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

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

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.

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.

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

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.

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

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)/(D^en/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.

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)

+

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).

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)

=

+

+

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.

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.

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

, /

-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

-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