20 min following equilibration.
Histology
Following recording animals were decapitated and the brains preserved in formalin (10%).
Sections (50 µm) were cut on a microtome and stained with NADPH-d to determine the cannula and probe placement. NADPH-d staining was performed as described by Hernandez-Chan et al.
(2011). Sections were placed in a 24-well plate and incubated for 3 h at 37° C in 0.1 M Tris–HCl buffer (pH 8.0), containing 0.6 mM β-nicotinamide adenine dinucleotide phosphate, reduced form
(Alfa Aesar), 0.81 mM nitro blue tetrazolium (Alfa Aesar), 0.3% Triton X-100 (Sigma), and 2% ethanol. After staining slices were mounted onto gelatinized slides and allowed to dry. Sections were dehydrated through graded alcohols (70–100%), cleared in xylene and cover-slipped.
HPLC-ECD
HPLC separation and electrochemical detection of ACh was performed using a Coulochem
III ECD with a 5011A platinum cell electrode (E1 = -100 mV, E2 = +600 mV). A Capcell Pak
MGII C18 column (75 × 1.5mm, 3 µm ø) coupled in series to a Thermo Scientific Dionex ACh-
SPR was kept at 35° C in a column heater (Figure 8.3). The mobile phase (100 mM H2PO4, 0.8 mM 1-octanesulfonic acid, pH 8.0 with phosphoric acid) was run through the system with a flow rate of 0.2 mL/min. A standard curve was created using known concentrations (1 nM - 10 µM) of
ACh chloride (Sigma Aldrich). Samples were then analysed using an injection volume of 15 µL.
One µM ACh chloride standards and blank aCSF were analysed between every 13 samples to
125 check for degradation of the system or contaminants. Butyrylcholine chloride was tested as a potential internal standard as butyrylcholine had been successfully used previously in ACh-SPR columns (Kehr et al., 1998). However, with the present system, butyrylcholine chloride and ACh had very similar, and often overlapping retention times, therefore the HPLC-ECD analysis proceeded without an internal standard.
Figure 8.3: Schematic of HPLC-ECD. The ACh-SPR contains two immobilized enzymes. Acetylcholinesterase (AChE) breaks down ACh to choline. Then choline oxidase converts choline to hydrogen peroxide. Hydrogen peroxide is detected using a platinum (5011A) electrode at +600 mV. Figure adapted from Damsma et al. (1987). Copyright Springer, used with permission.
Probe Recovery
Recovery of the probe was measured to correct for individual differences between probe membrane permeability and degradation of the probes between experiments. Microdialysis probes were immersed in 1 µM ACh chloride in aCSF solution and were perfused with aCSF at 0.5, 1, 2 and 4 µl/min at room temperature. Duplicate samples of 20 µl were collected at each flow rate prior to the use of the probe in a rat. Samples were kept on ice during collection and stored at -80°
126 C prior to analysis. The ACh concentration in recovery samples was detected using HPLC-ECD as described above. Recovery was expressed as a proportion of the initial 1 µM concentration.
Experimental samples were normalised to probe recovery.
Statistical Analysis
Analysis of the hippocampal EEG signal was performed off-line in Spike 2 (CED). Fast
Fourier Transformation, using overlapping Hanning windows (0.07 Hz), was calculated for 10 artefact free 15 s samples randomly chosen from baseline LIA activity and from 10 theta rhythm episodes 5 s following application of the tail clip for 15 s. The mean power as a percentage of baseline was calculated for the peak amplitude at five 3 Hz frequency bands (0-3, 3-6, 6-9, 9-12,
12-15 Hz). The mean frequency of the peak amplitude within each band was also calculated
(Fmax). A linear mixed model (LMM) analysis was performed on the electrophysiological power and Fmax data, with the treatment as a between-group factor and the frequency band as a repeated measure. The covariance structure with the lowest Akaike’s information criterion (AIC) was selected (McCulloch et al., 2008). The LMM was used in preference to a repeated measures
ANOVA due to the repeated measures and the potential for correlation in them to invalidate the
ANOVA assumption of sphericity (Smith, 2012). The behavioural scores were analysed using
Mood’s median test due to the non-parametric nature of the data (Sprent and Smeeton, 2007).
HPLC-ECD data were analysed using Chromeleon software (7.2, Dionex). The recovery of each probe was calculated and the final data were determined using the individual recovery profile for that trial. Data were analysed with the LMM analysis using the same method as the electrophysiological data.
127 RESULTS
Vestibular Deficiency
All sham animals were found to have no vestibular deficiency indicated by a score of zero at all days assessed (Table 8.1). A total of 7 BVL and 6 sham animals were assessed. BVL rats expressed strong vestibular syndromes at 1, 3, 7 and 30 days following transtympanic chemical lesion compared to the sham group (χ2=41, P ≤ 0.0001). The behavioural effects of the lesions were most severe at day 3, with a decrease in behavioural scores seen by day 30 (χ2=10.10, P ≤
0.05).
Table 8.1: Behavioural scores of animals prior to electrophysiological recording Day 1 Day 3 Day 7 Day 30
Sham 0 (0,0) 0 (0,0) 0 (0,0) 0 (0,0)
BVL 15 (12,17) 18 (11,22) 14 (3,17) 9 (3,12)
Vestibular loss was reflected by a strong bilateral vestibular syndrome in the BVL group at day 1, 3 7 and 30 compared to the sham group (P ≤ 0.0001). Data are expressed as median (range) with the maximum possible score being 24.
Electrode Placement
Microdialysis cannula tips were predominantly located within the dentate gyrus close to the base of the hippocampus (Figure 6.1A). PPT cannulae were located throughout the PPT and the surrounding tissue (Figure 6.1B). Cannulae located outside the PPT were removed from the analysis during xylocaine injection time points.
128 A B
C D
Figure 8.4: Probe and cannula tip locations. A) Hippocampal microdialysis probe tip placement. Sagittal view 2 mm lateral from Bregma. Red = BVL, Green = Sham. B) Section with hippocampal microdialysis probe track (red line), scale bar = 500 µm. C) PPT cannula placement. Sagittal view at 2 mm lateral from Bregma. Purple area is the PPT. Red = BVL, Green = Sham. D) Section with PPT cannula track (red line), scale bar = 500 µm.
129 Hippocampal Electrophysiology
Tail pinch manipulation did not produce consistent increases in the theta frequency as produced in Chapter 6 (Figure 8.5).
A A B B
Figure 8.5: Hippocampal response to tail pinch during urethane anaesthesia. A) Spectrogram and raw electrophysiological trace. Immediately after the tail pinch manipulation (red line) the amplitude decreased but there was no significant change in the frequency. B) A histogram FFT of the baseline data overlaid with data taken during the tail pinch. There was no shift in the theta range following application of the tail pinch.
An LMM analysis showed a significant effect in the maximum power related to the frequency band (Figure 8.6; Frequency band: F (4,53.8) = 19.2, P ≤ 0.0001; Treatment*Frequency band: F (4,53.8) = 2.9, P ≤ 0.05). Treatment (BVL, sham) and type of manipulation (tail pinch, shaker, xylocaine injection) did not have significant effects (P > 0.05). The frequency corresponding to the maximal peak power (Fmax) was not significantly different between the treatment groups (P > 0.05)
130 A B
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Figure 8.6: Hippocampal power distribution within frequency bands. FFT analysis determined the maximum power within five frequency bands. A) Response to tail pinch stimulation. B) Response to orbital shaker stimulation. C) Tail pinch stimulation following xylocaine lesioning of the PPT. D) Orbital shaker stimulation following xylocaine lesioning of the PPT. E) Tail pinch stimulation following recovery of xylocaine PPT lesions. Power is expressed as mean percentage of baseline control. Error bars are SEM.
HPLC-ECD Analysis
HPLC-ECD analysis of both ACh standards and experimental samples produced clear detection peaks with a retention time ~8 min (Figure 8.7). The limit of detection was 5 nM in a 5
131 µl injection volume (25 fmol). ACh standards produced a linear concentration curve (Figure 8.8; r2 = 0.99).
A B
C
Figure 8.7: HPLC-ECD output. ACh was detected at 8 ± 1 min. The injection artefact was detected at ~2 min. A) A chromatography trace of a 1 µM ACh chloride standard. B) A chromatography trace of a 10 nM ACh chloride standard. C) A chromatography trace of a tissue sample and detection of ACh at 8.094 min. The peak at 11.820 min is unknown.
132
Figure 8.8: Standard curve of known concentrations of ACh chloride.
Microdialysis recovery samples exhibited a non-linear decrease in recovered ACh concentration as flow rate increased (Figure 8.9). Each probe had a different recovery rate which was corrected for in the experimental data (Figure 8.10).
Figure 8.9: Relative recovery of microdialysis probes. Probe recovery decreased as flow rate (µl/min) increased.
HPLC-ECD analysis of microdialysis samples showed no significant effects of BVL or tail pinch and orbital shaker manipulations on hippocampal ACh release (Figure 8.10; Treatment: F
(1,9) = 3.0, P > 0.05; Manipulation: F (10,48) = 1.1, P > 0.05; Treatment*Manipulation: F (10,48)
133 = 1.5, P > 0.05). BVL rats generally showed lower ACh release than sham rats (Figure 8.10).
However, this difference was not statistically significant (Treatment: F (1,9) = 3.0, P > 0.05).
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x x x Figure 8.10: Acetylcholine release in BVL and sham rats. BVL rats had, on average, less ACh release compared to sham rats; however, this difference was not statistically significant. Error bars = SEM. (Xylo = during temporary xylocaine lesion of the PPT). Sham: n = 5; BVL: n = 6.
134 DISCUSSION No significant effects of BVL on ACh release were found following analysis of microdialysis samples. While ACh could be measured at normal hippocampal concentrations without the addition of acetylcholinesterase inhibitors, there was no change due to either BVL or in response to tail pinch or shaker manipulations. The limit of detection of 25 fmol was the same as a previous ACh microdialysis study utilising HPLC-ECD (Wise et al., 2002). The concentration of ACh released in the hippocampus was also similar to previous studies (1-4 pmol/min) (Mizuno et al., 1991). The lack of change during manipulations is likely due to habituation throughout the
20 min collection period. This is a problem with the temporal resolution of microdialysis. If samples could be collected and analysed during 30-60 s following stimulus onset, a clearer picture of ACh release during the stimulus could be obtained. Samples collected under 60 s using current microdialysis techniques would return samples either too small to be analysed by HPLC-ECD (1
µl) or ACh concentrations below the limit of detection (< 1 nM). One finding not affected by the limits of the microdialysis technique is the baseline measurements. BVL rats did not show any significant changes in ACh release during baseline periods under urethane anaesthesia compared to sham rats.
Increases in theta rhythm in response to a tail pinch following BVL were not replicated as seen in Chapter 6. Orbital shaker stimulation also failed to produce theta in the EEG trace. This is likely due to poor electrode placement. Electrodes were attached to the cannula and the microdialysis probe projected a further 2 mm. This would place the electrode in the cortex or upper layers of the hippocampus rather than in CA1 and the dentate gyrus as they were previously. Future experiments should record from a second part of the hippocampus to confirm theta. It is also possible that the tail pinch did not effectively elicit theta in this experiment, although anaesthesia dose and the tail clip were identical. A failure to elicit theta with the tail pinch would be reflected in the lack of change in ACh release. As mentioned previously, the temporal resolution of the
135 microdialysis protocol was likely responsible for the lack of change to ACh release following tail pinch manipulation.
ACh release in the hippocampus increases in awake animals during vestibular stimulation
(Horii et al., 1994). It was therefore expected that orbital shaker stimulation would increase hippocampal ACh release in sham rats. Compared to the earlier experiment by Horii et al. (1994), orbital shaker stimulation is an extremely mild form of vestibular stimulation. Horii et al. (1994) used both electrical stimulation of the round window and caloric stimulation during their experiment, both of which produced nystagmus and therefore activation of the VOR. The increase in ACh release may not have been wholly due to vestibular stimulation, and rather due partially to eye and facial movements.
In conclusion, BVL rats did not have different levels of ACh release in the hippocampus compared to sham rats. The temporal resolution of microdialysis makes analysing release of ACh difficult as chronic stimulation which is not affected by desensitization is required. Therefore, a system of pulses of stimulation may be needed to continually elicit theta under urethane anaesthesia. Another option would be to electrically or pharmacologically stimulate the PPT or
RPO to produce long lasting theta during the microdialysis collection period (Matulewicz et al.,
2013; Matulewicz et al., 2014).
136 9 GENERAL DISCUSSION This thesis examined changes to the hippocampal muscarinic cholinergic system following
BVL through a variety of techniques. Receptor density, receptor expression, cholinergic cell number, hippocampal field activity, and ACh release were all measured following BVL produced by sodium arsanilate intra-tympanic injection. Sodium arsanilate injections were found to be an effective and efficient means of producing complete BVL. Histological analysis demonstrated significant damage to the vestibular hair cells and the nerve fibres in the ampulla 30 days following injection. This was consistent with a previous study which found that sodium arsanilate produces large vacuoles in the sensory epithelia 7 days after injection, lesioning the vestibular hair cells
(Vignaux et al., 2012). Behavioural evidence of BVL and UVL was also analysed to assess the severity of the lesions. Rats with BVL consistently displayed head-bobbing and whole body circling behaviours, with altered tail hanging, righting reflexes, and hyperactivity. Rats with UVL displayed severe unidirectional head tilt towards the lesion side, barrel rolling behaviour, and the reflex to spin during a tail hang test. In the open field, BVL animals behaved significantly differently to sham rats at both 3 and 23 days post-lesion. BVL rats spent more time in, and exhibited more transitions into, the centre of the field, exhibited increased movement distance and velocity, and an increase in whole body rotations. These variables were so consistent among animals that it was possible to predict whether the rat had a BVL or not by day 23 with 100% accuracy using linear discriminant analysis and the leave-one-out method (Manly, 2005).
The density of M1 mAChRs decreased in both the hippocampus and striatum. This decrease was evident 30 days following BVL, but did not exist at an earlier 7 day time point. The number of neurons expressing M2/4 mAChRs increased, mirroring the M1 receptor changes. M2/4 receptors were present on a larger number of neurons in the hippocampus and striatum 30 days following
BVL, but not earlier. M2/4 receptor expression was also increased at day 30 on non-neuronal cells in the striatum.
137 Changes in hippocampal mAChR expression may disrupt theta, as seen following mAChR antagonist administration (Barnes and Roberts, 1991; Gdqbiewski and Eckersdorf, 1996).
Hippocampal theta rhythm (type 2) was elicited in BVL and UVL rats while under urethane anaesthesia using a tail pinch stimulus. Somatosensory tail pinch-induced theta was present in
BVL and UVL rats. In BVL rats, theta was potentiated, with the power of tail pinch-induced theta significantly larger in BVL animals compared to controls. Previous studies have found that theta
(type 1) is significantly reduced in power in awake BVL rats (Neo et al., 2012; Russell et al.,
2006). The present experiment shows that theta disruption following BVL is not due to an inability to generate theta, as tail pinch stimulation produces a pronounced theta response. It is likely that the loss of vestibular sensory signals, which normally generate type 2 theta (Shin, 2010), are the underlying cause of theta disruption in alert BVL rats. In human BVL patients, neck proprioceptive input has a larger effect on cortical activity compared to controls (Cutfield et al., 2014). The potentiation of theta in rats and cortical activity in humans may be due to changes in sensory integration areas early in the theta-generating pathway such as the PPT.
PPT cholinergic cells were counted using ChAT immunohistochemistry and stereology.
The number of ChAT-positive cells in BVL rats, and on the side contralateral to the lesion in UVL rats, was significantly increased. This suggests that BVL and UVL rats may have increased cholinergic output from the PPT. Increased cholinergic output from the PPT to the RPO is thought to enhance theta, as seen following direct carbachol administration to the RPO (Kinney et al.,
1998; Vertes et al., 1993). It is possible that increased cholinergic signalling from the PPT is producing the type 2 theta potentiation observed in BVL animals. Hippocampal theta rhythm is controlled by cholinergic and GABAergic neurons in the MS (Vertes and Kocsis, 1997). During bursts of theta, hippocampal ACh release is substantially increased (Dudar et al., 1979; Monmaur et al., 1997). ACh release is correlated largely with type 2 theta amplitude rather than frequency
(Monmaur et al., 1997). The increased type 2 theta power and increased number of ChAT-positive
PPT cells suggest that hippocampal ACh release would be increased in BVL rats. Conversely,
138 blocking M2/4 receptors in the hippocampus and striatum has been reported to increase local ACh release due to their function as an autoreceptor (Billard et al., 1995; Hersch et al., 1994; Stillman et al., 1996; Volpicelli and Levey, 2004). Our results suggested that an increase in M2/4 expression would decrease ACh release. Along with the decrease in M1 receptor density, this would result in a large change in both striatal and hippocampal cholinergic activity following BVL, with the total receptor balance shifting from excitatory post-synaptic M1 receptors towards inhibitory pre- synaptic M2 receptors. Neither of these hypotheses were found to be supported, with baseline hippocampal ACh release in BVL rats remaining unchanged from control sham rats. Tail pinch and rotational vestibular stimulation did not alter ACh release in either BVL or control sham rats, although this might have been due to the combination of the temporal resolution of the microdialysis and desensitization to the stimuli over time.
As baseline ACh release in the hippocampus was unchanged 30 days post-BVL, the increase in M2/4 and decrease in M1 receptors may be part of a compensatory mechanism to maintain normal ACh levels. That receptor changes occur 30 days, but not 7 days, following BVL illustrates a gradual shift in cholinergic function. In KO mice, removal of M2/4 autoreceptors increases ACh release (Quirion et al., 1995; Tzavara et al., 2003). Conversely, increased autoreceptor distribution is expected to inhibit ACh release into the hippocampus (Kuczynski and
Kolakowsky-Hayner, 2011; Richards, 1990). In the present experiment, M2/4 receptor distribution increased following BVL, yet the release of hippocampal ACh remained unchanged. Maintenance of stable extracellular ACh levels following BVL may be due to increased AChE. In mutant tilted mice, there was an increase in AChE density in the ventral DG (Kirby et al., 2013). No change in
AChE was seen elsewhere in the hippocampus (Kirby et al., 2013). Therefore, analysis of AChE in selective hippocampal subregions following BVL should be performed to determine changes in non-mutant rats, which may explain the stable ACh release seen in the present experiment.
While the present studies have found changes in mAChRs, theta generation, cholinergic cell numbers in the PPT and unchanged hippocampal ACh release following BVL, the exact
139 mechanism for the hyper-locomotor and spatial learning and memory deficits remains unknown.
The hippocampal muscarinic cholinergic system is certainly involved, however the cause of BVL behavioural changes are more complex than alterations to a single subtype of receptor.
Hippocampal NMDA receptors have been found to increase in density following BVL (Besnard et al., 2012), although other studies have found no change (Zheng et al., 2013). Dorsal hippocampal NMDA receptors are necessary for spatial working memory (McHugh et al., 2008), suggesting the increase in NMDA receptors following BVL is a compensatory response to progressive loss of vestibular input (Besnard et al., 2012). Another neurotransmitter that may be associated with BVL behavioural syndromes is GABA. GABAB receptors in the hippocampus inhibit interneurons to produce theta, alongside cholinergic excitation of pyramidal cells
(Hasselmo et al., 1996). Furthermore, systemic administration of a GABAB antagonist improves performance in the radial arm maze (Helm et al., 2005). Hippocampal microinjections of GABAA agonists impair spatial memory in a spatial delayed non-matching-to-position task (Mao and
Robinson, 1998; McHugh et al., 2008). Based on the role GABA receptors have in spatial memory,
BVL may lead to a compensatory down-regulation of both GABAA and GABAB receptors in the hippocampus, mirroring the response of NMDA receptors (Besnard et al., 2012). However, to date there is no study that has assessed GABA receptors in the hippocampus following BVL. In the striatum, dopamine receptors appear to play a role in the generation of behavioural syndromes following BVL (Stiles and Smith, 2015; Stiles et al., 2012). However, changes being limited to dopaminergic and cholinergic receptors in the striatum would be surprising. Striatal ACh release increases during the transition from declarative to procedural learning (Chang and Gold, 2003), as seen in BVL rats (Machado et al., 2014). Dopamine, glutamate, GABA and serotonin all affect
ACh release within the striatum (Calabresi et al., 2000; Ding et al., 2000; Gillet et al., 1985;
Scatton and Lehmann, 1982). However, these neurotransmitters can also have independent effects, through their respective receptors, on spatial memory in the striatum. In spatial memory tasks, microinjection into the striatum of either NMDA antagonists (Smith-Roe et al., 1999), GABA
140 antagonists (Salado-Castillo et al., 1996), or serotonin (Prado-Alcala et al., 2003) produce profound amnesic effects. Therefore, changes to any combination of these striatal receptors and neurotransmitters may produce hyperactivity and spatial memory disorders following BVL.
LIMITATIONS OF EXPERIMENTS Each experiment had its own limitations. The biggest limitation among these was the temporal resolution of the microdialysis, as stated previously. This prevented analysis of stimulation-induced hippocampal ACh release. Future analysis may be able to avoid this limitation by using pulses of stimulation which do not desensitize. Another limitation was performing microdialysis and recording field potentials under urethane anaesthesia. While vestibular stimulation appears to largely affect type 2 theta (Shin, 2010; Tai et al., 2012), which is best seen under urethane anaesthesia, it is possible to restrain awake animals and record vestibular-induced type 2 theta (Shin, 2010). The question remains as to whether the present results are applicable to awake animals with BVL, or if the potentiation of tail pinch-induced theta is only present under urethane anaesthesia. Repeating this study in awake animals would be likely to answer this question. Another assessment of type 2 theta can be performed by examining the REM sleep activity of BVL animals, measuring hippocampal theta and nystagmus. Lesions of the vestibular nuclei disrupt REM sleep (Perenin et al., 1972; Pompeiano and Morrison, 1965; Seguin et al.,
1973); however REM sleep has not been studied following BVL.
The labelling of ChAT-positive cholinergic cells was undertaken and cells within the PPT were quantified using stereology. However, the MS is the main cholinergic input to the hippocampus (Senut et al., 1989). It was initially planned to count both PPT and MS cells together; however, the PPT had priority due to its early location in the theta-generating pathway and link to type 2 theta (Kinney et al., 1998; Matulewicz et al., 2013; Nowacka et al., 2002). Locating and counting the cells of the PPT is more easily performed with sagittal sections as opposed to coronal, using the substantia nigra and superior cerebellar peduncle as border landmarks. Cutting each
141 hemisphere of the brain sagittally produced uneven and/or damaged sections in the centre of the brain (0.5 mm lateral to bregma), which is the location of the MS. Because of substantial damage to the MS-containing sections, MS ChAT-positive cells could not be counted using the stereological method.
The receptor labelling studies each had their own limitations. Autoradiography beta- imaging illustrated the density of M1 mAChRs throughout the dorsal hippocampus and striatum.
The ligand used to bind to these receptors, pirenzepine, has a high affinity for M1, but at high concentrations will bind to other mAChRs. This was avoided in the present experiment by reducing the concentration of the ligand below saturation (Flynn et al., 1997; Tayebati et al., 2004).
Due to the lower concentration and non-specific binding, maximum binding (Bmax) and affinity could not be calculated. Beta-imaging does not differentiate between receptors located on neurons and glia. Therefore, the decreased density in BVL animals may be due to a down-regulation of glial M1 receptors, rather than neuronal receptors. Flow cytometry does show the specific localization, differentiating between glia and neurons. However, in these experiments, it counted the number of cells with the M2/4 receptor present and could not determine the overall density of the receptors. Ideally, beta-imaging can be paired with flow cytometry to provide a clear analysis of the density, affinity, and localization of the receptor of interest. This was not possible in the current experiment as M1 receptor antibodies had not been tested in a flow cytometry application at the time of the study. Further, M2 receptors could not be measured with beta-imaging as the
3 specificity of the most suitable M2 ligand, [ H]AFDX-384, is low, with affinity pKi values of 8.22,
8.00, 7.51, 7.18, and 6.27 -logM at human M2, M4, M1, M3, and M5 receptors, respectively (Miller et al., 1991). For comparison, pirenzepine has over 10-fold greater affinity for M1 than any other mAChR, with pKi values of 8.04, 6.28, 6.8, 6.98, and 6.9 -logM for M1, M2, M3, M4, and M5, respectively (Hegde et al., 1997). Another limitation of the thesis is the lack of spatial memory behavioural testing; however spatial memory disruption following BVL in rats is a well- established phenomenon (Baek et al., 2010; Besnard et al., 2012; Brandt et al., 2005; Chapuis et
142 al., 1992; Dallal et al., 2015; Guidetti et al., 2008; Horn et al., 1981; Hufner et al., 2007; Machado et al., 2012a; Matthews et al., 1989; Neo et al., 2012; Ossenkopp and Hargreaves, 1993; Russell et al., 2003a; Russell et al., 2003b; Schaeppi et al., 1991; Schautzer et al., 2003; Smith et al., 2013;
Stackman and Herbert, 2002; Wallace et al., 2002; Zheng et al., 2012a; Zheng et al., 2009a; Zheng et al., 2004; Zheng et al., 2006; Zheng et al., 2007; Zheng et al., 2009b). The present study aimed to investigate how vestibular information is transmitted to the hippocampus through the theta- generating pathway, therefore measuring spatial learning and memory was irrelevant. It would also be repetitive to perform further spatial memory testing without significant changes to the testing protocol. It can be assumed, based on previous studies, that following BVL rats will develop spatial learning and memory disorders.
A significant limitation of vestibular lesion studies was the potential effect of auditory damage confounding the results. Sodium arsanilate intratympanic injections produce damage to cochlear hair cells (Anniko, 1976; Anniko and Wersall, 1976; Kaufman et al., 1992). The injection itself ruptures the tympanic membrane of both lesioned and sham ears, producing hearing impairment in all animals. However, sham animals recover as the membrane heals, and their hearing loss is also not as profound as lesioned animals as the cochlea remains intact (Anniko,
1976; Anniko and Wersall, 1976; Brown et al., 1935; Kaufman et al., 1992). Therefore, the present results cannot rule out auditory damage as a causative factor. However, in human patients, vestibular dysfunction is often paired with hearing loss (Agrawal et al., 2009). The combined effects of vestibular and auditory dysfunction are therefore clinically relevant.
FUTURE DIRECTIONS Determining how vestibular sensory information is projected to, and utilised by, the hippocampus is necessary to understand and treat cognitive disorders which arise from vestibular dysfunction. Rancz et al. (2015) used MRI and electrophysiology to examine the effects of vestibular stimulation in the rat brain. From the areas in which they recorded, the thalamus and
143 hippocampus had the shortest latency, which suggests these areas receive vestibular information before any other area measured. However, recordings from nuclei located early in the theta- generating, head-direction, and cerebello-cortical pathways, such as the PPT, RPO, MS, SUM,
PH, dorsal tegmental nucleus, lateral mammillary nucleus, and cerebellum, were not performed in this study (Rancz et al., 2015). Therefore, the pathway with the shortest latency from the VNC to the hippocampus was not determined. Future experiments should aim to use electrophysiology to conclusively determine the exact pathway(s) from the vestibular organs to the hippocampus, and whether individual vestibular organs activate separate parts of the hippocampus and other areas of the brain. Examining the responses to selective SCC and otolithic stimulation throughout the brain has already been performed by another member of our laboratory group (Zhang et al., 2013b); however, the results have not yet been fully analysed by the authors. The analysis of these data may determine which of the vestibular-hippocampal pathways provide the shortest latency, and whether the individual SCCs and otoliths have a preference for a single pathway, e.g. SCC information may have a preference for the head-direction pathway, while information from the otoliths may have a preference for the theta-generating pathway (Zhang et al., 2013b).
Changes in receptor and neurotransmitter expression throughout the theta-generating pathway following BVL have not been studied. The MS and PH are considered necessary for theta generation yet neither has been examined following BVL. Quantification of the cells which release the neurotransmitters involved in the generation of theta within each of these areas is needed. This would assess the impact BVL produces throughout the theta-generating pathway. Specifically, this could be achieved by counting cholinergic and GABAergic cells in the MS (Vertes, 2005), and glutamatergic and histaminergic cells in the PH (John et al., 2008).
Further analysis of theta is required in BVL animals. Type 2 theta is difficult to elicit in rodents without urethane anaesthesia, however, it is present during passive rotation if the animal is restrained (Shin, 2010). To determine if non-vestibular-induced theta is potentiated in alert animals following BVL, a follow-up study should be performed assessing different forms of
144 sensory stimulation on restrained BVL and sham rats. REM sleep type 2 theta is necessary for contextual memory consolidation (Boyce et al., 2016). It is therefore likely that disturbances in
REM sleep are related to the cognitive impairment observed following BVL. An analysis of theta during REM sleep in BVL rats is needed to build on studies which found REM sleep disturbance following VNC lesions (Perenin et al., 1972; Pompeiano and Morrison, 1965; Seguin et al., 1973).
The spatial working memory of BVL rats following cholinergic treatment should be assessed in a future study. Pharmacologically targeting the PPT or hippocampus to increase the cholinergic response using AChE inhibitors or carbachol may improve spatial memory performance. Inhibition of AChE improves spatial memory performance (Sweeney et al., 1990).
AChE has been shown to be increased in the ventral DG of mutant tilted mice (Kirby et al., 2013).
Whether this occurs in non-mutant BVL rodents needs to be examined. Improving cognitive performance in BVL patients would significantly improve quality of life (Brandt et al., 2005;
Grabherr et al., 2011; Schautzer et al., 2003). Therefore, if the rodent study was successful, a human study examining BVL patients’ spatial memory performance following the administration of cholinergic drugs should be undertaken. Prior to this, a questionnaire could be utilised to assess self-reported spatial memory performance in BVL patients following the administration of cholinergic drugs for other disorders.
CONCLUSION Rats with BVL have significant behavioural changes 30 days following the lesion procedure. These behavioural symptoms are paired with changes to the hippocampal theta- generating pathway, with the number of cholinergic PPT neurons increasing. This was associated with an increase in theta power during non-vestibular sensory stimulation, a tail pinch, in BVL animals. However, the release of ACh within the hippocampus remained unchanged, although there were changes to the distribution of muscarinic cholinergic receptors. M1 receptors were down-regulated in both the striatum and hippocampus following BVL, while the number of neurons expressing M2 receptors increased in both regions. Further research is needed to assess
145 the effects of BVL on other neurotransmitters in the theta pathway, as well as research on awake animals. If receptor changes lead to the spatial working learning and memory deficits which occur following BVL, a pharmacological therapy could be developed to increase the quality of life for patients with BVL. Further to this, neurodegenerative syndromes which may be linked to vestibular dysfunction such as AD, could be treated earlier in the disease progression by assessing vestibular function and treating the associated cognitive disorders.
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188 11 APPENDICES
A) ETHOVISION STATISTICAL ANALYSIS
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Figure 11.1: EthoVision analysis, non-significant results. Rats were placed in an open field 3 and 23 days post-BVL. Recordings were analysed with EthoVision tracking software.
189 Table 11.1: Multiple linear regression where distance moved (centre point) was the dependant variable (day 3). Unstandardized Standardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 10418.91 1394.49 .00 Turn Angle -95.36 18.70 -.85 .00 2 Constant 8988.64 1217.82 .00 Turn Angle -82.28 15.48 -.73 .00 Highly Mobile 117.55 43.77 .37 .03 3 Constant 5100.76 1939.49 .03 Turn Angle -53.97 17.52 -.48 .02 Highly Mobile 107.41 36.06 .34 .02 Distance Moved (nose) 0.15 0.07 .36 .05 4 Constant 1439.31 1540.24 .38 Turn Angle -28.74 12.70 -.26 .06 Highly Mobile 98.60 22.31 .31 .00 Distance Moved (nose) 0.18 0.04 .44 .00 Outer Zone (centre) 1.19 0.32 .31 .01 5 Constant -1433.98 1639.70 .42 Turn Angle -12.29 11.66 -.11 .33 Highly Mobile 41.49 28.46 .13 .20 Distance Moved (nose) 0.13 0.04 .31 .01 Outer Zone (centre) 1.87 0.37 .49 .00 Velocity (centre) 635.12 254.81 .34 .05 6 Constant -3097.74 444.88 .00 Highly Mobile 28.78 25.98 .09 .31 Distance Moved (nose) 0.14 0.04 .33 .01 Outer Zone (centre) 2.15 0.26 .56 .00 Velocity (centre) 787.05 211.73 .42 .01 7 Constant -3394.49 360.17 .00 Distance Moved (nose) 0.12 0.03 .28 .01 Outer Zone (centre) 2.31 0.22 .60 .00 Velocity (centre) 964.52 140.37 .52 .00 8 Constant -3448.64 199.36 .00 Distance Moved (nose) 0.06 0.02 .14 .04 Outer Zone (centre) 2.48 0.13 .65 .00 Velocity (centre) 1161.36 89.61 .62 .00 Relative Angular Velocity -56.67 12.93 -.15 .00
190 9 Constant -2469.33 362.58 .00 Distance Moved (nose) 0.04 0.02 .10 .04 Outer Zone (centre) 2.59 0.10 .68 .00 Velocity (centre) 1166.33 62.20 .62 .00 Relative Angular Velocity -45.80 9.71 -.12 .00 Outer Zone (nose) -0.90 0.31 -.08 .03 10 Constant -2416.97 172.75 .00 Distance Moved (nose) 0.04 0.01 .10 .00 Outer Zone (centre) 2.65 0.05 .69 .00 Velocity (centre) 1233.22 32.90 .66 .00 Relative Angular Velocity -51.91 4.80 -.14 .00 Outer Zone (nose) -1.07 0.15 -.09 .00 Centre Zone (centre) -16.23 3.50 -.07 .01 11 Constant -2599.93 117.36 .00 Highly Mobile -11.67 3.65 -.04 .03 Distance Moved (nose) 0.03 0.01 .07 .01 Outer Zone (centre) 2.73 0.04 .71 .00 Velocity (centre) 1329.98 36.02 .71 .00 Relative Angular Velocity -59.09 3.63 -.16 .00 Outer Zone (nose) -1.03 0.09 -.09 .00 Centre Zone (centre) -17.10 2.09 -.07 .00 12 Constant -2251.51 61.99 .00 Highly Mobile -8.32 1.17 -.03 .01 Distance Moved (nose) 0.03 0.00 .07 .00 Outer Zone (centre) 2.60 0.02 .68 .00 Velocity (centre) 1284.89 12.38 .69 .00 Relative Angular Velocity -63.44 1.23 -.17 .00 Outer Zone (nose) -0.75 0.05 -.07 .00 Centre Zone (centre) -18.67 0.65 -.08 .00 Absolute Angular Velocity -0.20 0.03 -.05 .01
191 13 Constant -2223.87 22.16 .00 Highly Mobile -8.60 0.41 -.03 .00 Distance Moved (nose) 0.03 0.00 .07 .00 Outer Zone (centre) 2.59 0.01 .68 .00 Velocity (centre) 1286.86 4.29 .69 .00 Relative Angular Velocity -63.14 0.43 -.17 .00 Outer Zone (nose) -0.72 0.02 -.06 .00 Centre Zone (centre) -18.69 0.22 -.08 .00 Absolute Angular Velocity -0.22 0.01 -.05 .00 Contracted -0.24 0.05 .00 .04 Note: R2 = .72 for Model 1, ΔR2 = .12 for Model 2, ΔR2 = .06 for Model 3, ΔR2 = .06 for Model 4, ΔR2 = .02 for Model 5, ΔR2 = .00 for Model 6, ΔR2 = .00 for Model 7, ΔR2 = .02 for Model 8, ΔR2 = .00 for Model 9, ΔR2 = .00 for Model 10, ΔR2 = .00 for Model 11, ΔR2 = .00 for Model 12, ΔR2 = .00 for Model 13.
Table 11.2: Multiple linear regression where time contracted was the dependant variable (day 3).
Standardized Unstandardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 24.45 5.22 .00 Relative Angular Velocity 3.92 1.52 .63 .03 Note: R2 = .40 for Model 1.
Table 11.3: Multiple linear regression where time stretched was the dependant variable (day 3).
Unstandardized Standardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 137.14 41.34 .01 Moving .11 .04 .68 .02 Note: R2 = .46 for Model 1.
192 Table 11.4: Multiple linear regression where time in the outer zone (nose point) was the dependant variable (day 3).
Unstandardized Coefficients Standardized Coefficients Model B Std. Error β P value 1 Constant 1143.52 14.66 .00 Intermed Zone -1.13 .12 -.95 .00 (nose) 2 Constant 1085.57 16.32 .00 Intermed Zone -1.09 .07 -.92 .00 (nose) Not Moving .20 .05 .25 .00 3 Constant 1018.21 24.60 .00 Intermed Zone -1.10 .05 -.93 .00 (nose) Not Moving .28 .04 .35 .00 Moving .05 .02 .16 .01 Note: R2 = .90 for Model 1, ΔR2 = .06 for Model 2, ΔR2 = .02 for Model 3.
Table 11.5: Multiple linear regression where time in the centre zone (nose point) was the dependant variable (day 3).
Standardized Unstandardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 8.22 2.50 .01 Intermed Zone (Centre) .20 .03 .93 .00 2 Constant -12.51 7.47 .13 Intermed Zone (Centre) .17 .02 .78 .00 Velocity (nose) 2.14 .75 .30 .02 Note: R2 = .86 for Model 1, ΔR2 = .07 for Model 2.
Table 11.6: Multiple linear regression where time in the centre zone (centre point) was the dependant variable (day 3).
Standardized Unstandardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 1.81 1.80 .34 Centre-Intermed Transitions 1.00 .23 .81 .00 Note: R2 = .66 for Model 1.
193 Table 11.7: Multiple linear regression where meander was the dependant variable (day 3).
Unstandardized Coefficients Standardized Coefficients Model B Std. Error β P value 1 Constant 41.72 29.53 .19 Clockwise -11.13 2.16 -.85 .00 Rotation Note: R2 = .73 for Model 1.
Table 11.8: Multiple linear regression where time mobile was the dependant variable (day 3).
Standardized Unstandardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 814.54 49.99 .00 Not Moving -.96 .17 -.88 .00 2 Constant 637.72 39.79 .00 Not Moving -.71 .09 -.65 .00 Outer-Intermed Transitions 5.60 .99 .48 .00 Model 2 P = 0.0001 Note: R2 = .77 for Model 1, ΔR2 = .18 for Model 2.
Table 11.9: Multiple linear regression where time not moving was the dependant variable (day 3).
Unstandardized Standardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 717.52 79.20 .00 Mobile -.81 .14 -.88 .00 2 Constant 681.09 43.03 .00 Mobile -.84 .07 -.92 .00 Treatment 110.83 21.80 .41 .00 Note: R2 = .77 for Model 1, ΔR2 = .17 for Model 2.
194 Table 11.10: Multiple linear regression where clockwise rotation was the dependant variable (day 3).
Unstandardized Standardized Coefficients Coefficients Model B Std. Error β P value 1 Constant -4.95 2.40 .07 Total Rotations .89 .12 .92 .00 2 Constant -4.44E-15 .00 1.00 Total Rotations 1.00 .00 1.04 .00 Counter-Clockwise Rotation -1.00 .00 -.41 .00 Note: R2 = .84 for Model 1, ΔR2 = .16 for Model 2.
Table 11.11: Multiple linear regression where frequency of outer-intermediate zone transitions was the dependant variable (day 3).
Standardized Unstandardized Coefficients Coefficients Model B Std. Error β P value 1 Constant -.18 2.72 .95 Centre-Intermed Transitions 2.91 .34 .94 .00 2 Constant .12 2.12 .96 Centre-Intermed Transitions 2.03 .41 .66 .00 Centre Zone (nose) .28 .10 .37 .02 Note: R2 = .87 for Model 1, ΔR2 = .06 for Model 2.
Table 11.12: Multiple linear regression where total rotations was the dependant variable (day 3).
Unstandardized Standardized Coefficients Coefficients Model B Std. Error β P value 1 Constant 7.52 1.78 .00 Clockwise Rotation .95 .13 .92 .00 2 Constant 4.44E-15 .00 1.00 Clockwise Rotation 1.00 .00 .96 .00 Counter-Clockwise Rotation 1.00 .00 .40 .00 Note: R2 = .84 for Model 1, ΔR2 = .16 for Model 2.
195 B) PUBLISHED RESEARCH
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201 C) CONFERENCE PROCEEDINGS Aitken P, Zheng Y, Benoit A, Philoxéne B, Le Gall A, Denise P, Smith PF, Besnard S (2015).
Vestibular lesions affect the expression of M1 acetylcholine receptors in the hippocampus. In
Spring Hippocampal Conference. Taormina.
Aitken P, Zheng Y, Benoit A, Philoxéne B, Le Gall A, Denise P, Smith PF, Besnard S (2015). The effect of vestibular loss on the hippocampal cholinergic system. In Audiology Spring Symposium.
University of Auckland: Auckland.
Aitken P, Zheng Y, Smith PF (2015). Bilateral vestibular lesions increase response of non vestibular-induced theta rhythm in rats. In Australasian Winter Conference on Brain Research.
Queenstown.
Aitken P, Zheng Y, Smith PF (2016). Bilateral vestibular lesions increase sensitivity to non- vestibular induced theta rhythm in rats. In 29th Bárány Society meeting. Seoul, Korea. Journal of
Vestibular Research: 26 (1-2) pp 155-260.
Aitken P, Benoit A, Zheng Y, Philoxéne B, Le Gall A, Denise P, Smith PF, Besnard S (2016).
Acetylcholine muscarinic receptor changes in the hippocampus following bilateral vestibular loss.
In 29th Bárány Society meeting. Seoul, Korea. Journal of Vestibular Research: 26 (1-2) pp 155-
260.
202 D) COPYRIGHT PERMISSIONS
Dear Phillip,
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Table 3. Comparison of muscarinic receptor subtype proteins (immunocytochemistry), mRNAs (in situ hybridization), and pharmacological - binding sites (autoradiography) in select regions of rat brain. Levey AI, Kitt CA, Simonds WF, Price DL, Brann MR (1991). Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies. J Neurosci 11: 3218-3226.
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DOI: 10.3233/NRE-130866 Citation: NeuroRehabilitation, vol. 32, no. 3, pp. 437-443, 2013
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