Gaze-Stabilizing Deficits and Latent Nystagmus in Monkeys With Early-Onset Visual Deprivation: Role of the Pretectal NOT

Michael J. Mustari,1 Ronald J. Tusa,1 Andrew F. Burrows,2 Albert F. Fuchs,3 and Christine A. Livingston2

 1Yerkes Regional Primate Research Center and Department of Neurology, Emory University, Atlanta, Georgia 30322;  2Department of Anatomy and Neuroscience, University of Texas Medical Branch, Galveston, Texas 77555; and  3Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mustari, Michael J., Ronald J. Tusa, Andrew F. Burrows, Albert F. Fuchs, and Christine A. Livingston. Gaze-Stabilizing Deficits and Latent Nystagmus in Monkeys With Early-Onset Visual Deprivation: Role of the Pretectal NOT. J. Neurophysiol. 86: 662-675, 2001. We studied the role of the pretectal nucleus of the optic tract (NOT) in the development of monocular optokinetic nystagmus (OKN) asymmetries and latent nystagmus (LN) in two monkeys reared with binocular deprivation (BD) caused by binocular eyelid suture for either the first 25 or 55 days of life. Single-unit recordings were performed in the right and left NOT of both monkeys at 2-3 yr of age and compared with similar unit recordings in normally reared monkeys. We also examined ocular motor behavior during electrical stimulation of the NOT and during pharmacological inactivation and activation using GABAA agonists and antagonists. In BD animals a large proportion of NOT units was dominated by the contralateral eye, in striking contrast to normal animals where 100% of NOT units were sensitive to stimuli delivered to either eye. In the 55-day BD animal no binocularly sensitive neurons were found, while in the 25-day BD animal 60% of NOT units retained at least some binocular sensitivity. Differences in direction sensitivity were also observed in BD animals. We found that 56% of units in the 55-day BD monkey and 10% of units in the 25-day BD monkey responded preferentially to contraversive visual motion. In contrast, only 5% of the NOT units encountered in normally reared monkeys respond preferentially during contraversive visual motion, the rest were most sensitive to ipsiversive visual motion. NOT neurons of BD monkeys showed a wide range of speed sensitivities similar to that of normal monkeys. Unilateral electrical stimulation of the NOT in BD animals induced a conjugate nystagmus with slow phases directed toward the side of stimulation. When we blocked the activity of NOT units with muscimol, a potent GABAA agonist, LN was abolished. In contrast, LN was increased when spontaneous activity of the NOT was enhanced with bicuculline, a GABAA antagonist. Our results indicate that the NOT in BD monkeys plays an important role in the OKN deficits and LN generation during monocular viewing. We hypothesize that the large proportion of units dominated by the contralateral eye contribute to the development of monocular OKN asymmetries and LN.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In our previous paper (Tusa et al. 2001), we described the eye movement behavior of monkeys reared with brief periods of early binocular deprivation of vision (BD). The presence of monocular optokinetic nystagmus (OKN) asymmetry and latent nystagmus (LN) suggested to us that there might be a problem in the neural properties of the nucleus of the optic tract (NOT). The NOT contains neurons that are sensitive to visual motion, typically, toward the side of recording (Hoffmann et al. 1988; Mustari and Fuchs 1990). The visual sensitivity of units in the NOT is derived from contralateral retinal (Hoffmann and Stone 1985) and ipsilateral striate and extrastriate visual cortical inputs (Baleydier et al. 1990; Hoffmann et al. 1992; Mustari et al. 1994). Many units in the primate NOT have very large receptive fields appropriate for encoding full-field visual motion to support optokinetic eye movements (Hoffmann et al. 1988; Mustari and Fuchs 1990; see Fuchs and Mustari 1993 for review). Lesions of the NOT of rabbits (Collewijn 1975b), cats (Precht and Strata 1980), and primates (Kato et al. 1988; Schiff et al. 1990) eliminate the slow buildup in OKN eye velocity, toward the side of lesion, during horizontal full-field visual motion. The slow buildup in eye velocity during OKN is thought to be due to the charging of a velocity storage mechanism (Raphan et al. 1977). The NOT could provide inputs to the velocity storage mechanism through projections to the medial vestibular nucleus (MVN) and nucleus prepositus hypoglossi (NPH) (Belknap and McCrea 1988; Büttner-Ennever et al. 1996a; Magnin et al. 1983; Mustari et al. 1994). Earlier studies have shown that NOT unit responses are affected by lesions of the visual cortex or disruptions of binocular vision early in development (see Schor 1983 for review). For example, NOT units may lose their ability to respond to high-velocity visual motion or small-sized stimuli after lesions of the visual cortex (Hoffmann 1982). NOT units lose their binocularity when animals are reared with early onset strabismus (Cynader and Hoffmann 1981; Distler and Hoffmann 1996) or monocular deprivation (Hoffmann 1983). Behaviorally, such animals exhibit an asymmetric OKN, like that found in human infants (see Schor 1993 for review).

We hypothesize that monocular OKN deficits and LN in BD monkeys are due to a defect in the NOT. To adequately test this hypothesis, we conducted single-unit studies recording studies in the NOT of BD monkeys. A preliminary report of this study has appeared previously (Mustari et al. 1995).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have described most of the methods used in this study in detail elsewhere (Mustari and Fuchs 1990), so only a brief description is provided here. All of the work described here was approved by our respective Institutional Animal Care and Use Committees.

We prepared animals for single-unit recording experiments as described below. Single-unit activity was recorded from the NOT in the two deprived (25- and 55-day BD) and the three normal monkeys when they were 2-3 yr old. Eye movements were measured with an implanted scleral search coil (Judge et al. 1980) and an electromagnetic induction method (Fuchs and Robinson 1966). All surgical procedures were performed under aseptic conditions with the monkey under deep anesthesia (halothane 1.5%).

During single-unit recording sessions, the monkeys sat in a sound-dampened and light-proof enclosure facing a tangent screen onto which the target spot and other visual stimuli were rear-projected. The monkeys were rewarded for fixating a stationary target spot while other visual test stimuli were moved on the tangent screen, for making accurate saccades to target steps and for smoothly tracking a sinusoidally oscillating target. Visual stimuli were either random dot patterns subtending a large portion of the visual field (70° × 50° or 100° × 100°) or a small-diameter target spot (0.1°). Visual test stimuli were moved in various directions at various speeds (1-200°/s) with either sinusoidal or constant velocity wave shapes.

Extracellular single-unit potentials were recorded with tungsten microelectrodes by conventional methods (Mustari and Fuchs 1990). Target- and eye-position signals, unit discharge, and other relevant signals were saved on analog or digital-audio tape for subsequent computer digitization and quantitative off-line data analysis. The eye and target positions were digitized at 1 kHz. We used a window discriminator (Bak Electronics) to provide a transistor-transistor logic (TTL) acceptance pulse that accurately indicated the time of each action potential. The rising phase of the acceptance pulse was recorded with an accuracy of 10 µs with an interrupt driven process.

Single-unit recording experiments were performed in different ways in the 25- and 55-day BD animals. The 55-day BD monkey had difficulty suppressing nystagmus and holding good fixation, even during binocular viewing. Therefore we immobilized the eye contralateral to the NOT recording site. This immobilization was performed when the animal was 3 yr of age. We used a retrobulbar injection of 15 units of botulinum toxin-A (BTX; provided by Dr. Alan Scott, Smith-Kettlewell Institute, San Francisco and administered by Dr. Ray Gariano) to immobilize the eye. The immobilization reduced eye velocities to less then a few degrees per second and lasted for the entire time we performed single-unit recording experiments in the 55-day BD monkey. This allowed us to perform accurate visual receptive-field testing. Each eye was tested for visual sensitivity during viewing of a stationary or moving visual stimulus. We measured the eye movements in the mobile eye during single-unit recording and LN testing in this animal. When the animal was returned to his cage, the eyelid over the immobilized eye was held closed by a single mattress suture (8-0 nylon). This ensured that the cornea stayed protected in the event that the Botulinum interfered with blinking. We did not check for potential adaptation of LN during this monocular viewing condition. However, extensive behavioral testing was performed prior to retrobulbar injections (see Tusa et al. 2001). Because the 25-day BD animal had little trouble fixating or suppressing nystagmus during binocular viewing (see RESULTS), a retrobulbar BTX block was unnecessary. Single-unit data from the NOT of normally reared monkeys, obtained in previous and ongoing studies (Mustari and Fuchs 1990), were used for comparative purposes.

To pharmacologically activate or inactivate the NOT, we placed microinjections of a GABAA antagonist (bicuculline methiodide) or GABAA agonist (muscimol) into the NOT. Reversible activation or inactivation, through modulation of GABAA circuits, was achieved by delivering small (0.2 µl) injections of bicuculline (2%) or muscimol (2% in sterile physiological saline) at the center of the NOT. For control injections, we used similar volumes of sterile saline. For all injections, we replaced our standard tungsten single-unit recording electrode with a glass pipette of relatively small tip diameter (15 µm). Injections were delivered slowly over the course of about 2 min by applying short-duration (10-50 ms), low pressure pulses (20-50 psi) with a pico-pump (W.P.I. PV-830).

We marked the location of our recording sites by placing electrolytic lesions (10 µA D.C. for 10 s) on several electrode tracks in each animal. At the conclusion of the experimental series, the animal was given a lethal dose of barbiturate and perfused transcardially, with saline followed by 10% Formalin. Frozen sections were cut in a coronal stereotaxic plane every 50 µm, mounted on microscope slides, and stained with cresyl violet for histologic reconstruction of electrode tracks and unit locations.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Location of recording

We were able to use well-established functional landmarks (Mustari and Fuchs 1990) to verify that we were recording in the NOT including localizing following-omnipause neurons (FOPNs) immediately dorsal to the NOT (Mustari and Fuchs 1990; Mustari et al. 1997). At the end of our recording experiments, histological sections were stained to localize electrode tracks as described in METHODS. Figure 1 shows examples of lesions (arrows) placed on two different rostral NOT tracks in the 55-day BD monkey. These lesions were placed either at the depth where we recorded direction-selective units (medial track) or 1 mm deep to these units (lateral track).



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Fig. 1. Photomicrograph of a 50-µm-thick cresyl violet-stained section cut in the coronal stereotaxic plane. The stereotaxic level corresponds to A0.5 in the atlas of Snider and Lee (1973). Two microlesions (arrows) were placed on different representative electrode tracks at the depth (medial track) or 1 mm deep (lateral track) to the site where direction-selective nucleus of the optic tract (NOT) units were recorded. The approximate location of the NOT at this level is indicated by the dotted lines on the right (see Mustari et al. 1994). NOT, pretectal nucleus of the optic tract; IV, trochlear motor nucleus. Scale bar = 1 mm.

We recorded from the NOT on each side of the brain stem in both the 25- and 55-day BD monkeys. We tested units for ocular dominance, visual speed sensitivity, direction selectivity, receptive-field size, and spontaneous activity in the light and dark. Unfortunately, in some cases, we lost isolation before completing all testing.

Direction selectivity

NOT units of BD monkeys, like those of normally reared animals, were differentially sensitive to the direction of visual motion. Figure 2 (left column) shows examples of directional tuning curves obtained in normally reared and in BD monkeys (25 or 55 day). We tested direction selectivity by presenting a large-field random-dot pattern moving in eight different directions, separated by 45°, either to a fixating monkey or immobilized eye. We estimated the preferred direction of each unit by fitting a curve to the average firing rate measured at each direction (Mustari and Fuchs 1990; Wallman and Velez 1985). The point where the fitted curve reached a maximum was considered the preferred direction. Figure 2 shows the preferred directions for our population of NOT neurons in polar histogram form (Fig. 2, right column). Included in the plot for the 55-day BD case are seven neurons that we classified as purely ipsiversive. For these cells, we used the results obtained during horizontal motion to assign them to the ipsiversive group because isolation was lost before pure vertical visual motion was tested. In normally reared monkeys, most (95%; n = 44) NOT units prefer ipsiversive visual motion. Ipsiversive units also were most common (90%; n = 38) in the 25-day BD monkey. However, a striking difference in direction preference was observed in the 55-day BD monkey where 56% (n = 35) of NOT units responded preferentially during contraversive visual motion.



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Fig. 2. NOT unit direction selectivity: direction preference was tested by moving a large-field visual stimulus in 8 different directions (left column) while the monkey fixated a stationary target spot located at the center of the tangent screen. Tic mark indicates 10 spikes/s. Dashed circles in the left column indicate the spontaneous firing rate level for each neuron. The preferred direction of the unit was taken as the point where the curve fit to the data reached a maximum. A: directional preference of NOT units recorded in 2 normal monkeys. B: similar testing and data obtained from the 25-day binocularly deprived (BD) monkey. C: directional tuning recorded as above for the 55-day BD monkey; however, here the eye was immobilized by retrobulbar blockade with botulinum toxin A (see METHODS). Right column, polar histograms for the 3 test groups with number of units indicated on the horizontal axis. The number of units in each histogram sector is indicated on the 0° axis. Ipsiversive direction is toward the right (0°).

The directional tuning for individual NOT units was relatively broad in both normally reared and BD monkeys. We estimated the broadness of directional tuning by computing a bandwidth that takes into account average firing rate in both the excitatory and inhibitory directions (Albright 1984). Bandwidth was estimated as the width at half-maximum firing rate. Bandwidths ranged from relatively narrow as shown for the unit illustrated in the middle left column of Fig. 2 to broad (Fig. 2, top left column) in both normally reared and BD monkeys. Figure 3 shows the bandwidth data for normally reared and BD monkeys. The bandwidth histograms showed considerable overlap with means of 126 ± 32° (mean ± SD), 140 ± 53°, and 110 ± 20° for normal, 25-day BD, and 55-day BD, respectively. We performed a one-way ANOVA (Tukey test) and found no significant difference in the bandwidth distributions of normal versus BD (either 25 day or 55 day) NOT units.



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Fig. 3. Histograms of bandwidths taken from tuning curves like those shown in Fig. 2. Bandwidth was estimated as the width (in degrees) of the response vectors exceeding the value centered between maximal and minimal responses. Bandwidth tuning means A, 126 ± 32; B, 140 ± 53; C, 110 ± 20.

We used the vectors associated with maximum and minimum firing to construct a directionality index for each unit. The degree of direction selectivity for a given unit can be described by the ratio of activity in the maximum and minimum directions (max - min)/(max). Units were judged as highly direction selective if the above ratio was greater than 0.5 and direction biased if the value exceeded 0.25. If a unit had appreciable spontaneous firing rate, this could be accounted for by computing a second index (max - min)/max - spont). A value greater than 1 with this index indicated strong directional tuning (not illustrated). As illustrated in Fig. 4, the majority of units were highly direction selective or biased. This remained the case when spontaneous firing rate was taken into account (2nd index). Furthermore, the degree of direction selectivity was similar in normally reared and BD monkeys. We performed a one-way ANOVA and found no significant difference in the directionality indexes of normal versus BD NOT units.



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Fig. 4. Directional index computed for direction-selective NOT units as illustrated in Fig. 2. Histogram bars represent the percentage of units in each group. The number of units tested in each condition is indicated under the abscissa. The directionality index computed for all units was the difference between maximum and minimum responses divided by the maximum response. An index value = 1, indicates 100% direction selectivity. A: normal monkeys. B: 25-day BD. C: 55-day BD.

Ocular dominance

We tested the ocular dominance preference of NOT units in our BD monkeys by presenting stimuli moving in the optimal direction, over a similar range of velocities (see Visual speed sensitivity below) to those tested during binocular viewing. We measured the average firing rate in the preferred direction, tested through each eye, to assign units to one of five different ocular dominance groups. Units driven exclusively by the contralateral (contra) or ipsilateral (ipsi) eye were placed in group 1 or 5, respectively. Units biased toward either the contralateral or ipsilateral eye were placed in groups 2 or 4, respectively. NOT units driven equally by either eye were placed assigned to group 3. To place a unit into one of these groups, we calculated an ocular dominance index (OD) from the ratio (contra - ipsi)/(contra + ipsi). Units in OD group 1 (contra) had OD indexes >0.95. Units with OD index near 0.0 ± 0.2 were assigned to group 3. Other units were assigned to groups 2 or 4. Units biased toward the ipsilateral eye had negative values using the above index. We encountered no units driven exclusively from the ipsilateral eye in our monkeys. In the normal monkey, all NOT units can be driven from either eye (Hoffmann 1992; Inoue et al. 2000; Mustari and Fuchs 1990). As shown in Fig. 5, NOT units in BD monkeys had a strikingly abnormal distribution of ocular dominance. In the 55-day BD monkey, all units (n = 31) were driven exclusively by the contralateral eye. In the 25-day BD monkey, 62% of NOT units were dominated by the contralateral eye with some responding only to stimulation of the contralateral eye. However, binocular units were present in this animal, and a few of these units even showed a bias in favor of the ipsilateral eye (group 3). The pattern of ocular dominance in BD animals is clearly biased toward the contralateral eye. We performed a one-way ANOVA (Tukey test) and found that the ocular dominance distributions of normal and BD units were significantly different (P < 0.001).



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Fig. 5. NOT unit ocular dominance preference. Units were 1st tested during binocular viewing. The optimal stimulus was determined and then used to test the unit's response during monocular viewing. Assignment to an ocular dominance group was determined using a ratio of response obtained during visual stimulation presented to each eye (see text). Units that responded only during stimulation of one eye were placed in group 1 (contra-eye) or group 5 (ipsi-eye). Units with equal responses during stimulation presented to either eye, were placed in group 3. Units with a bias toward the contra- or ipsilateral eyes were placed in group 2 or group 4, respectively. Open histogram bars, normal; shaded bars, 25-day BD; dark bars, 55-day BD.

Visual speed sensitivity

We tested the speed sensitivity of NOT units in BD monkeys by moving a large-field visual stimulus in the unit's preferred direction over a series of speeds from 1 to 200°/s while the monkey fixated a stationary target spot. In Fig. 6, we illustrate representative speed tuning curves from each of our monkeys. We tested the speed sensitivity of 50 neurons in 3 normal monkeys, and 35 neurons in BD monkeys (n = 15, 55 day BD; n = 20, 25 day BD). As in normally reared monkeys, NOT units of BD monkeys responded over a wide range of stimulus speeds (Fig. 6). Some units showed firing rates that increased with stimulus speed over most of the range tested; others preferred or were tuned to a specific speed. In all animals, we found clear examples of NOT units that were most sensitive to either high (>50°/s) or low visual speeds (Fig. 6). We conclude that BD monkey NOT units retain sensitivity to high-speed visual motion (i.e., >50°/s). However, the peak firing rate at the optimal speed tended to be lower in the 55-day BD animal compared with normal animals.



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Fig. 6. Examples of speed tuning for NOT units of normal (A), 25-day BD (B), and 55-day BD monkeys (C). Speed tuning was tested by moving a large-field visual stimulus in the preferred direction for each unit. The average firing rate over at least 10 cycles of such testing was used to plot the individual tuning curves shown. Data from different representative units are indicated by different symbols.

Initiation of LN

To measure the onset time of LN accurately, we placed ferro-electric liquid crystal shutters that close within 35 µs (Display Tech) in front of both eyes of the 25-day animal. We performed this testing when the animal was 3 yr old. Sample data from this testing are shown in Fig. 7. After the animal fixated a 0.1° stationary red target spot on a stationary 100° by 100° random dot field, the shutter in front of one eye was closed. Neither the fixation target nor random dot pattern were moved during this testing. To estimate the latency of LN initiation, we used the point where eye velocity exceeded baseline by 3°/s. LN was initiated at relatively short latency averaging 82.2 ± 9.0 ms (11 trials). Unfortunately, for our earlier experiments with the 55-day BD animal, we did not have the liquid crystal shutters, so data regarding the latency of LN are not available for that animal.



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Fig. 7. Initiation of latent nystagmus (LN) in the 25-day by closure of a shutter placed in front of the right eye. The vertical dashed line indicates shutter closure time. In A, a single example of LN initiation and subsequent slow phases are shown. In B, we show the average LN eye velocity trace from 11 trials taken from the time region indicated by the box in A. Onset time of LN was 82.2 ms (arrowhead), which was defined when eye velocity increased by 3°/s over baseline. He, horizontal eye position; He, horizontal eye velocity; Sh, shutter synch signal (the trace deflects up when the shutter closes).

Unit response during LN

If the NOT plays a role in LN, we would expect units there to be modulated during LN. Figure 8 illustrates the response of a unit recorded in the right NOT during LN. This unit had a large receptive field and preferred contraversive visual motion. For this testing, the monkey (25-day BD) viewed a 100° × 100° stationary random dot pattern and a stationary fixation spot rear-projected on a tangent screen. The unit illustrated in Fig. 8 discharged during each LN slow phase to the right (Fig. 8A), when visual motion was contraversive (leftward). Like all NOT units tested (n = 25), this one showed no response in association with the quick phases of nystagmus, whether ipsiversive (as here) or contraversive. Furthermore, units with ipsiversive direction preference were inhibited during LN. To verify that unit response during LN was visually contingent and not related to the eye movement per se, we blinked off the stationary visual stimulus during LN (Fig. 8A). Following the blink, unit discharge ceased, even though slow-phase eye movement continued. Furthermore, when the next slow phase started, NOT unit discharge did not resume at the expected latency (Fig. 8A, arrow). Finally, when the stationary visual scene was turned back on during a slow phase, the unit response resumed at its characteristic visual latency (55 ms). In Fig. 8B, NOT unit responses for 21 successive LN slow phases have been aligned on the start of each slow phase. Because slow phases vary in duration, the response duration in some rasters is shorter than in others. The peristimulus time histogram (Fig. 8B) for times later then 200 ms after slow-phase start decrements due to variable duration slow-phase events. The latency between slow-phase start time and the onset of unit discharge illustrated in Fig. 8 averages 55 ± 12 ms.



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Fig. 8. NOT unit discharge during LN associated with monocular viewing through the left eye. A: several successive slow phases of LN are shown. The stationary visual stimulus was shuttered off (bottom trace) during the 3rd and 4th slow phases shown. The unit ceases its discharge during the time the stimulus is off (see text). Arrow indicates the expected time (visual latency), after slow phase start, for the unit to discharge if the visual stimulus was present. B: data from 21 successive slow phase events aligned (vertical line) on slow-phase start time. The unit discharge associated with each slow-phase event is shown in raster form along with the average firing rate histogram below.

Spontaneous firing rate

Because LN can be initiated during viewing of a featureless scene or ganzfeld (Tusa et al. 2001), we checked whether NOT unit discharge was associated with luminance level. For this testing, we measured the resting firing rate during binocular viewing of a ganzfeld and compared this activity to that recorded in complete darkness. We restricted this testing to pretectal units that were direction selective (n = 15). In the absence of visual motion, some NOT units had higher firing rates in the light then in darkness; other units showed little difference in firing rate in these two conditions. However, we did not test the effects of luminance on NOT unit response during stimulation with moving visual stimuli.

Electrical stimulation of NOT

In earlier studies of normally reared monkeys, we found that stimulus trains (50-250 Hz) of low current (10-80 µA) biphasic pulses (200 µs duration) delivered at the recording site of direction-selective units elicited an optokinetic-like nystagmus with ipsiversive slow phases (Mustari and Fuchs 1990). Similarly, when we delivered comparable stimulation in BD monkeys, we also generated an optokinetic-like nystagmus with slow phases directed toward the side of stimulation. For example, electrical stimulation applied for 10 s in the 25-day BD monkey produced an ipsiversive slow-phase nystagmus with slow-phase velocities reaching well over 100°/s (Fig. 9). Because this stimulation was delivered during binocular viewing in the light, the animal may have been able to suppress some of his nystagmus by fixating on stationary contours in the large-field random dot pattern. This is indicated by the fall-off in eye velocity during the electrical stimulation. We found the same type of ipsiversive (slow-phase) response during electrical stimulation in the 55-day BD monkey (not illustrated). Therefore, despite the increased percentage of contraversive units in the NOT of BD monkeys, stimulation of their NOTs still produced nystagmus with ipsiversive slow phases.



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Fig. 9. Nystagmus elicited by delivering a train of electrical stimulus pulses in the right NOT of a 25-day BD monkey. Note that immediately following the start of the electrical stimulus train (thick black bar, bottom trace) that rightward slow phase nystagmus is generated. Top trace, horizontal eye velocity; middle trace, vertical eye velocity; bottom trace, electrical stimulus artifact (filled rectangle).

Effect of NOT inactivation on LN

If the NOT plays an important role in LN, silencing its output should produce an effect opposite to that of electrical stimulation, i.e., LN should be reduced or abolished. Rather then use permanent lesions to test this hypothesis, we injected muscimol (a potent GABAA agonist) into the NOT to reversibly block this area. For this testing, the monkey viewed a stationary small target spot centered on a stationary large-field (100° × 100°) random dot pattern.

Covering the left eye of the 55-day BD monkey produced a strong LN with leftward slow phases (Fig. 10A). The left eye was the mobile eye in these experiments. The right eye had been previously immobilized with botulinum injections (see METHODS). After we blocked the left NOT with a small (0.2 µl) injection of 2% muscimol, the direction of nystagmus reversed (Fig. 10B). This reversal in the direction of nystagmus took about 3 min to reach a maximum velocity and lasted for over 1 h. When we tested the animal the following day during right eye viewing, the usual pattern of leftward slow-phase nystagmus was observed. When we performed a bilateral muscimol blockade of the NOTs in the 55-day BD animal, LN was virtually eliminated (Fig. 10C).



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Fig. 10. Effects of muscimol injection in the NOT on LN associated with monocular viewing (55-day BD monkey). LN during right eye viewing is severely reduced following left NOT blockade. A: preinjection. B: postinjection; bottom row, postunilateral injection. C: effects of injections of muscimol in the NOT bilaterally (55-day BD monkey).

Figure 11 shows examples of our muscimol experiments in the 25-day BD monkey. In the 25-day BD day animal, LN was strongest during left eye viewing conditions (e.g., Fig. 11A). Prior to injection, a brisk LN with typical nonconstant velocity slow phases was produced (Fig. 11A). During binocular viewing (Fig. 11B), gaze was stable because the monkey could fixate on components in the stationary scene. During right eye viewing, only a modest LN was present with slow phases toward the left (Fig. 11C). After muscimol injection of the right NOT, LN during left eye viewing was abolished (Fig. 11D). Furthermore, OKN could not be elicited toward the side of muscimol injection (not illustrated). The normally weak LN associated with right eye viewing became much stronger following muscimol injections in the right NOT (Fig. 11F). So we conclude that the muscimol produced a functional inactivation of the NOT.



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Fig. 11. Effects of unilateral muscimol injection in the NOT on LN associated with monocular viewing (25-day BD monkey). LN during left eye viewing is completely abolished following right NOT blockade. Top row, preinjection; bottom row, postinjection. Viewing conditions as indicated.

After bilateral muscimol blockade in the 25-day BD monkey (Fig. 12B), LN was virtually abolished. Here we show the initiation of LN by including the shutter trace representing closure of a liquid-crystal shutter (upward deflection), placed in front of the right eye (Fig. 12, bottom trace). In the preinjection condition (Fig. 12, top panel), the monkey is able to hold a steady fixation during binocular viewing. When the shutter in front of the right eye was closed, a brisk LN was present. However, after simultaneous muscimol injection into both the right and left NOTs, LN does not occur during monocular viewing (Fig. 12, bottom panel). Instead, there is a series of saccades either to the left or right with drifts back to the center of the screen. The time constant of decay in velocity of these drifts averages 300 ms, a value close to that reported for the oculomotor plant. In summary, we found that muscimol blockade of the NOT virtually eliminated LN in both the 55-day BD animal (Fig. 10C) and 25-day BD monkey (Fig. 12B), supporting the hypothesis that the NOT is necessary for LN.



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Fig. 12. Effects of bilateral muscimol injection on LN (25-day BD monkey). A: preinjection LN is initiated by closing a shutter in front of the right eye (bottom trace, LCD shutter synch trace). B: postinjection, when the shutter in front of the right eye is closed LN fails to occur. Rather, gaze instability is present during monocular viewing (see text).

Figure 13 shows the results of delivering bicuculline methiodide (0.2 µl) into the center of the right NOT in the 25-day BD monkey. Bicuculline injections were not performed in the 55-day BD animal. During this testing, the monkey faced a tangent screen with stationary visual stimuli including a centrally located target spot and large-field random dot pattern. Prior to the injection, viewing by the left eye produced LN with slow phases to the right, whereas viewing through the right eye produced LN with slow phases to the left (Fig. 13, A and C). During binocular viewing, gaze was stable (Fig. 13B). After bicuculline injection in the right NOT, slow phases were always ipsiversive (rightward) regardless of the viewing condition (Fig. 13, D-F). Furthermore, slow phases of the pharmacologically evoked nystagmus were virtually constant velocity (e.g., Fig. 13D), unlike the decreasing velocity slow phases of preinjection LN (see Fig. 13A). Therefore bicuculline-evoked nystagmus resembled normal OKN. This result compliments that obtained with electrical stimulation (described above), showing that unilateral NOT activation in the BD monkey produces a nystagmus with ipsiversive slow phases as in normally reared monkeys. During binocular viewing postinjection, there was some decrease in the slow-phase eye velocity, perhaps due to attempted fixation. Control injections of sterile saline of the same volumes used in bicuculline or muscimol experiments produced no change in LN or OKN.



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Fig. 13. Effects of unilateral bicuculline methiodide injection in the right NOT. A single injection of 200 nanoliters of bicuculline produced nystagmus with ipsiversive slow phases. The slow phases of bicuculline-evoked nystagmus typically had nearly constant velocity slow phases. During monocular viewing, LN added with the bicuculline evoked nystagmus. Top row, preinjection; bottom row, postinjection, conditions as indicated.


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In this study, we have tested the hypothesis raised in our previous paper that monocular OKN (MOKN) asymmetry in BD monkeys may be due to a loss of binocular cells in the NOT (Fig. 11, Tusa et al. 2001). We also made several observations in different experiments, which support our hypothesis that the NOT is involved in the production of LN. These observations include the alteration of response properties of NOT neurons in BD monkeys during LN and the amelioration or enhancement of LN with different pharmacological manipulations of the NOT.

Unit discharge in BD monkeys

BINOCULARITY. In normally reared monkeys, all NOT neurons are binocularly driven with approximately equal influence from either eye (Hoffmann et al. 1988; Mustari and Fuchs 1990). In our BD monkeys, however, units were either driven exclusively from the contralateral eye (55-day BD) or biased toward the contralateral eye (25-day BD). Indeed, loss of NOT binocularity in cats is commonly associated with monocular deprivation or strabismus, and in such animals, monocular OKN is asymmetric with weak or absent temporalward responses (see Hoffmann et al. 1996). Our results indicate that the degree of monocular OKN asymmetry could be associated with the degree of binocularity of the NOT. This is because monocular OKN asymmetry was more prominent in our 55-day BD animal compared with the 25-day BD animal. This was coincident with a complete shift in ocular dominance to the contralateral eye in the 55-day BD animal and only a partial shift in the 25-day BD animal.

Loss of NOT binocularity could be due to defective visual cortical input representing, at least, the ipsilateral eye. The fact that we found neurons with high-speed sensitivity in the NOTs of our BD monkeys indicates that, at least, some cortical inputs are preserved. This is because high-speed sensitivity of NOT units has been shown to depend on cortical inputs, at least, in cats (Hoffmann 1982). Currently, nothing is known regarding the cortical connections of the NOT in BD monkeys. However, it might be expected that BD monkeys have a decreased proportion of binocular neurons in all cortical areas projecting to the NOT. In addition, monocular cortical neurons representing the ipsilateral eye may be completely or partially suppressed, at cortical or subcortical levels. Further studies will be required to determine which of these possibilities applies in BD monkeys.

If the afferent limb of the optokinetic system is driven by only one eye, then a nystagmus might be expected to develop when one eye is covered similar to latent nystagmus. This is because such monocular stimulation will necessarily result in an asymmetric activation of NOT units on each side of the brain (see Tusa et al. 2001, Fig. 11). This may be associated with an increased firing rate of NOT neurons in the light, leading to LN. When we checked the resting firing rate of direction-selective NOT units in the light and dark, we did find some units that responded significantly more when the visual stimulus was turned on. Although we cannot rule out that direction-selective units play a role in generating LN, we do know that LN occurs during monocular viewing of a featureless scene, presumably devoid of retinal image motion (Tusa et al. 2001). It is possible that nondirection-selective luminance-sensitive units, for example, like those thought to play a role in pupillary control, may also contribute to the initiation of LN. However, we concentrated our unit studies on direction-selective NOT units. Latent nystagmus also has been reported in monkeys raised with artificial strabismus (Kiorpes et al. 1996). This study focused on cortical visual motion processing and smooth pursuit. They found an abnormal pattern of ocular dominance for units in the middle temporal (MT) visual area, especially in animals with LN. Although Kiorpes and colleagues did not perform brain stem recordings, their results are important in identifying a different type of binocular visual deprivation from our BD approach that produces some LN and reduced cortical binocularity. Our model for LN argues that NOT binocularity would be compromised in such animals.

DIRECTION SELECTIVITY. In the normal monkey, the majority of NOT units are direction selective with over 95% preferring ipsiversive visual motion (Fig. 2). However, in the BD monkeys, units with a preference for contraversive visual motion were more frequently encountered. This was most obvious in the 55-day BD monkey, where 56% of the NOT units preferred contraversive visual motion. It seems unlikely that the directionality of NOT units changed as a result of BD. It is more likely that there was a dropout of normal ipsiversive cells, which raised the proportion of contraversive cells.

Contraversive units do not seem to contribute to the OKN because there is no temporalward response during monocular stimulation. It is possible that these contraversive units could maintain LN for as long as monocular viewing continues. Such contraversive visual units are robustly activated during LN (Fig. 8). For example, a slow phase that moves the eye to the right over a stationary visual scene produces net leftward visual motion. For units recorded in the right NOT, during LN associated with left eye viewing (rightward slow phases), net visual motion is to the left. This is analogous to the situation seen during smooth pursuit of a small target spot moving across a stationary large-field visual pattern. For example, an ipsiversive NOT unit will respond during the contraversive direction of smooth pursuit over a stationary background because the net direction of large-field visual motion remains ipsiversive (Mustari and Fuchs 1990). The response of NOT units during LN could comprise a positive feedback system, where LN slow phases produce visual motion, which produces even stronger NOT activation. This would occur whenever there is a bias for one eye.

OCULAR FOLLOWING, OPTOKINETIC AND LATENT NYSTAGMUS INITIATION. Our studies of LN onset latency provide an important perspective on potential contributing neural pathways. When monocular viewing was initiated by closing a shutter placed in front of the eye, LN occurred with a latency as short as 70 ms. Considering that there was little visual motion while the monkey was actively fixating, such a short LN onset latency suggests the involvement of rather direct visual-motor connections. The shortest latency, visually elicited, eye movement is known as the "short latency ocular following response" (OFR). The OFR has a latency of about 50 ms and is enhanced by triggering a step of large-field visual motion 20-50 ms after a saccade. The OFR uses at least some of the same circuits as OKN (Inoue et al. 2000). The initial rapid jump in eye velocity of OKN has a latency of 95 ms. The difference in OFR and OKN latencies can be accounted for by the way in which visual stimuli are typically presented in these paradigms. OKN is normally elicited by suddenly illuminating a moving stripped drum (see Fuchs and Mustari 1993 for review). In contrast, the OFR is elicited with a step of visual motion of an already illuminated but stationary pattern. Kawano and colleagues (1994) have shown that the OFR is delayed when a moving pattern is suddenly illuminated, resulting in OKN-like latencies (i.e., 95 ms). Therefore we suggest that the LN onset times we observed indicate that LN employs visual afferent pathways very similar to those of the OFR and OKN. These pathways could involve direct retinal projections to the contralateral NOT or cortical-NOT circuits (see Mustari et al. 1994 for review). We know from our work and others (Mustari and Fuchs 1990; Schiff et al. 1988) that electrical stimulation of one NOT produces a nystagmus with slow phases directed toward the side of stimulation. The onset of slow-phase eye velocity produced by electrical stimulation is comparable to that produced by the sudden onset of visual motion (Inoue et al. 2000; Kawano et al. 1992; Mustari and Fuchs 1990) or closing a shutter in front of one eye in our BD monkeys (see Figs. 9 and 12A).

PHARMACOLOGICAL EXPERIMENTS. We found complimentary results in our reversible activation and inactivation studies in BD monkeys. When we placed unilateral injections of bicuculline into the NOT of the 25-day BD animal, a nystagmus with ipsilaterally directed slow phases was produced. Possibly, this occurred because we disinhibited the NOT, producing a net increase in firing rate of the injected NOT and an imbalance between the output of the two NOTs. In contrast, if one NOT is inactivated, a nystagmus with slow phases directed away from the side of injection develops. This nystagmus occurs possibly because the injected NOT is silenced, thus leaving the opposite NOT with a relatively increased net firing rate. Finally, if we block both NOTs simultaneously, LN is virtually abolished for the duration of the blockade.

We have not yet tested other oculomotor sites for their potential roles in LN. For example, the dorsolateral pontine nucleus (DLPN) might be expected to play a role in LN. This is because the DLPN has been shown in single-unit recording and lesion studies to play a role in smooth pursuit (Mustari et al. 1988; Suzuki and Keller 1984; Suzuki et al. 1990; Thier et al. 1988), ocular following (Kawano et al. 1992), and optokinetic nystagmus (May et al. 1988; Mustari et al. 1988). Further studies of the visual motion processing areas of the brain stem and extrastriate cortex associated with the superior temporal sulcus of animals raised with impoverished binocular experience are crucial to developing an understanding of mechanisms responsible for development of NOT binocularity, optokinetic following, LN, and gaze stability.


    ACKNOWLEDGMENTS

The authors acknowledge the expert technical assistance offered by S. Usher and H. McMullen. We also acknowledge the contribution of Dr. Ray Gariano for placing the retrobulbar injections of botulinum toxin to immobilize the eye. We express our appreciation to J. R. Economides for help in preparing figures for the manuscript.

This work was supported by National Institutes of Health Grants EY-06069, EY-00745, RR-00165, and RR-00166.


    FOOTNOTES

Address for reprint requests: M. J. Mustari, Yerkes Regional Primate Research Center, Dept. of Neurology, 954 Gatewood Rd. NE, Emory University, Atlanta, GA 30322 (E-mail: mjmustar{at}rmy.emory.edu).

Received 6 November 2000; accepted in final form 10 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society