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Firing characteristics of central vestibular neurons in response to angular rotation in the head-restrained rat.

Ariana R. Andrei,

Department of Neurology & Neurosurgery, McGill University, Montreal. Canada.

October 2005

A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science.

© AR Andrei, 2005

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TABLE OF CONTENTS

ABSTRACT... 4

RESUME ... 5

ACKNOWLEDGEMENTS... 6

CHAPTER 1: GENERAL INTRODUCTION & LITERATURE REVIEW 1. GENERAL INTRODUCTION... 7

II. INTRODUCTION TO THE VESTIBULAR SySTEM... ... ... .... ... ... 9

Semicircular canal orientation... .... ... ... .... 9

Projections of the vestibular nerve... ... ... .... ... ... 10

The vestibular nuclei... ... ... ... .... ... ... .... 10

The vestibuloocular reflex... 12

The role of vestibular inputs for spatial orientation... 13

III. PROPERTIES OF PRIMATE CENTRAL VESTIBULAR NUCLEJ... .... .... ... ... ... 15

Vestibular Only (VO) neurons... 15

Position Vestibular Pause (PVP) neurons... 16

Eye Head (EH) neurons... .... 17

Burst Tonie (BT) neurons... 17

Eye Position (EP) neurons... 18

Other neurons... ... .... ... ... ... ... ... 18

IV. PREVIOUS STUDJES OF RAT CENTRAL VESTIBULAR NUCLEJ... 19

Responses to sinusoidal rotation... ... ... ... ... ... 19

Responses to optokinetic stimulation... ... ... ... 20

IV. PROPERTIES OF GUINEA PlO CENTRAL VESTIBULAR NUCLEI... 21

V. PROPERTIES OF GERBIL CENTRAL VESTIBULAR NUCLEI... 22

CHAPTER 2: FIRING CHARACTERISTICS OF CENTRAL VESTIBULAR NEURONS IN RESPONSE TO ANGULAR ROTATION IN THE HEAD- RESTRAINED RAT 1. INTRODUCTION... ... ... ... 24

II. MATERIALS AND METHODS Animais and surgery. ... ... ... .... ... ... ... 27

General procedure... ... ... ... ... ... .... ... 28

Data acquisition... ... ... ... ... ... ... 30

Analysis of the vestibuloocular reflex (VOR)... 31

Analysis of neuronal discharges.... ... ... ... ... ... 31

III. RESULTS... 35

Measurement of the vestibuloocular reflex using video- oculography... ... 35

General trends and classification of units... ... ... 36

Behaviour ofpure vestibular neurons during VORL••••....••• 37

Behaviour ofpure vestibular neurons during VORF...•.. 38

Behaviour of oculomotor-related neurons during VORL.... 39

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Comparisons of vestibular-only units versus oculomotor

related uni/s... ... ... ... ... ... ... ... 41

IV. DISCUSSION... 42

Summary of findings... ... ... ... ... ... ... ... 42

Vestibuloocular reflex: comparisons with other studies in rat... ... 43

Vestibular-related neurons: comparisons with other studies in rat... ... .... ... ... ... .... ... .... ... ... ... ... .... 45

Vestibular-related neurons: comparisons with studies in alert mammals... ... ... ... ... .... ... .... ... ... ... ... 48

A. Primates. .... ... ... ... ... 49

B. Guinea Pigs & Gerbils... ... .... ... 51

Considerations.... ... ... ... ... .... .... ... ... .... 52

V.FIGURES & TABLES... 54

CHAPTER 3: GENERAL DISCUSSION 1. IMPLICATIONS FOR UPSTREAM SySTEMS... 67

II. FUTURE DIRECTIONS... .... .... ... .... ... ... .... ... ... ... ... ... 70

REFERENCES... 72

APPENDIX 82 1. ANIMAL ETHICS CERTIFJCATE... ... ... ... .... ... ... ... ... 83

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ABSTRACT:

Characterization of vestibular-related neurons in awake, head-restrained rats.

Although an extensive body of literature exists describing the properties of cells found in the primate vestibular nuclei (VN), the rat vestibular nuclei have not been explored in a comparable manner in order to allow for meaningful comparisons. Previous single-unit experiments in rat VN did not track eye movements and were performed in anesthetized or paralyzed preparations. This study characterizes the properties of vestibular-related cells in awake, behaving rats, using standard single-unit methodology, and video oculography to monitor eye movements, thus providing the tirst description that is comparable to the primate literature. Male, Long-Evans rats were head restrained and sinusoidally rotated at frequencies ranging from O.l-l.OHz, reaching a maximum velocity of 100 deg/sec. Eye position sensitivity was assessed by recording cell activity as the rat tixated at different locations. We show the presence of cells that are sensitive only to vestibular stimulation, equivalent to vestibular-only cells in primates. These cells have no eye sensitivity, and show a moderate increase in head velocity sensitivity with increasing stimulus frequency. Additionally, we show the presence of cells with eye movement-related sensitivities that bear a close resemblance to primate eye-head neurons.

These results suggest that the rat vestibular nuclei may contain a similar cross section of cells as those found in the primate vestibular nuclei. These results shed light on the type of information in the vestibular nuclei that is available for other upstream systems, and is particularly relevant to spatial orientation, which has been shown to depend on vestibular input.

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RÉSUMÉ:

Caractérisation des neurones vestibulaires chez le rat éveillé tête fixe

Au contraire des Primates chez qui les propriétés des neurones vestibulaires ont été largement étudiées in vivo, il existe encore trop peu de données chez le Rat pour permettre une comparaison interspécifique. En effet chez cette espèce, les enregistrements des neurones vestibulaires ont été réalisées sur des préparations de rats anesthésiés ou paralysés, et sans que les mouvements de l'oeil ne soient pris en compte, interdisant ainsi une description du rôle de ces cellules dans les réflexes vestibulo-oculaires. Dans cette étude, nous avons enregistré chez des rats éveillés les réponses électrophysiologiques des neurones vestibulaires en réponse à des rotations atteignant 100 deg/sec et des fréquences comprises entre 0,02 et 1Hz. Nous avons également enregistré les mouvements des yeux à l'aide d'une technique de vidéo-oculographie. Nos résultats montrent la présence de différentes sous-populations de cellules distinguées en fonction de la sensibilité de leur activité à différentes excentricités de l'oeil. La majorité des cellules sont de type

« vestibular-only », sensibles uniquement aux rotations de la tête. Ces neurones présentent une faible augmentation de leur sensibilité en réponse à des stimulations de fréquences croissantes. Nous rapportons également la présence de cellules sensibles à la fois aux rotations de la tête et à la position de l'oeil, présentant des réponses comparables aux cellules « Eye-Head » décrites chez les Primates.

Ces résultats qui permettent pour la première fois une comparaison directe des réponses in vivo des neurones vestibulaires du Rat et des Primates suggèrent une

organisation comparable du système de ces deux espèces. Enfin, nos données permettent de préciser le type d'information que les neurones vestibulaire envoient vers d'autres structures également impliquées dans l'orientation spatiale de l'animal..

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ACKNOWLEDGEMENTS

For my father, Mr. Cezar Andrei, who always supported me in whatever dream 1 was running after at the time, and Dr. Angel Alonso who remembered what it was like to not know anything, and offered immeasurable assistance during my loneliest days at the MN!.

1 would like to thank Dr. Kathleen CuIlen, my supervisor, for letting me take on a project that was clearly over my head. Thank you for always pushing and believing in me. l've leamed so much from you that goes weIl and beyond science. The most important thing being that if you want them to come true, you have to fight for your dreams with everything you've got.

1 would also like to express my gratitude to Dr. Oum Hassani, and Dr. Barbara Jones who shared their secrets with me, and without whom this project would not have been possible.

l've also had the pleasure ofworking (and playing) with sorne of the most wonderful people these past few years. The immensely talented Mr. Walter Kucharski, who built aIl the equipment for this project, but more importantly, who became one of my dearest friends. My fellow labbies, who saw me through sorne of the darkest days and to whom 1 am forever grateful; Dr. Mathieu Beraneck, whose scientific prowess came to my rescue on so many occasions; Ms. Jessica Brooks, whose powers of resiliency constantly impressed; Ms. Meaghan McCluskey who showed me the ropes when 1 first arrived, and introduced me to AB; Dr. Soroush Sadeghi - the man, the mystery, the legend - there's no room for aIl you've done for me; and Ms. Marion Van Hom - my twin that took twenty sorne years to meet. Pardon my sappiness, but 1 love you aIllike family .

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Chapter 1

General Introduction &

Literature Review

The farther back you can look, the farther forward you are likely to see.

- Winston Churchill

1. General Introduction

Where are you right now? Perhaps you are sitting at a desk, or drifting to sleep on the couch. Now think a little bigger. Think about the building you are in, the street outside, the neighbourhood, the city, the country, the continent and so on. Regardless of whether you are zooming in and out on an enormous imaginary map or whether you have thousands of these litde maps strung together, what is certain is that you have a definite conception of where you are in relation to a multitude of things. Your brain has

integrated available sensory and cognitive cues and translated them into a palpable feeling - a sense of direction, ifyou will. But how does the brain do this? What information does it need? Does this happen at the single neuron level, or is it the work of a whole network that leads to this conscious percept? Neurophysiologically, these are enormous questions that could take decades to answer. The present study addresses a smaller problem within this theme.

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In the last twenty or so years, studies in the rat have discovered, what appear to be, neural correlates for the sense of direction. In order to navigate accurately within an environment, two general pieces of information are required: 1) information about one's current location and 2) information about the direction that one is moving. Two cell types have been characterized whose activity best correlates with these components. Place cells, found in the hippocampus and various parahippocampal structures, show activity that correlates with the animal's location relative to its environment, irrespective of bodily posture (see Shapiro and Eichenbaum, 1999 for review). Head direction cells, found in several nuclei of the thalamus as weIl as a multitude of other connected structures, show activity that corresponds to the head's orientation relative to the environment (see Taube, 2003 for review).

The present study will focus on the nature of the sensory input that is available to construct a head direction cell. TheoreticaIly, to generate an output like that observed in head direction ceIls, two pieces of information are required - information about the environment via sens ory cues (vision, olfaction etc), and information about the movement of the head via other sensory cues (vestibular, proprioception, motor efference copy etc).

As we shall see, there is ample evidence that the vestibular system is required to maintain head direction cell activity, but the nature ofthat vestibular input remains a mystery. This introduction will review the vestibular system from the end organ to the brainstem, provide evidence for its role in head direction cell activity and finish with the behaviour of individual neurons within in the vestibular nuclei.

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Il. Introduction to the Vestibu/ar System

The vestibular system is a sensory system sensitive to movements of the head.

The end-organ consists of the three semicircular canals, oriented at approximately 90 degrees to each other that detect angular rotations of the head in three dimensions, as weIl as the two otolith organs (the saccule and utric1e) that sense omni-directionallinear accelerations. The vestibular system is necessary for detecting the movement of the head and maintains several postural and eye reflexes. As weIl, it seems to be required for the more complex task of spatial orientation and navigation.

Semicircular canal orientation

When the head is situated in line with the axis of rotation, it experiences only angular accelerations and thus stimulates only the semicircular canals. The two vertical canals are oriented at 90 degrees to one another. In humans the horizontal semicircular canal is oriented 25 degrees upward from earth-referenced horizontal plane during normal upright posture (Blanks et al, 1975). This is similar to what is observed in rhesus and squirrel monkeys whose horizontal canals are oriented 22 and 18 degrees upwards, respectively (Blanks et al. 1985), as weIl as in cat whose horizontal canal is oriented 23 degrees upwards (Blanks et al. 1972). To achieve maximal activation of the horizontal canal and minimal activation of the vertical canals, the head should be oriented 15 degrees nose down in rhesus and squirrel monkeys (Blanks et al. 1985) and 21 degrees nose down in the cat (Blanks et al. 1972). This is somewhat different in rats in which the horizontal canal is oriented 35 degrees upward, and maximal activation of the horizontal canal is achieved with a head position of 43 degrees nose down, relative to an earth-referenced horizontal plane (Blanks and Torigoe, 1989). It is important to note that

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head direction cells (HDC) seem to be active mainly for horizontal (relative to earth) movements. But given the natural orientation of the canals, pure horizontal movements activate all of the semicircular canals, so the input to HDCs could originate from neurons that receive projections from any of the three canals.

Projections of the vestibular nerve

The vestibular nerve is formed from the bipolar neurons whose cell bodies' reside in Scarpa's ganglion. The afferent fibres that come from the vestibular end organs branch as they enter the brainstem (see Barmack, 2003 for review). One bundle

containing thinner axons projects upwards to the uvula-nodulus of the cerebellum, while the bundle containing thicker axons enters the vestibular nuclear complex in the

brainstem. These two structures comprise the first sites of processing of vestibular information by the central nervous system, the latter ofwhich is the subject ofthis study.

The vestibular nuc/ei

The vestibular end organ sends projections primarily to the vestibular nuclei.

There are four "classical" vestibular nuclei (VN) that receive direct projections from the vestibular nerve: the medial vestibular nucleus (MVN); the inferior vestibular nucleus (IVN); the lateral vestibular nucleus (L VN) and the superior vestibular nucleus (SVN).

The horizontal canals send afferents mainly to the MVN and the L VN, while the SVN receives input primarily from the vertical canals (reviewed in Barmack, 2003).

Response properties of neurons within the vestibular nuclei were originally described by Duensing and Schaefer (1958) and classified based on the location of cell with respect to the midline and its directional selectivity to horizontal head movement.

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Type 1 responses indicate that the cell is sensitive to head movements ipsilateral to the recording site. For example, a cell recorded on the left side of the brain, that is sensitive to leftward, counterclockwise rotations, would be classified as type 1. Type II indicates contralateral sensitivity, while Type III responses indicate bilateral sensitivity and Type IV indicates no sensitivity to either contra- or ipsilateral rotation.

The vestibular nuclei send and receive projections to and from an array of different brain areas. VN sends ascending projections to the thalamus, which then sends projections to cortical areas involved in vestibular processing (insular cortex), as weIl as to vermis and flocculus ofthe cerebellum (reviewed in Barmack, 2003). VN sends descending projections to the spinal cord via the lateral vestibulospinal tract, and mediate the vestibulocollic reflexes that stabilize the head (reviewed in Barmack, 2003). Within the brainstem, VN provides ascending inputs to the III, IV and VI cranial nuclei that control the contraction of extraocular muscles, and mediate the vestibuloocular reflex, which will be discussed shortly.

The vestibular nuclei are the site of convergence of multiple sensory inputs. In addition to the afferent projections from the vestibular nerve, the vestibular nuclei receive visual information from a variety of cortical and brainstem structures, as weIl as

proprioceptive information from the spinal cord. This multimodal convergence allows the vestibular nuclei to mediate postural reflexes, such as the vestibulocollic reflex that functions to stabilize the head on the neck, as weIl as eye reflexes such as the

vestibuloocular reflex that functions to stabilize gaze. Moreover, this convergence of inputs means that individual neurons within the vestibular nuclei can be sensitive to more than one kind of stimulus. For example, neurons can have eye movement sensitivity as

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weIl as the expected head movement sensitivity. This will be discussed in more detail in a subsequent section.

The vestibuloocular reflex

This present study makes substantial advantage of this reflex to elicit eye movements, so sorne words should be said about it. The vestibuloocular reflex (VOR) functions to maintain a stable image on the retina during head movements. The reflex is mediated by a three neuron arc, first described by Lorente de No' in 1933. The arc consists of afferent projections from the vestibular end organs to intemeurons in the vestibular nuclei, which in tum project to extraocular motoneurons that contract the eye muscles to move the eye in the opposite direction as the head (reviewed by Cullen and Roy, 2004). This results in eye movements that are equal in amplitude and speed, but opposite in direction to the head movement.

This reflex is readily elicited using sinusoidal rotation, but several other forms of the VOR have also been described, including linear and torsional VORs (see Hess and Dieringer, 1991; Wong and Sharpe, 2005, respectively). The VOR is quantified by measuring its gain (gain = -peak eye velocity/ peak head velocity), and determining the phasic relationship between the eye and head velocity. For example, a gain ofunity means that the eye movement is directly proportion al to the head, but in the opposite direction, while a phase of -5 degrees, means that the peak eye velocity lags the peak head velocity by 5 degrees. There have been other several studies of the rat VOR, but the present study is the first known investigation to quantify eye movements using video oculography, rather than the invasive scleral search coil technique (Fuchs and Robinson, 1966).

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The raie of vestibular inputs for spatial orientation

Aside from mediating reflexes, the vestibular nuclei have been implicated in higher order cognitive processes that correlate to the sense of direction. Head direction cells have been extensively described in the rat and can be found within the lateral mammillary nuclei, the anterior dorsal and lateral dorsal nuclei of the thalamus, retrosplenial granular cortex, and postsubiculum (see Taube 1998 for review). The activity of these cells correlates with the orientation of the head in the horizontal plane in body in space coordinates. That is, these cells are sensitive to the relationship of the head with respect to the environment, rather than the head on the body. The vestibular system is a likely candidate to provide the necessary head movement information to these cells and has been the subject of many studies. (It should be noted that since the vestibular nuclei encode information in a head-fixed reference frame, their output must be modified at sorne point to explain the difference in the coordinate reference frames between the vestibular nuclei and the head direction cells.)

Severallesion studies have implicated the vestibular system in the generation and maintenance of he ad direction cell activity. Permanent lesions of the vestibular labyrinth were shown to eliminate HDC activity in the anterior dorsal thalamic nuclei (Stackman and Taube, 1997), and temporary inactivation of vestibular hair cells with tetrodotoxin eliminated directional activity of neurons in the postsubiculum (Stackman et al, 2002).

Regardless of the type of lesion employed, it is clear that eliminating vestibular inputs, adversely affects the direction al sensitivity of several populations ofHDCs. The ability to navigate back to a starting location using only self-motion cues (also known as de ad reckoning) has also been shown to be impaired following vestibular lesions (Wallace et

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al, 2002), indicating that vestibular signaIs play an important role in navigation when other sensory cues are absent.

Although there is ample evidence that vestibular input is necessary for the generation and maintenance of a head direction signal, it remains unc1ear precisely what vestibular information is actually being received by the head direction cell network. One of the main reasons it has been so difficult to elucidate what signaIs are actually being supplied by the vestibular system to the head direction cell network, is that thus far it has not been possible to isolate vestibular input to these cells. Most previous work in the field has been done in freely moving animaIs, so it is impossible to discriminate which self-motion cues (proprioception, motor efference copy, optic flow or vestibular inputs) are being used by the cells to generate their output. Studies that have attempted to isolate vestibular inputs, have found that tight restraint (wrapped tightly in a towel), followed by manual passive rotation abolished directional activity (Taube et al, 1990), while loosely holding the animal and rotating it caused severe attenuation of activity (Taube, 2003). To complicate matters further, second order vestibular neurons in the primate vestibular nuc1ei, that are thought to feed into the HDC network, have recently been found to show severe attenuation in their activity during voluntary movement (Roy and Cullen, 2001;

see section entitled Properties of Primate Vestibular Nuclei Neurons). The paradox lies in trying to reconcile the fact that while vestibular input is necessary for establishing directionality, the head velocity signal does not seem to be available during active movement and when it is available (during passive movements) it is precisely then that HDC activity dec1ines!

Previous studies of rat vestibular nuc1ei have failed to monitor eye movements (see section entitled Previous Studies of Rat Vestibular Nuclei Neurons), so the behaviour of neurons could not be compared to those studied in primates. It remains unknown how the

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behaviour of primate vestibular nuclei compares to that of rat. Therefore, it is unclear how much one can infer about rat vestibular nuclei, based on finding obtained in the primate. The present study aimed to rectify this situation, by characterizing neurons in the rat vestibular nuclei in relation to both head and eye movements.

III. Properties of Primate Vestibu/ar Nuc/ei Neurons

Before delving into what is known about the rat vestibular nuclei, we will begin by reviewing what has been leamed from the primate. Primate vestibular nuclei neurons have been the subject of a great number of studies. This review will focus on the types of cells found within the vestibular nuclear complex, classified according to their response properties during various conditions. As a population, these cells have been shown to have a broad range of behaviours that correlate with eye movements, head rotations or a combination of the two. The ability to train primates to perform a variety of eye and head movements has allowed cell activity in the vestibular nuclei to be correlated with a variety ofnatural and controlled eye and head movement conditions - sorne ofwhich are impossible to achieve in a rodent model.

Vestibular Only (VO) Neurons

Vestibular only cells receive input from the eighth nerve emanating from the vestibular end organs, and as expected their activity correlates with movement of the head (Scudder and Fuchs, 1992). The type ofhead movement they are sensitive to is

dependent from which vestibular end organ the afferent innervating the cell originates from (i.e. vertical, horizontal semicircular canal etc.). VOs are believed to be responsible

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~ ...

for mediating the vestibulocollic reflex (VCR) and project to the cervical spinal cord (reviewed in Cullen and Roy 2004). During passive whole body rotation, VOs are sensitive to ipsilateral headlbody movements, and have no eye movement sensitivity whatsoever (Cullen and McCrea, 1993; Scudder and Fuchs, 1992). In contrast, during active head on body movements VOs show a ~ 70% reduction in their response to head velocity, while knowledge of self-generated passive head movements does not affect their response (Roy and Cullen, 2001).

Position Vestibular Pause (PVP) Neurons

As their name states, PVP neurons are sensitive to head velocity, oppositely directed eye position, and in addition, show pausing behaviour for sorne saccades. A type 1 PVP would show sensitivity to contralateral eye position, ipsilateral head velocity during whole body rotations, and pause for ispilaterally directed saccades (reviewed in Cullen and Roy, 2004). A type II PVP would show oppositely directed sensitivities.

PVPs are the intemeurons thought to mediate the VOR. Most PVPs send excitatory projections to the contralateral abducens nucleus or medial rectus portion of the oculomotor nucleus, and sorne send inhibitory projections to the ipsilateral abducens nucleus (reviewed in Cullen and Roy, 2004). During active head movements, PVPs modulate their activity depending on the gaze control strategy being employed. For example, when the goal is to redirect the gaze, an active VOR would be

counterproductive, and accordingly PVP activity pauses just before gaze shift onset, and resumes following the completion of the gaze shift (reviewed in Cullen and Roy, 2004), thus preventing an active VOR from interfering with the desired eye movement.

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Eye-Head (EH) Neurons

EH neurons are sensitive to both eye and head velocity in the same direction (i.e.

ipsilateral). During sinusoidal rotation, EH units will modulate their activity dependent on he ad velocity, and will burst for quick phase eye movements in the same direction as their head sensitivity. This differentiates them from PVPs whose eye and head sensitivities are in opposite directions. EH neurons also have proportionally higher eye velocity sensitivity and less eye position sensitivity than PVPs (Scudder and Fuchs, 1992). EH neurons can be characterized using smooth pursuit and VOR cancellation paradigms, allowing for the isolation of eye and head movements, respectively (reviewed in Cullen and Roy, 2004).

Smooth pursuit involves visually tracking of a moving target with the head fixed in space, while VOR cancellation involves rotating the head-fixed animal on a tumtable while it fixates on a target that moves with the tumtable. EH neurons are thought be the primary neurons that mediate smooth pursuit eye movements in the primate, but are also believed to play a role in the VOR and VCR (reviewed in Cullen and Roy, 2004). During active head on body movements, EH neuron activity can be described as a summation of their sensitivity to head movement during whole body rotation in the dark and their sensitivity to gaze during smooth pursuit (Roy and CuIlen, 2003).

Burst Tonie (BT) Neurons (a/so known as Burst Position)

BT neurons are sensitive to eye position and velocity, but unlike any of the other cell types thus far discussed, they have no head velocity sensitivity. These cells burst in the ON direction, and pause for the OFF direction, and most are sensitive for ipsilateral eye movements (Scudder and Fuchs, 1992). BT neurons resemble abducens motor

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neurons, but their bursts are more pronounced, while their tonic firing is more irregular (Scudder and Fuchs, 1992).

Eye Position (EP) Neurons

As their name states, EP units have eye position sensitivity, usually eye velocity sensitivity, but no head sensitivity as tested using the aforementioned VOR cancellation paradigm (Scudder and Fuchs, 1992). Unlike, burst tonic ceIls, EP neurons do not burst for ON direction saccades, although sorne do pause for OFF direction saccades (Scudder and Fuchs, 1992).

Other Neurons

Neurons within the vestibular nuclei exhibit a wide spectrum of sensitivities. The types ofunits described above only mentions the most commonly encountered varieties.

It should be note d, though, that many cells show sensitivities that are best described as a mixture or variation on the above mentioned cell types, but are encountered less

frequently and in insubstantial numbers. For example, Vestibular Pause (VP) cells behave just like PVPs, except that they have no eye position sensitivity during fixation (Fuchs and Kimm, 1975). Also described in the literature are cells that have vestibular and eye position sensitivity but without the eye velocity sensitivity that would classify them as EH units, as weIl as cells that show increased activity for eye movements to both the left and the right (Scudder and Fuchs, 1992).

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IV. Previous Studies olRat Vestibular Nuc/ei Neurons

Responses to sinusoidal rotation

There have been a limited number of studies that have examined the

responses of rat central vestibular neurons to sinusoidal rotation. AlI of the se studies were conducted in anesthetized or paralyzed preparations, conditions that are known to cause differential responses in the vestibular nuclei when compared to the alert preparation.

Additionally, none ofthese studies were able to accurately quantify eye movements, so neuronal activity could only be correlated with the movement of the head according to the aforementioned Duensing and Schaefer (1958) nomenclature. When considered in respect to the extensive description of cell types in the primate VN based on responses to both he ad and eye movements, it is evident that this classification scheme is quite limited.

That said, there are several important properties of rat VN that can be ascertained from these studies.

First, type 1 versus type II neurons within the vestibular nuclei have generally been found to occur in equal proportions (Kubo et al. 1975; Lannou et al. 1979;

Hammann and Lannou, 1987). In another study, Lannou et al. (1982) reported a slight variation, fin ding 60% type l, and 40% type II neurons. It is unlikely that changes in alertness states of the animal would affect these cell proportions.

These neurons were tested using sinusoidal rotational stimuli in the dark with frequencies ranging from 0.025Hz, up to 0.35Hz, at various peak velocities. The present study extends the range of stimuli reaching up to 1.0Hz. Neuronal activity was primarily quantified based on its phase relationship with head acceleration. The phase (relative to he ad acceleration) of neuronal responses tended to lag the head as the stimulus frequency

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was increased (Hammann and Lannou, 1987; Lannou et al. 1979). More specifically, the majority of neuronal responses were approximately in phase with head velocity (Kubo et al. 1975; Lannou et a1.1982). (Recall that velocity lags acceleration by 90 degrees.)

The sensitivity (gain) of neurons to angular acceleration of the head has also been reported. Several studies found that the gain decreased with increasing head

acceleration (Lannou et al. 1982; Lannou et al. 1979), and with increasing frequency (Hammann and Lannou, 1987). For example, Lannou et al. (1979) reported a mean gain of 2.3 at 0.05Hz, whereas at O.2Hz, the mean gain dropped down to 0.4. In addition, type l neurons tended to have greater sensitivities than type II neurons (Lannou et al. 1982).

Responses to optokinetic stimulation

Despite the lack of eye movement recordings, rat vestibular nuc1ei neurons have also been tested with visual stimuli. Optokinetic stimulation involves the rotation of a large visual pattern occupying a major portion of the visual field - usually consisting of alternating black and white vertical stripes projected onto a large screen and rotating at relatively low velocities (under 55 deg/sec). The stimulus consists solely of the moving visual grating, with no vestibular stimulation whatsoever. It has been found that vestibular nuc1ei neurons in the anesthetized brown rat (DA/HAN) respond to optokinetic

stimulation in the complete absence ofvestibular stimulation (Cazin et al. 1980; Lannou et al. 1980), indicating there are cells in the rat VN responsive to eye movements,

although this does not necessarily mean that the neurons in question would respond to eye movements not induced by an optokinetic stimulus. For example, primate vestibular-only neurons respond to optokinetic stimulation, but do not respond to other types of eye movements (Waespe and Henn, 1979). Thus, on the basis of these studies we may

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r-..

ascertain that neurons within the rat vestibular nuc1ei, like those of other species, respond to optokinetic stimulus. To date, there has been no qualitative or quantitative description of rat vestibular nuc1ear neurons in response to any other eye movements.

However, there have been several studies in other rodents that have used

methodology that c10sely resembles that used in the primate studies. These studies may provide insight into the types of responses that we may find in the rat vestibular nuc1ei, and will be considered in the subsequent sections.

V. Properties of Guinea Pig Vestibular Nuclei Neurons

Unlike, the rat vestibular nuc1ei, individual neurons in the guinea pig VN have been characterized according to both eye and he ad sensitivities, in both the alert and anesthetized preparations.

Findings from the alert preparation will be described first. The occurrence of type l and type II neurons was considerably more variable than that found in the rat. For example, Ris et al. (1995) reported 79% type land 8% type II neurons, while Ris and Godaux (1998) found 47% type l, and 25% type II neurons. In relation to eye movements, Ris et al. (1995) described neurons that were sensitive to eye position, neurons that paused for saccades in either direction, neurons that burst for contralaterally directed saccades, and neurons that were sensitive to both eye position and saccades.

Qualitatively, these results from the guinea pig resemble the properties of neurons in the primate vestibular nuclei Given that phylogenetically guinea pigs are more closely related to rats than to primates, it is reasonable to hypothesize that a similar array of neurons may be found in the rat vestibular nuclei.

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The anesthetized preparation illustrates the profound differences in the vestibular system that exist between the alert and anesthetized conditions. When stimulated at 0.2Hz, neurons from the vestibular nuclei of anesthetized guinea pigs showed a severe attenuation in their mean resting discharge - compare 4l± 24.7 spikes/s (Ris et al 1995) in the alert condition versus 10.9±16.5 spikes/s for type 1 neurons, and 5.9±8.7 spikes/s for type II neurons (Curthoys et al. 1988; Curthoys et al. 1 988b) in the anesthetized condition. In addition, while the sensitivities of type 1 neurons were comparable to those in the alert preparation, the sensitivity to head rotation of type II neurons was

dramatically decreased. Given the differences in the properties of guinea pig vestibular nuclei neurons between the alert and anesthetized conditions, we expect that the results of the present study will differ considerably from those of previous studies in rat.

VI. Properties ofGerbil Vestibular Nuclei Neurons

Kaufman et al (2000) have been the only group thus far to examine the properties of individual neurons within the gerbil vestibular nuclei. Using sinusoidal rotations, they recorded from vestibular nuclei (as weIl as other adjacent structures) and recorded eye movements using the scleral search coil technique (Fuchs and Robinson, 1966).

Neurons were grouped into those with eye related sensitivity and those without (equivalent to vestibular-only cells in the primate), and then subcategorized according to the Duensing and Schaefer (1958) nomenclature. This study is notable because its methodology closely resembles those of the present study and the qualitative descriptions of cell types found within these structures are particularly interesting. Kaufmann et al (2000) described neurons that responded only to head rotations and neurons with eye

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~_ ..

/

related activity, sorne ofwhich responded to contralateral rotation, burst for ipsilateral quick phases, and paused for contralateral quick phases. These properties, again, resemble those of neurons found within the primate vestibular nuclei.

Based on the findings of these studies in the primate, guinea pig and gerbil, we expect to find a comparable subset of neurons in the rat vestibular nuclei with similar properties, exhibiting sensitivity to both eye and head movements. Aside from being the first characterization of vestibular nuclear neurons in the rat in this manner, our results will help to elucidate the potential roIe that the vestibular nuclei play in the generation of upstream head direction cells. If neurons in the rat vestibular nuclei do resemble those of these other species, considerable questions will remain as to the origin of the head

position signal, the availability of a head velocity signal during active movements, and the site of the coordinate reference frame transformation that must occur to generate the observed head direction cell signal. If, on the other hand, the properties of neurons in the vestibular nuclei of the rat are, in fact, different, the solution to one or more of these questions may be revealed.

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Chapter 2

Firing characteristics of central vestibular neurons in response to angular acceleration in the head- restrained rat

1. INTRODUCTION

In order for any organism to navigate within an environment, it requires information about its current whereabouts within that environment, as weIl as the direction in which it is heading. Two populations of cells within the rat have been identified in these past twenty years that correspond to these variables, and could be the neural correlates for the sense of direction. Place ceIls, found within the hippocampus and parahippocampal structures, modulate their activity depending on the animal's current location within the environment (see O'Keefe, 1993 for review), while head direction cells, found in a variety of structures, modulate their activity in response to the head's orientation with respect to the environment (see Taube, 2003, for review). The prevailing mystery remains, though, as to what sensory and/or cognitive information is required to generate such complex neural activity. TheoreticaIly, to produce a signallike that found in a head direction celI, information about the current position of the head must be

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integrated with infonnation referenced to the external environment. That is, a reference change must occur that modifies infonnation about relative movements of the he ad with respect to the body and converts it into infonnation about the movement of the head relative to the outside world.

The most obvious source of infonnation about the movement of the head is the vestibular system. There are severallines of evidence that point to the vestibular system as having an important role in the development ofupstream he ad direction cell activity.

First, there have been severallesion studies that have shown that an intact vestibular system is required to maintain the directional sensitivity ofhead direction cells (Stackman et al. 2002; Stackman and Taube, 1997). Anatomically, a potential pathway from the vestibular nuclei to structures housing he ad direction cells has been mapped out (Taube, 2003). The medial vestibular nucleus sends projections to the nucleus prepositus hypoglossi which projects to the dorsal tegmental nucleus (McCrea and Baker, 1985), which in turn has extensive projections to the structures housing head direction cells (Liu et al. 1984). A direct pathway from the medial vestibular nucleus to the dorsal tegmental nucleus also exists, but it is much less prominent than the aforementioned pathway (Liu et al. 1984). The prevailing hypothesis is that the stability of directional sensitivity ofhead direction cells during rapid shifts in body movements, results from the change in activity within the vestibular nuclei (Brown et al. 2002). For this proposed circuitry to work, input from the horizontal semicircular canals (who se responses are in phase angular head velocity) must be integrated to provide a measure ofhead position. Neurons within the dorsal tegmental nucleus have been shown to have asymmetric responses to angular head velocity - that is, neurons will modulate for movements in one direction, but not the other (Sharp et al. 2001). These responses have been hypothesized to be an important stage in

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the transfonnation of the signaIs from the horizontal semicircular canals into he ad direction cell signaIs, and a model has been proposed as to how this may occur (Bassett and Taube, 2001).

However, the basis of this line of reasoning rests on the underlying assumption that neurons within the vestibular nuclei encode a consistent horizontal head velocity signal and that this signal is available to be integrated at all times. However, this does not seem to be the case. The vestibular nuclei are the site of convergence and processing of many sensory and motor signaIs, and individual neurons are sensitive to both eye and head movements (see Cullen and Roy, 2004 and Bannack, 2003 for review). Vestibular- only neurons are the most likely cell type to project to upstream head direction cells, since integration of their responses will generate pure head position infonnation, unlike other cell types whose responses also contain additional extraneous eye-related infonnation.

The head velocity sensitivity of this class of neurons, however, has recently been shown to exhibit severely attenuated activity during active movements (Roy and Cullen, 2002).

This suggests that during voluntary movements, neurons within the vestibular nuclei do not reliably encode head velocity, and therefore their responses cannot be integrated to yield accurate infonnation about head position. But given that no previous study has ever characterized the behaviour of vestibular nuclei neurons in the alert rat, it is impossible to know whether these findings in the primate also apply to the rat, or are species specific.

The main goal of the present study was to characterize the behaviour of individual

neurons in the vestibular nuclei of the alert rat during passive rotations and compare these results with those from other species.

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Il. MATERIALS & METHODS

Animais and surgery

Experimental data were obtained from adult male Long-Evans rats (Charles-River, St. Constant, Quebec, Canada) weighing between 300-600g. All experimental procedures conformed to the guidelines of the Canadian Council on Animal Care, and were approved by the McGill University Animal Care Committee. Surgi cal procedures are similar to those previously described by Lee et al. (2004). Throughout the experiments, animaIs were maintained on a 12/12-h lightldark schedule with the lights on during the day. Food and water were available ad libitum except during experiments, when animaIs were offered food treats periodically. For surgery, animaIs were anesthetized with a mixture of ketamine, xylazine, acepromazine, and atropine (50/5/1/0.05 mg/kg, Iml/kg, intra-

muscular), and maintained during the surgery with a lower dose (0.3 ml/kg) of the mixture when necessary. AnimaIs were then placed in an adapted stereotaxic frame (David Kopflnstruments, Tujunga, CA) for the implantation ofthe head restraint device and recording weIl. Using aseptic technique, the top of the skull was exposed and several small holes were drilled in 4 plates of the skull. A larger hole was drilled for the

recording site over the vestibular nuclei approximately 9.5-11.5mm posterior and 0.5- 2.0mm lateral to Bregma (Paxinos and Watson, 1986). Machined stainless steel screws (Small Parts, Logansport, IN) were inserted into the smaller holes and affixed into place with an adhesive luting cement (Parkell, Farmingdale, NY). A U-shaped frame was mated to its mounting bracket with screws, which itself mounted to the stereotaxic frame, was positioned on the head and affixed in place using dental acrylic. The V-frame

allowed animaIs to be immediately positioned in stereotaxic coordinates when head-fixed.

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The recording weIl, situated in the middle of the V-frame, was then sealed with sterile bone wax. AnimaIs were allowed to recover for 2 days before experimental sessions commenced.

Genera/procedure

On a recording day, the animal was briefly «1 minute) anesthetized using isoflurane gas (4% induction), while the head was secured. After such short anesthesia, animaIs recovered to normal state of alertness in 5-10 minutes, and it has been previously reported that the VOR recovers and remains stable after 30 minutes (Kaufman et al.

2000). Experiments began at least 30 minutes after the initial head restraint. AnimaIs were seated in a natural resting posture on a Plexiglas platform. The platform was fitted with a stainless steel arch that fixed the head and prevented the animaIs from twisting their bodies. Otherwise, the setup allowed for free movement of the body and limbs. To reduce stress, animaIs were familiarized with Plexiglas container prior to surgery, and were offered food treats as positive reinforcement during experiments. AnimaIs, eventually, leamed to sit calmly in the setup, with minimal struggling.

The Plexiglas platform was mounted on a tumtable fitted with a movable electrode carrier (David KopfInstruments, Tujunga, CA), and an ISCAN video eye tracking system (lSCAN, Burlington, MA). The tumtable could be manually rotated 360 degrees in the horizontal plane (Figure 1). A fine positioning system allowed the

animal's head to be centred in line with the table's axis of rotation, ensuring that aIl movements were purely angular and did not contain any translational component.

Neuronal sensitivities to horizontal head velocity were determined by rotating the animal

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at various frequencies and velocities «0.1 to I.5Hz; velocities up to 100 deg/s). During experiments, animaIs were situated in the middle of an enclosed recording room with the lights on and had a view of the recording instruments and experimenter. The paucity of spontaneous eye movements in the rat made determining neuronal eye sensitivity

difficuIt. It was found, though, that by using small amplitude and low velocity

movements, the animal' seye would move and remain stable for ~ l minute at different eccentricities in the orbit before reverting back to primary position. This technique was used to determine neuronal sensitivity to eye position, while neuronal responses to VOR quick phases were used to acquire data related to eye velocity. A second paradigm was used in an attempt to separate the head and eye sensitivities. In this configuration, an independently lit box, lined with a bold black and white stripe pattern (designed by Dr.

Mathieu Beraneck), was placed over the recording setup. At very low frequencies

«O.OIHz) and velocities, the stable visual field helped to suppress eye movements, but cell sensitivities were found to drop so low in either condition at these frequencies that this method could not be used to parse out the differential contributions of eye and head to neuronal activity. At higher frequencies with the box in place, the eye movement amplitude was comparable to that observed in the open visual field condition, while the phase of the response was advanced - similar to the resuIts described by Fuller (1985) with the placement of a Ganzfeld. AnimaIs tended to react to the placement of the box and unit isolation was frequently lost and unrecoverable, so there is considerably less data available from this paradigm.

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Data acquisition

Extracellular single-unit activity was recorded using enamel-coated tungsten

microelectrodes (2-10 Mn impedance, Frederick-Haer). Electrodes were housed in a 25- gauge guide tube that was used to penetrate through the dura matter and cerebellum, following the application of a drop of lidocaine to the area to minimize discomfort. The guide tube was positioned with respect to the reference point marked on the skull during surgery. The electrode was advanced along the Z-axis into place with a miniature hydraulic drive (Narishige). Spike potentials were amplified (gain = 80dB) and filtered (band-pass 300 to 10k Hz). Unit isolation was continuously monitored to ensure that the level of a windowing circuit was set at an appropriate level to generate a pulse that coincided with the rising phase of a spike. This pulse was then sent to the event channel of a peripheral device (CEDI401) that logged the times of the action potentials. Eye position was monitored using a video-based eye tracking system (lSCAN, Burlington, MA). A digital camera with an infrared CCD sensor was positioned such that one eye was in focus. The camera was adapted for use with the rat eye by attachment of a macro lens.

The infrared light source mounted to the camera, was positioned to illuminate the eye in such a way as to minimize shadows and maximize contrast between the iris and pupil.

The output from the camera was sent to a personal computer running ISCAN eye tracking software, which calculated the amount of eye movement by subtracting the movement of the pupil from that of the light source reflection. This method ensures that slight

movements of the head or camera would not be misinterpreted as eye movements.

Calibration of the eye position signal was similar to that described by Stahl et al. (2000).

Briefly, the camera was rotated 10 degrees around the eyeball while it was ensured that

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the animal was not making any voluntary eye movements (these were observed on a peripheral monitor). The outputted ISCAN eye position value was calibrated into actual degrees of eye movement by the following equation:

Eye movement (deg) =(asin ((ISCAN eye position value-X)/Rp)*(180/ n)), Where X is the outputted ISCAN eye position value with the camera at zero position, and Rp is the comeal curvature calculated from the ISCAN eye position values obtained by moving the camera out to different eccentricities, by the following equation:

Rp= (X I-X2)/(Den/180),

Where XI and X2 are the ISCAN eye position values with the camera at two different positions, and D is the total angular difference between these two positions measured in degrees. For example, if the camera was moved 5 degrees to the left and 5 degrees to the right of the primary, zero position, then D would equallO degrees, while XI and X2 would be the ISCAN values of the eye position obtained at these two camera positions.

Head position was monitored via an optical tracker (US Digital, Vancouver, WA) mounted to shaft of the tumtable, and could track 360 degree movements accurately within less than 0.5 degrees. The voltage signal outputted by the device was calibrated by rotating the animal to points ofknown eccentricity.

The he ad and eye position signaIs, as weIl as the single unit activity were

simultaneously recorded on Digital Audio Tape (DAT) tape (20kHz maximum sampling frequency), which could be replayed and retriggered off-line.

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Analysis of the vestibuloocular reflex (VOR)

The response of the VOR has typically been quantified in terms ofits gain and phase with respect to head velocity. The gain of the response was determined by using the following model:

(peak eye velocity) = -1 • Gain • (peak head velocity)

+

bias

A gain ofunity would indicate that the eye moved with the same velocity as the head, but in the opposite direction. The bias term compensates for the differences in the position of the eye and head. The phase of the response was determined by a model that estimated the phase as the latency that gave the best fit between eye and head velocity as

determined by the variance-accounted-for (V AF). A phase of 0 degrees indicates that the eye and head velocities were in-phase, while positive values indicate that the eye led the head velocity, while negative values indicate that the eye lagged the head velocity. Gain and phase analysis were conducted on segments that did not contain quick phases. Quick phases were removed with a customizable algorithm.

Analysis of neuronal dis charges

Prior to analysis, eye and head position signaIs were digitally low-pass filtered using a 51 st order finite-impulse-response filter with a Hamming window and cutoff frequency set at 10Hz. All analysis was performed using custom algorithms (Matlab;

Mathworks Inc., Natick, MA). Eye and head velocity signaIs were calculated by digitally differentiating the position signaIs. A spike density function was used to represent the neuronal firing rate, in which a Gaussian function (standard deviation of 1 Oms) was convolved with the spike train (Roy and Cullen, 2003).

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A neuron's response to eye position was quantified by a regression analysis used to detennine the relationship between the mean eye position and the mean neuronal firing rate during periods of steady fixation. Significance of eye position sensitivity regression analysis was determined according to the Z distribution. A least-squared regression analysis was used to establish each neuron's phase shift relative to head velocity (phase, degrees), resting discharge rate (bias, spikes/s), and head velocity sensitivity (g).

The sensitivity and phase of the cells' responses to sinusoidal rotation were calculated using an approach similar to that used by other studies (Roy and Cullen, 2003).

First, firing rates and mean eye positions were calculated during periods of steady fixation. A regression analysis (Figure 5A) was then performed to determine each neurons eye position sensitivity (slope = kfixation) and its resting discharge rate (y intercept

= bias). Vestibular-only units, by our definition, did not show significant correlations between eye position and firing rate and upon further inspection did not modulate their activity during VOR quick phases. Subsequently, the horizontal head velocity sensitivity (g) was determined during VORL using the following model:

FR (t)

=

bias

+

k • eye position (1)

+

g • head velocity (1)

The neuron in Figure 5 (unit 19.2), for example had resting discharge of83.l spikes/s, and a horizontal head velocity sensitivity coefficient (g) of 0.32 spikes/s)/deg at 0.5Hz stimulus frequency. At this frequency, neuronal modulation led horizontal head velocity by 4.9 degrees (27 ms). The eye position parameter k, was inc1uded as a secondary measure to ensure that the unit had no eye sensitivity. For all units c1assified as vestibular-only, k ~ 0, and thus this parameter effectively dropped out of the model equation. The same model was used to quantify properties of oculomotor-related units, except that kt- O.

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Only unit data from intervals between quick phases of vestibular nystagmus and/or saccades were included in this analysis. To avoid fitting neuronal responses as cells were driven to eut-off, only segments during which the firing rate was greater than 5 spikes/s were included in the optimization. Spike trains were analyzed separately to determine whether neurons paused or burst for saccades. In instances when the neuron did burst, the resting discharge (bias, spikes/s) and eye velocity sensitivities (r,

(spikes/s)/(deg/s» were also estimated during saccades.

The ability of the linear regression analysis to model neuronal discharges, was quantified by calculating the variance-accounted-for (V AF) for each regression equation.

The VAF was computed as {l- [var(est - fr)/var(fr)]}, where est is the modeled firing rate, and fr is the actual firing rate (see Roy and Cullen, 2001).

The nature of our rotational stimulus precluded the possibility of rotational stimuli with absolutely consistent frequency and velocity. A wide distribution offrequencies and velocities were utilized. The relationship between the frequency and velocity employed was linear with little variation. For example, each sample with a 0.5Hz stimulus

frequency had a peak velocity of approximately 100 deg/s, while lower frequencies had lower velocities. For analysis, results were grouped based on the frequency of

stimulation. Stimuli ofless than 0.2Hz were placed in the O.lHz bin; 0.2-0.39Hz stimuli in O.3Hz bin; 0.4-0.7Hz in the 0.5Hz bin; 0.75-1.2Hz in the 1.0Hz bin; and 1.25-1.7Hz stimuli in the 1.5Hz bin. Statistical significance throughout was determined using paired Student's t-tests, unless otherwise stated.

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III. RESULTS

Measurement of the vestibu/oocular reflex using video-oculography

Rotations in the horizontal plane consistently evoked the vestibuloocular reflex (VOR), producing eye movements in the opposite direction to he ad movements. Previous studies of rat VOR have employed the invasive magnetic scIeraI search coil technique (Fuchs and Robinson, 1966) to measure eye movements. This method has been shown to interfere with the normal movements of the eye in the mouse (Stahl et al. 2000). The present study reports the first recording of rat VOR with the use of a completely un- invasive video-based technique. Figure 2 illustrates a typical VOR response elicited using a sinusoidal rotation stimulus at ~ 1.0Hz. The sharp changes in eye velocity represent the vestibular nystagmus quick phases that act to reposition the eye in the orbit once it reaches extreme eccentricities. The VOR response was measured over the range of stimulus frequencies of 0.1- 1.5Hz. The peak stimulus velocity varied with the individual frequency tested (see METHODS).

VOR responses in the light from three male Long-Evans rats were used in this study. Figure 3A shows a Bode plot of the gains measured across the frequencies tested.

It can be seen that the mean gain does not change considerably across the frequency range tested. Across aIl frequencies the mean gain was 0.64 ± 0.03 SEM. At 0.1 Hz, the mean gain was 0.74 ± 0.06 SEM, while at 1.0Hz, the mean gain was 0.56 ± 0.04 SEM.

lndividual responses varied considerably within each frequency bin. For example, the 0.5Hz bin contained responses ranging from 0.42 aIl the way up to 1.1. It has been previously reported that the VOR response gain could vary considerably depending on the alertness level of the animal (Quinn et al. 1998), and this high variability reported here

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, / - -

could be attributed to such a cause. Figure 3B illustrates the phase relationship between the eye and the head movement across frequency bins. The phase of the VOR shows a consistent increase in lag time as frequency increases. At the lower frequencies tested the eye is approximately in phase with the head velocity (at 0.1 Hz, eye lags by 0.41 ± 0.17 SEM degrees), while at higher frequencies, the eye lags the head velocity by more (at 1.0Hz, eye lags by 3.5± 0.77 SEM degrees). See D1SCUSS10N for comparisons with other studies of rat VOR.

General trends and classification of units

ln addition to quantifying the rat VOR, we also recorded from vestibular related neurons in the brainstem. The present methodology allowed for the identification of units with vestibular sensitivity, and! or oculomotor sensitivity. Neurons were classified based on their firing behaviour during horizontal sinusoidal rotation in the light (VORd, horizontal sinusoidal rotation with a fixed visual environment (VORF), fixation, and during quick phases of the vestibular nystagmus. Neurons were initially identified based on their responses to horizontal sinusoidal rotation, therefore, any units that had no or very low sensitivities to he ad rotation would not have been detected. Units were classified according to the Duensing and Schaffer (1958) nomenclature. A summary of the

response types observed is represented in Table 1. A total of 12 type 1 units were recorded. Of these, 9 were sensitive to only vestibular stimulation, and 3 showed

oculomotor-related activity. Only one type II unit was encountered. This type II unit was sensitive to head rotations, but showed no sensitivity to eye movements. The distribution of resting firing rates for all cell types described is shown in Figure 4. Type 1 neurons with only vestibular sensitivity had a mean resting discharge rate of 34.8 spikes/s ±21. 7

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SD. The type II vestibular-only neuron had a very similar resting discharge rate of 37.0 spikes/s. In contrast, neurons with vestibular and oculomotor sensitivity had a mean discharge rate of 15.0 spikes/s ±7.8 SD. A two-sample Student's t-test revealed that the difference between type 1 vestibular-only units and oculomotor-related units is significant (p = 0.03).

Behaviour of pure vestibular neurons during VORL

Neurons that were classified as vestibular-only neurons 1) modulated their activity during sinusoidal rotation, 2) had no eye position sensitivity and 3) did not change their firing pattern for saccades during vestibular quick phases (n = 9). Figure 5 illustrates the firing behaviour ofa typical vestibular-only cell during VORL . The ceIl's activity increased during ipsilateral rotations, had no significant eye position sensitivity and showed no consistent change in activity during saccades in either direction. Rotations in the contralateral direction correlated to a drop off of activity, but failed to drive the unit to cut off. This was typical of most vestibular-only units recorded, although sorne units did get driven to cutoff during contralateral rotations. Table 2 summarizes the parameter values and phase relationships calculated for aIl vestibular-only neurons during VORL

(Table 2A) at different frequencies of stimulation. The mean sensitivity to horizontal head velocity during VORL was not significantly different between frequencies (Figure 6A, black circles). This data, though, is comparable to that observed in the gerbil (Kaufman et al, 2000), in which sensitivities during rotations up to 1 Hz fell between 0.2 to 0.5

(spikes/s)/deg, with no significant difference between frequencies. There was no measurable significant difference between phase leads across frequencies (Figure 6B, black trace), although visual inspection reveals a rising trend, indicating increasing phase

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lead with increasing frequency. Data from the type II neuron recorded was comparable to those of the type 1 neurons.

During VORL it is not possible to obtain a measure of pure head velocity

sensitivity for any neurons since passive whole body rotation, produce both eye and head movements. Therefore eye and head velocities are not independent, but rather are equal in amplitude and opposite in direction, thus cannot be estimated separately (Roy and Cullen, 2003). We attempted to measure pure head velocity sensitivity with the use of a fixation box.

Behaviour of pure vestibular neurons during VORF

Previous studies in the primate that have employed paradigms during which the animal cancelled its VOR by fixating on a target that moved in synchrony with the tumtable, thereby producing no eye movements and allowed the unit's sensitivity to head rotation to be measured in the absence of eye movements. This level of behavioural training was not possible in our rodent mode!. We, however, attempted to attenuate eye movements with the use of the VORF box. The VORF box provided a fixed visual field that effectively neutralized any optic flow information, inducing a visual-vestibular conflict that should act to suppress eye movements during low frequencies of vestibular stimulation. It was found that for the lowest frequency bin (O. 1Hz), the VORF box did work to effectively suppress eye movements. For example at O.lHz, the mean gain of the VOR was 0.7 ± 0.06 SEM, with the VORF box in place, eye movements were virtually aboli shed and the gain dropped to ~ zero. At 0.5Hz we observed that the VORF box reduced the gain of the VOR by approximately 40%. This is comparable to findings in the mouse, in which the VORF box almost completely aboli shed eye movements at 0.1 Hz,

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~-­

/

and caused an approximately 30% attenuation at 0.5Hz (Dr. Mathieu Beraneck,

unpublished observation). However, the fixation box was difficult to employ effectively.

Regardless of the time alIowed for adaptation to the box, rats consistently reacted to the placement of the box, and on most occasions this resulted in the loss ofunit isolation.

Table 2B summarizes the sensitivities and phase relationships estimated during the VORF condition across frequencies. OveraIl, there was no significant difference in the mean gain (Figure 6A, gray circles) or phase (Figure 6B, gray circles) between frequency bins. The differences in sensitivity between the VORL and VORF conditions, were also not significantly different (p = 0.31), nor do they appear to be different following visual inspection (Figure 6A). For the lowest frequency bin, where the VORF box effectively suppressed eye movements, we can claim to have estimated the true sensitivity of neurons to horizontal head velocity independent of eye movements.

Behavior of oculomotor-related neurons during VORL

Neurons classified as oculomotor-related exhibited a significant relationship between their firing rate and eye position during fixation (n = 3). AdditionaIly, aIl of the oculomotor-related neurons found, also exhibited a clear modulation of firing activity during quick phases of the VOR. No oculomotor-related neurons that consistently paused for saccades in either direction were found. Figure 7 shows a typicaI response of an oculomotor-related celI in response to eye position sensitivity (Figure 7 A) and during VORL (Figure 7B). This neuron increased its activity during head rotations in the ipsilateral direction, burst for saccades in the ipsilateral direction (arrows in Figure 7B), and was completely silenced during head rotations in the contralateral direction. The behaviour of this celI most closely resembles that of the eye-head cell type described in

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