INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The vestibuloocular reflex (VOR) generates eye movements during head perturbations such that subjects are able to maintain their line of sight or gaze on a stationary object (see Leigh and Zee 1999 for review). The gain and phase of the VOR in the light (VORl) and dark (VORd) have been well characterized for a range of head-perturbation frequencies in humans and non-human primates (e.g., Barr et al. 1976; Correia et al. 1985; Das et al. 1998; Minor et al. 1999; Paige 1983). These studies have shown that the gain of the VOR is higher when viewing a stationary target in the light than when imagining a target in complete darkness, suggesting cooperation between the VOR and visually mediated eye movements. A more dramatic example of visual-vestibular interaction is the considerable plasticity, including modification of VOR gain, that is observed when residual retinal slip signals remain during head rotations (Hirata and Highstein 2001; Ito 1972; Lisberger et al. 1983, 1994; Miles and Eighmy 1980; Miles and Lisberger 1981; Raymond and Lisberger 1996). The source of neuronal signals necessary for modifying the gain of the VOR has long been a subject of interest (e.g., du Lac et al. 1995; Maekawa and Simpson 1973; Raymond and Lisberger 1998; Yakushin et al. 2000). In a recent study (Raymond and Lisberger 1998), it has been suggested that climbing fiber inputs to the cerebellum that produce complex spike activity of horizontal gaze-velocity Purkinje cells (HGVP) in the flocculus/ventral para-flocculus carry necessary and sufficient information to support visual modification of the VOR at high-frequency (>5 Hz). Simple spike activity was not modulated during high-frequency VOR adaptation suggesting that mossy fiber inputs did not influence high-frequency VOR gain adaptation. In contrast, during VOR adaptation at low frequencies of head rotation (<2 Hz), both the complex spike and the simple spike activity were modulated and therefore either could support VOR gain adaptation. The source of the simple spike modulation was not specifically identified in this study but could have involved vestibular and visual-mossy fibers.

We chose to examine the potential role of visual mossy fibers in short-term modification of VOR gain at low frequency. The dorsolateral pontine nucleus (DLPN) is a major source of visual mossy fibers to the vestibulo-cerebellum (Glickstein et al.1994). The DLPN is known to receive visual cortical inputs from the extrastriate cortex (Distler et al. 2002; Glickstein et al. 1980, 1994; May and Andersen 1986), including areas middle temporal (MT) and medial superior temporal (MST), which are specialized for visual motion processing (Andersen et al. 1990; Maunsell and Newsome 1987). In turn, the DLPN sends mossy fiber projections to the contralateral ventral paraflocculus and dorsal paraflocculus (Glickstein et al. 1994; Nagao 1997) and vermal lobule VI and VII (Brodal 1979, 1982; Langer 1985). Single-unit recording (Kawano et al. 1992; Mustari et al. 1988; Suzuki and Keller 1984; Suzuki et al. 1990; Thier et al. 1988) and lesion studies (May et al. 1988) demonstrate that DLPN neurons carry appropriate signals to play a role in the initiation and maintenance of smooth pursuit, optokinetic and ocular following eye movements. However, it is unknown whether the DLPN, which provides retinal image motion (slip) signals to the vestibulocerebellum, could also play a role in visual-vestibular behavior. Therefore in this study, we examined the role of the DLPN in VOR gain adaptation during low-frequency head rotations by comparing gain adaptation before and after temporarily inactivating the DLPN with muscimol.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Behavioral data were collected from three normal juvenile rhesus monkeys (Macaca mulatta), weighing 3-7 kg. A detailed description of our surgical procedures can be found in earlier publications (Mustari and Fuchs 1990; Mustari et al. 1997; Mustari et al. 2001). Only a brief description is provided here. Surgical procedures were carried out under aseptic conditions using isoflurane anesthesia (1.25-2.5%) to stereotaxically implant a head-stabilization post and recording chambers (Crist Instruments). In the same surgery, a scleral search coil was implanted underneath the conjunctiva of one eye using the technique of Judge et al. (1980). All procedures were performed in strict compliance with National Institutes of Health guidelines and the protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Emory University.

During all experiments, monkeys were seated in a primate chair with the head stabilized in the horizontal stereotaxic plane. Eye movements were detected and calibrated using standard electromagnetic methods (Fuchs and Robinson 1966) using precision hardware (CNC Electronics, Seattle, WA). Motion of the laser spot was controlled by a two-axis mirror galvanometer setup (General Scanning, Watertown, MA). All of our monkeys were extensively trained to perform a fixation task and track a small diameter (0.2°) target spot moving in sinusoidal or step-ramp trajectories. Vestibular stimulation was provided by a servo-controlled 60 ft-lb DC torque motor (Neurokinetics) that oscillated the chair sinusoidally about the vertical axis. All stimulus generation was computer controlled with custom Labview software and hardware (National Instruments). Eye, head and target position feedback signals were processed with anti-aliasing filters at 200 Hz using 6-pole Bessel filters prior to digitization at 1 kHz with 16-bit precision. Velocity arrays were generated by digital differentiation of the position arrays using a central difference algorithm in Matlab (Mathworks, Natick, MA).

We first located the DLPN by its stereotaxic location relative to the oculomotor nucleus and by finding neurons that were modulated for motion of either a large-field (75 × 75°) stimulus or during smooth-pursuit of a small diameter (0.2°) target spot moving (±10°; 0.1-0.75 Hz) over a dark background (May et al. 1988; Mustari et al. 1988). In muscimol injection experiments, we used a small-diameter (<50 µm) micropipette whose tip was positioned at the depth where smooth-pursuit-related neurons were recorded. Injections (0.5 µl; 2%) were delivered using a picoliter pump (WPI-PV830) connected to the micropipette to provide timed pressure pulses allowing for a gradual delivery of muscimol over several minutes (Mustari et al. 2001). The efficacy of muscimol injections was confirmed by measuring the gain of smooth pursuit during step-ramp tracking (May et al. 1988). Smooth pursuit measurements were taken 15 min after the injection and in some experiments also at the conclusion of gain modification experiments, 2 h post injection. We estimate that our injections blocked most of the DLPN based on the volume and concentration of our muscimol injections in comparison with those in published studies describing the spatial extent and duration of inactivation after muscimol injection (Arikan et al. 2002).

Adaptive changes of the VOR gain were produced by sinusoidal whole body rotation (0.2 Hz, 30°/s) for 2 h while the monkey viewed an earth-stationary visual surround with magnifying (×2) or minifying (×0.5) lenses (Designs for Vision) that produced either increases or decreases of VOR gain. We characterized the gain of the VOR by testing in complete darkness (VORd) and while the monkey viewed a visual stimulus (VORl). We measured VORd before and after adaptation, at the adapting frequency.

We used the initial acceleration and steady-state smooth pursuit velocity during step-ramp tracking (velocity of 20°/s) to characterize the quality of smooth pursuit. Pursuit initiation was taken as the time that average eye speed reached 3 SD above the pretrial value during fixation. Initial acceleration was calculated as the average eye acceleration in the first 100-ms period of pursuit to the step-ramp stimulus. Average steady-state velocity was defined as the region where eye velocity reached a plateau, typically taken between 200 and 300 ms after pursuit initiation. At least 10 trials of rightward or leftward step-ramp tracking were averaged to quantify initial acceleration and steady state velocity. VORd gain was calculated as the ratio of peak eye velocity to peak head velocity, measured in darkness. The peak eye and head velocities over the oscillation period were determined by fitting sinusoids to each half-cycle. Average gains and their SDs were calculated separately for rightward and leftward head rotations from 10 cycles.

DLPN injection sites were confirmed histologically using standard methods for Nissl staining as described in our earlier studies (Distler et al. 2002; Mustari et al. 1988, 1994, 2001).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Smooth pursuit after muscimol injections in the DLPN

After unilateral injection of muscimol in the DLPN, we always observed deficits in the monkey's ability to generate and maintain smooth pursuit of a target moving toward the side of injection (ipsilesional). Unilateral inactivation of the DLPN did not produce nystagmus. Figure 1 illustrates representative smooth pursuit deficits for one of our monkeys. We measured the initial acceleration (1st 100 ms) and average steady-state smooth pursuit speed to characterize the quality of smooth pursuit in all of our monkeys (Table 1). Prior to muscimol injection, steady-state eye speed was close to target speed (20°/s) for leftward and rightward trials (Fig. 1, A and C). After muscimol injection in the right DLPN, eye speed for ipsilesional pursuit (rightward) was reduced to only 32-61% of preinjection values (Fig. 1, B and C). The impairments observed during ipsilesional smooth pursuit were statistically significant (P < 0.01). In all injections for three monkeys, initial acceleration of ipsilesional smooth pursuit was reduced by 35-68%. For example, in monkey C, the average initial eye acceleration during ipsiversive smooth pursuit was significantly reduced (P < 0.01; t-test) from 131.8 to only 41.6°/s². This corresponds to a postinjection performance reduction of 68%. There were no significant deficits in pursuit in the contralesional direction. We also observed deficits in the monkey's ability to generate and maintain vertical smooth pursuit (Table 1). In Fig. 1C, we show that even 2.5 h after muscimol injection, there was still a clear deficit in ipsilesional initial acceleration (e.g., 35.9°/s²) and steady state velocity (4.8°/s) of smooth pursuit. Further details regarding the values of initial acceleration and steady-state velocity are shown in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Effects of right dorsolateral pontine nucleus (DLPN) inactivation by muscimol on smooth pursuit for monkey C. Left: ipsiversive and contraversive tracking in control (A) and DLPN inactivation experiments (B), respectively. Ten individual trials (left) and average (right) of smooth eye velocity as a function of time for target motion at 20°/s are shown. Upward deflections show rightward eye velocity.  and - - -, average eye velocity for control and inactivation data; · · · , eye velocity 2.5 h after muscimol injection.

Visual modification of the VOR

Prior to adaptation, VORd gain ranged between 0.89 and 0.96 in our three animals. VORl gain was significantly increased when the animals viewed a stationary target (range: 0.98-1.06). Immediately on viewing through the ×2 or ×0.5 lenses, VORl gain during 0.2-Hz, 30°/s head rotations showed significant increases (41-43%) or decreases (39-63%; Fig. 2, C and D). The observed changes were always symmetric. After 2 h of lens viewing, the VORd gain was tested to measure the degree of adaptation. VORd gain showed significant and appropriate changes in both the ×2 and ×0.5 adaptive conditions (Fig. 2, E and F). The VORd gain after ×2 adaptation was increased by 23-32% and 24-32% for ipsi- and contraversive eye velocities. Likewise, after ×0.5 adaptation, the gain was reduced by 22-48% and 23-43% for ipsi- and contraversive eye velocities. These values served as controls for our muscimol experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effects of right DLPN inactivation on the vestibuloocular reflex (VOR) for monkey C. From top to bottom (A-F), the traces are eye and head velocity during sinusoidal head rotation in darkness (A), viewing a stationary surround in the light (B), viewing a stationary surround through ×2 lenses before adaptation (C), viewing a stationary surround through ×0.5 lenses before adaptation (D), in darkness after ×2 adaptation (E), in darkness after ×0.5 adaptation (F).  and - - -, head and eye velocities, respectively. Eye velocities are inverted and saccades have been removed.

During muscimol injection experiments, we first verified the efficacy of DLPN inactivation using smooth-pursuit tracking criteria and then tested visual-vestibular interaction. Before adaptation experiments commenced, we found that postinjection VORd gain (Fig. 2A) was not significantly different from preinjection gain values (t-test; P > 0.93). Likewise, the VORl gain was not significantly different (t-test; P > 0.86) between pre- and postinjection conditions when viewing a stationary target (Fig. 2B). In the ×2 and ×0.5 viewing conditions immediately after muscimol injection, the VORl gain increased (40-43%) and decreased (32-68%) symmetrically in spite of strictly unilateral DLPN inactivation. These values were not significantly different from preinjection values (t-test; P > 0.43; Fig. 2, C and D). After 2 h of lens viewing, the VORd gain associated with ×2 adaptation conditions was increased by 25-31% and 26-32% for ipsi- and contralesional eye velocities. These values were not significantly different from preinjection adaptation values (t-test; P > 0.75; Fig. 3B). Likewise, after 2 h of ×0.5 viewing, the VORd gain was reduced by 24-48% and 23-48% for ipsi- and contralesional eye velocities. These were similar to preinjection adaptation values (t-test; P > 0.61; Fig. 3C). Importantly, even after 2 h of adaptation, there was still a clear deficit in ipsilesional smooth pursuit indicating that our DLPN block was stable throughout the adaptation period (Fig. 1C). Our results demonstrate that after unilateral DLPN inactivation, there was a strong ipsilesional smooth pursuit deficit, but no effect on the visual-vestibular behaviors we measured (Fig. 2) or short-term VOR adaptation.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Bar graphs quantifying parameters of smooth pursuit and VOR adaptation by showing the mean and SD of ipsiversive and contraversive initial eye acceleration for smooth pursuit (A), VOR gain adaptation at ×2 (B) and ×0.5 (C) conditions. *, statistically significant (P < 0.01) effects of unilateral DLPN inactivation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The major finding in this study is that VOR adaptation proceeds symmetrically in the presence of a unilateral DLPN inactivation that produces an asymmetric deficit in smooth pursuit. It is important to note that it has been demonstrated already that DLPN lesions also impair optokinetic nystagmus (OKN) associated with large-field visual image motion (May et al. 1988). It is possible that this impairment relates to the pursuit component of OKN (see Fuchs and Mustari 1993 for review). We assume that our muscimol injections silenced neurons that carry signals related to either large- or small-field (foveal/parafoveal) visual motion. This is because DLPN neurons with either large or small visual receptive fields are found mixed together through out the full extent of the nucleus (Mustari et al. 1988; Suzuki et al. 1990; Thier et al. 1988).

Our result suggests that short-term gain adaptation of the VOR may not depend on the DLPN. Furthermore, even though we found a significant deficit in smooth pursuit after unilateral DLPN inactivation, there was no change in the VORd or VORl gain prior to adaptation. This suggests that visual enhancement of the VOR depends on pathways other than those mediated by the DLPN.

Previous chemical lesion studies involving the DLPN have shown that unilateral lesions caused deficits in smooth-pursuit eye movements directed toward the side of lesion (May et al. 1988). In those studies, reversible or permanent lesions were produced either with lidocaine-HCl or the excitotoxin, ibotenic acid. In our study, we made reversible lesions in the DLPN using muscimol. Muscimol is likely to be effective in silencing DLPN output because neurons there have significant concentrations of GABA_A receptors (Brodal 1988; Mihailoff 1990). Our DLPN inactivation experiments produce ipsilesional smooth pursuit deficits that are comparable to those reported in earlier studies (May et al. 1988). The fact that our smooth-pursuit deficits were stable throughout the entire duration of our short-term adaptation experiments demonstrates the efficacy and specificity of our reversible lesions. Studies by Arikan and Colleagues (2002) indicate that the zone of inactivation after a muscimol injection comparable to ours is likely to be several mm across. Furthermore, the zone of inactivation, as determined by single-unit recording, persists over the course of several hours. Similar durations of effective blockade after muscimol injections have been reported in the monkey nucleus of the optic tract (NOT) (Mustari et al. 2001; Yakushin et al. 2000). Our current results indicate that we were able to produce an effective block during the full time period we used for rapid adaptation of the VOR. Nevertheless, it is possible that some neurons in the DLPN remain active. Because our smooth-pursuit deficits are as large as any reported regardless of the type of unilateral lesion employed, we would argue that if the DLPN played a significant role in short-term adaptation of the VOR that we would see some asymmetry in the adapted VOR. Finally, in every case, we found that smooth pursuit was back to normal values the day after our injections.

We expect that our unilateral muscimol injections produced a significant disruption in visual slip information sent to the ventral paraflocculus. Studies by Rambold and colleagues (2002) indicate that the macaque ventral paraflocculus plays a role in both smooth pursuit and adaptation of the VOR. Our studies were specifically designed to determine whether interrupting visual signals carried by DLPN derived mossy fibers would effect visual-vestibular behavior. It should also be noted the DLPN neurons may carry partially formed commands for smooth pursuit (Mustari et al. 1988; Thier et al. 1988) and ocular following response (Kawano et al. 1992) to the ventral paraflocculus. These eye movement systems are thought to rely on similar pathways (see Kawano et al. 1994). Current modeling studies are consistent with the idea that the cortical projections from areas MT and MST to the DLPN play a role in smooth pursuit but not necessarily in the VOR (see Tabata et al. 2002 for review).

By examining the phase relationship of simple- and complex-spike firing in Purkinje cells recorded during conditions that would result in VOR gain increase or decrease, Raymond and Lisberger (1998) have argued that only complex spikes carry the necessary information to drive visually guided motor learning at high-frequency (>5 Hz). However, at lower frequencies, either mossy-fiber-driven simple spikes or climbing-fiber-driven complex spikes could play a role in VOR adaptation. One mechanism could involve correlated activity arriving over visual climbing fibers and vestibular mossy fibers. These signals would not be entirely coincident due to the significantly different visual and vestibular latencies carried in visual climbing fibers compared with vestibular mossy fibers (see du Lac et al. 1995; Quinn et al. 1998; for review). Correlated activity of complex spikes carried over climbing fibers and simple spikes carried in vestibular parallel fibers could induce high-frequency adaptation. It is possible climbing fiber inputs mediate their effect by evoking long-term depression in Purkinje cells (Ito 1972). Simple-spike modulation produced by correlated activity of vestibular and visual inputs carried over mossy fiber pathways could potentially induce low-frequency adaptation through different mechanisms. Our studies do not address the site of motor learning in the VOR (see du Lac et al. 1995; Hirata and Highstein 2001; Hirata et al. 2001; for review). Our studies only consider one potential source of visual motion information that could provide error signals for visually guided motor learning in the VOR.

Results obtained in our muscimol inactivation studies indicate that the DLPN, which provides visual signals to the vestibulo-cerebellum through the mossy fiber inputs, does not appear to contribute to VOR adaptation during low-frequency head rotation. However, it is possible that other basilar pontine nuclei such as the nucleus reticularis tegmenti pontis (NRTP) could provide retinal image slip information via mossy fibers to guide modification of the VOR at low frequency.

There is strong direct evidence that visual climbing fibers are essential for adaptation of the VOR produced in a visual-vestibular mismatch paradigm. The most important source of visual climbing fibers (carrying retinal image motion signals) is derived from the dorsal cap of the inferior olive. This structure receives visual projections from the accessory optic system and pretectal NOT (Buttner-Ennever et al. 1996; Fuchs and Mustari 1993; Mustari et al. 1994; see for review). These projections carry direction- and velocity-sensitive visual-motion information (Hoffmann and Distler 1989; Mustari and Fuchs 1989, 1990). Yakushin and colleagues (2000) provided evidence that the primate NOT plays a role in VOR adaptation during low-frequency head rotation. They demonstrated that after unilateral muscimol inactivation of NOT, VOR gain could not be adaptively reduced during short-term adaptation experiments similar to those in this study. Importantly, they also found that the adapted VOR state could not be maintained after NOT lesions. These findings support the argument that NOT-derived signals are essential for VOR plasticity. We found no such effects after unilateral DLPN inactivation. This probably reflects fundamental differences in the efferent pathways of the NOT and DLPN. The NOT has a strong projection the dorsal cap of the inferior olive, the sole source of visual climbing fibers to the cerebellum. In contrast, the DLPN can only influence the cerebellum over mossy fiber pathways. These results taken together with the constraining findings of Raymond and Lisberger (1996) indicate that the visual climbing fiber pathways from the accessory optic system and NOT play an essential role in VOR adaptation at both high- and low-frequency head rotation.

Anatomical studies indicate the DLPN projects most heavily to the ventral paraflocculus (Glickstein et al. 1994; Nagao 1990, 1997). Recent lesion studies indicate that the ventral paraflocculus contributes to both smooth pursuit and VOR adaptation (Rambold et al. 2002). Therefore it is reasonable that visual mossy fiber input to ventral paraflocculus from DLPN plays a role in smooth pursuit but may not necessarily play a role in adaptation of the VOR.

In summary, our results suggest that mossy fiber inputs to the ventral paraflocculus from the DLPN contribute to smooth pursuit but not to VOR adaptation during low-frequency head rotation. Perhaps another structure such as the NRTP or climbing fiber inputs from the dorsal cap of the inferior olive to either part of the floccular complex participate in the short-term VOR adaptation. Further studies will be necessary to determine the potential role of the bilateral DLPN and NRTP in VOR adaptation.