Saccade-Related Inhibitory Input to Pontine Omnipause Neurons: An Intracellular Study in Alert Cats

Kaoru Yoshida,1 Yoshiki Iwamoto,1 Sohei Chimoto,1 and Hiroshi Shimazu2

 1Department of Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575; and  2Department of Neurophysiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Yoshida, Kaoru, Yoshiki Iwamoto, Sohei Chimoto, and Hiroshi Shimazu. Saccade-Related Inhibitory Input to Pontine Omnipause Neurons: An Intracellular Study in Alert Cats. J. Neurophysiol. 82: 1198-1208, 1999. Omnipause neurons (OPNs) are midline pontine neurons that are thought to control a number of oculomotor behaviors, especially saccades. Intracellular recordings were made from OPNs in alert cats to elucidate saccade-associated postsynaptic events in OPNs and thereby determine what patterns of afferent discharge impinge on OPNs to cause their saccadic inhibition. The membrane potential of impaled OPNs exhibited steep hyperpolarization before each saccade that lasted for the whole period of the saccade. The hyperpolarization was reversed to depolarization by intracellular injection of Cl- ions, indicating it consisted of temporal summation of inhibitory postsynaptic potentials (IPSPs). The duration of the saccade-related hyperpolarization was almost equal to the duration of the concurrent saccades. The time course of the hyperpolarization was similar to that of the radial eye velocity except for the initial phase. During the falling phase of eye velocity, the correlation between the instantaneous amplitude of hyperpolarization and the instantaneous eye velocity was highly significant. The amplitude of hyperpolarization at the eye velocity peak was correlated significantly with the peak eye velocity. The time integral of the hyperpolarization was correlated with the radial amplitude of saccades. The initial phase disparity between the hyperpolarization and eye velocity was due to the relative constancy of peak time (~20 ms) of the initial steep hyperpolarization regardless of the later potential profile that covaried with the eye velocity. The initial steep hyperpolarization led the beginning of saccades by 15.9 ± 3.8 (SD) ms, which is longer than the lead time for medium-lead burst neurons. These results demonstrate that the pause of activity in OPNs is caused by IPSPs initiated by an abrupt, intense input and maintained, for the whole duration of the saccade, by afferents conveying eye velocity signals. We suggest that the initial sudden inhibition originates from central structures such as the superior colliculus and frontal eye fields and that the eye velocity-related inhibition originates from the burst generator in the brain stem.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Saccades are rapid eye movements that shift the line of sight. The brain stem neural circuit that generates saccadic eye movements, the saccade generator, contains two essential classes of neurons, medium-lead burst neurons (MLBNs) and omnipause neurons (OPNs). MLBNs exhibit a high-frequency burst of spikes immediately before and during saccades (Büttner et al. 1977; Cohen and Henn 1972; Keller 1974; King and Fuchs 1979; Luschei and Fuchs 1972; Nakao et al. 1990). They consist of excitatory and inhibitory burst neurons (EBNs and IBNs). In the horizontal burst generator, EBNs project to the ipsilateral abducens nucleus to excite agonist motoneurons (Igusa et al. 1980; Sasaki and Shimazu 1981; Strassman et al. 1986a), and IBNs project to the contralateral abducens nucleus to inhibit antagonist motoneurons (Hikosaka and Kawakami 1977; Hikosaka et al. 1978; Scudder et al. 1988; Strassman et al. 1986b; Yoshida et al. 1982). OPNs exhibit steady discharge during fixation and cease firing before and during saccades in all directions (Cohen and Henn 1972; Evinger et al. 1982; Keller 1974; Luschei and Fuchs 1972). Stimulation of the OPN region interrupts ongoing saccades, suggesting that OPNs tonically inhibit MLBNs during fixation and that a pause of tonic discharge of OPNs removes this inhibition during saccades (Keller 1974). In agreement with this suggestion, OPNs have been shown to project to the EBN and IBN regions and make direct inhibitory connections with these burst neurons (Curthoys et al. 1984; Nakao et al. 1980; Strassman et al. 1987). These findings suggest that a pause of OPN activity is important for the generation of saccades. However, the mechanism of OPN pause induction and how the onset and the duration of the pause are controlled are still unknown. Robinson (1975) proposed an attractive model that suggested an inhibitory trigger signal initiates a pause of tonic discharge of OPNs, and once OPNs are suppressed, EBNs and IBNs are allowed to begin discharging in response to excitatory input. Assuming an inhibitory connection to OPNs from IBNs (or from EBNs through inhibitory interneurons), the OPNs are kept silenced as long as the burst neurons continue firing (cf. Keller 1979, 1980; Scudder 1988, for review, Fuchs et al. 1985).

The synaptic events that cause the cessation of OPN activity during saccadic eye movements are not known. In particular, whether their pause in activity is caused by a removal of tonic excitatory input (disfacilitation) or by an increase in inhibitory input (postsynaptic inhibition) is still uncertain. Because visually evoked saccades are abolished by combined ablation of the superior colliculus (SC) and the frontal eye field (Schiller et al. 1980), the input that initiates an OPN pause probably originates from these structures. The rostral pole of the SC contains fixation cells that tonically discharge during fixation and cease firing before and during saccades in cats (Munoz and Guitton 1989, 1991) and in monkeys (Munoz and Wurtz 1992, 1993). Stimulation of the SC (King et al. 1980; Raybourn and Keller 1977), especially at its rostral part (Paré and Guitton 1994), induces monosynaptic excitation of OPNs. These findings may favor the possibility that a pause in OPN activity is caused by disfacilitation. On the other hand, electrical stimulation of the SC suppresses OPN spikes after initial excitation in monkeys (Raybourn and Keller 1977) and in cats (Kaneko and Fuchs 1982; King et al. 1980). It is well known that the SC contains many cells that exhibit burst discharge for saccades and project to the brain stem reticular formation (Berthoz et al. 1986; Moschovakis et al. 1988b; Munoz and Guitton 1986; Munoz et al. 1991; Scudder et al. 1996a). These findings imply that burst discharge of SC neurons may contribute to production of a pause in OPN activity by postsynaptic inhibition.

Another, probably more important, question is what patterns of afferent discharge cause a pause in OPN activity. The information will be indispensable in understanding how the onset and the duration of the OPN pause are controlled. Anatomic studies (Ito et al. 1984; Langer and Kaneko 1984, 1990) have revealed various origins of cells projecting to the OPN region, including the SC and areas of saccade-related neurons in the midbrain and pontomedullary reticular formation. However, little is known about the discharge patterns of cells identified as directly connecting with OPNs.

The present study was undertaken to investigate membrane potential changes that underlie the cessation of OPN activity by means of intracellular recording techniques. OPN recordings in alert cats allowed us to clarify the synaptic events associated with saccades and to evaluate contributions of the above two possible mechanisms (disfacilitation and active inhibition) to the production of a pause. Moreover, our intracellular recordings allowed us to estimate the temporal pattern of afferent discharge impinging on OPNs because the changes of membrane potentials reflect total afferent inputs. Our results have shown that a pause of OPN activity is caused by inhibitory postsynaptic potentials (IPSPs). To determine the signal carried by inhibitory afferents to OPNs, we quantitatively analyzed the time course and the magnitude of saccade-related IPSPs and correlated them with the concomitant eye movement parameters. On the basis of these analyses, possible origins of inhibitory inputs to OPNs will be discussed with reference to previously studied saccadic activity of SC cells and brain stem burst neurons.

Preliminary reports of a part of this study appeared previously (Iwamoto et al. 1997; Yoshida et al. 1996).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation

Experiments were performed with four adult cats. Each animal underwent the following surgical procedures under pentobarbital sodium anesthesia and aseptic conditions. A heating pad was placed under the body to control the body temperature, and heart and respiratory rates were monitored for the duration of surgery. A coil of Teflon-coated stainless steel wire was implanted beneath the insertions of the four recti of the right eye to measure eye movements. The tympanic bulla on each side was opened, and a silver ball electrode was placed on the round window to stimulate the vestibular nerve. Stainless steel tubes were embedded in a block of dental acrylic attached securely to the frontoparietal skull with bone screws for insertion of stereotaxic bars, permitting painless immobilization of the animal's head during the intracellular recording sessions. An opening (5-7 mm diam) was made in the posterior part of the parietal bones overlying the cerebellar vermis, and the dura was removed to allow recording-electrode access through the cerebellum to the brain stem. A cylindrical chamber, made from a plastic microcentrifuge tube, was placed over the opening and fixed to the skull with dental acrylic. All experimental protocols complied with the guidelines of the University of Tsukuba policy on the humane care and use of laboratory animals.

Recording conditions

During recording sessions, the animal was placed in the stereotaxic apparatus mounted on a turntable that could be rotated about the earth's horizontal and vertical axes, together with the magnetic field-generating coils. The head was fixed in a 26.5° nose-down position with the interauricular midpoint on the axes of rotation and the right eye in the center of the magnetic field. The body was restrained gently with a cloth bag. The animals were kept quiet without any signs of distress or discomfort for the duration of recording. If the animals appeared to be getting bored, experiments were stopped, or a small piece of food or milk was given during a break in the recording. This procedure was effective in keeping the animals alert. Eye movements were measured by a magnetic search-coil system with a resolution of <0.1° and a bandwidth ranging between DC and 300 Hz (Fuchs and Robinson 1966). Calibration of the eye movement recording was made by assuming that the gain of the vestibuloocular reflex (VOR) evoked by sinusoidal head rotation in the light was equal to 1.0 (Iwamoto et al. 1990).

Intracellular recording from OPNs was collected while the animal was alert. Saccades were induced usually by a visual stimulus such as experimenter's hand, food and novel objects. Glass micropipettes filled with 4 M NaCl, instead of KCl, solution were used for intracellular recording because, if the tip of the micropipette is broken in the course of insertion, leak of high concentrations of potassium would exert noxious effects on brain tissues. These micropipettes were adequate for recording postsynaptic potentials (Eccles 1964). The microelectrodes had an electrical resistance of ~10 MOmega . A conventional input stage (Nihon Kohden MEZ-8300) was used for recording and passing current through the microelectrode. Intracellular potentials (bandwidth DC to 5 kHz), and eye position signals were monitored continuously on a computer screen and oscilloscopes. The same signals were recorded on a magnetic tape for later off-line analysis.

Data analysis

Data analysis was performed on a Macintosh computer with the software Spike2 (CED, Cambridge, UK) and homemade programs. The membrane potential and eye position signals were digitized and sampled every 1.0 ms. Instantaneous eye velocity signals were obtained by calculating the slope of the line fit to position samples contained in a 5-ms moving window centered on the current time point. The radial eye velocity was computed from horizontal and vertical eye velocity by use of the Pythagorean theorem. The onset of saccades was defined as the time when the radial eye velocity reached 15°/s. The onset of the saccade-related membrane potential change in OPNs was determined by a visually manipulated cursor; initial change was so steep that there was little variation of measurement among experimenters. The amplitude and the time of peak of the potential change were determined on a smoothed trace that had been obtained using a five-point running average of the data to filter synaptic noise.

Histological studies

A sharpened metal needle was chronically attached to the edge of the opening of the skull at its midline with dental acrylic as the reference of the insertion point of the glass micropipettes. The location of the tip of the intracellular microelectrode was measured with reference to the tip of the metal needle by the scale of a micromanipulator attached to the stereotaxic frame. When all recording sessions were completed, a thin tungsten microelectrode was inserted, with the aid of the micromanipulator, into the approximate center of the area where OPNs had been recorded. After confirming that extracellular spikes of OPNs still could be recorded in this region, an electrolytic lesion was made by passing cathodal currents (50 µA, 30 s) through the tungsten microelectrode. At the end of the experiment, the animals were killed with an overdose of pentobarbital sodium and perfused with saline followed by 3 L of fixative containing 2% paraformaldehyde and 1.6% glutaraldehyde in 0.1 M phosphate buffer. The marked spots were histologically studied in Nissl-stained serial sections (100 µm in thickness). Sites for intracellular recordings were reconstructed with reference to the marked spots. They were distributed in the region near the midline and rostral to the abducens nucleus, similar to those reported in previous studies in cats (Curthoys et al. 1981; Evinger et al. 1982).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of impaled OPNs and general features of saccade-related membrane potential changes

Neuronal activity was recorded near the midline of the pons at the level immediately rostral to the abducens nucleus. The midline of the brain stem was estimated by recording vestibular-induced monosynaptic volleys in the medial longitudinal fasciculus (MLF) (cf. Iwamoto et al. l990). The location of the abducens nucleus was determined by recording negative and positive field potentials induced after stimulation of the contralateral and ipsilateral vestibular nerve, respectively, and by recording units exhibiting characteristic discharge patterns associated with eye movements (Fuchs and Luschei 1970). Extracellular spikes of OPNs were identified by cessation of tonic discharge before and during saccades in all directions (Fig. 1A) as described previously (Cohen and Henn 1972; Evinger et al. 1982; Keller 1974; Luschei and Fuchs 1972). During advancement of the microelectrode, the OPN spikes first recorded were usually negative in polarity. Further advancement of the electrode tip on the cell revealed an initial positivity of spikes. Then the electrode was advanced slightly or positive currents were passed through the recording microelectrode, thereby impaling the cell.



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Fig. 1. Identification of an omnipause neuron (OPN). A: discharge pattern of extracellular spikes associated with saccades before impalement. B: intracellular recording of the membrane hyperpolarization and a pause of spikes associated with a saccade. C: saccade-related hyperpolarization after inactivation of spike generation mechanism. In A-C, Hor and Ver indicate horizontal and vertical eye positions, respectively.

The impaled cells were identified further as OPNs by their characteristic changes in the membrane potential associated with saccades. A steep hyperpolarizing deflection of the membrane potential occurred before each saccade and completely suppressed ongoing spikes (Fig. 1B). Hereafter, the membrane potential change in the hyperpolarizing direction will be simply called the "hyperpolarization" for the convenience of description. Figure 1B shows that the OPN resumed firing in midcourse of the decay phase of the hyperpolarization when the membrane potential reached the firing threshold. The membrane potential of OPNs during intersaccadic intervals was about -40-50 mV for a period after impalement. Usually, it gradually declined in the course of recording, probably due to partial deterioration of the membrane, and was maintained at a steady level of about -30 mV. Most measurements of the amplitude of saccade-related hyperpolarization for quantitative correlation analysis were taken at the steady level (about -30 mV) of the membrane potential. Although spike generation mechanisms were inactivated at this membrane potential level, a steep hyperpolarization was induced for all saccades (Fig. 1C, see also Fig. 3). Intracellular recordings were obtained from 23 OPNs. The maximum duration of holding the cell was ~20 min in this study.

IPSPs in OPNs associated with saccades

Synaptic mechanisms of the saccade-related hyperpolarization in OPNs were examined by Cl- ion injection into the cell by passing hyperpolarizing currents through the recording microelectrode. Figure 2 shows examples of saccade-related membrane potential changes recorded in an OPN before (Fig. 2A) and after injection of Cl- ions (Fig. 2B). Before injection, the membrane potential showed a steep hyperpolarizing deflection followed by a relatively slow return to the previous steady potential level. After injection of Cl- ions, the saccade-related hyperpolarization was reversed to a depolarizing potential (Fig. 2B). Both the hyperpolarization and depolarization preceded the onset of concurrent saccades and lasted approximately the duration of the saccades. Figure 2C shows extracellular potentials recorded in the vicinity of the cell as a control. These results indicated that the saccade-related hyperpolarization consisted of temporal summation of the IPSPs that were produced continuously before and during the whole period of saccades. In agreement with these findings, we noted that rapid fluctuation of the membrane potential increased during the period of saccade-related hyperpolarization (Figs. 1C and 2A), indicating an increase in synaptic bombardment. The hyperpolarizing IPSPs were in no case spontaneously reversed to a depolarizing potential unless Cl- ions were iontophoretically injected into the cell.



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Fig. 2. Saccade-related inhibitory postsynaptic potentials (IPSPs) in an OPN. A: saccade-related hyperpolarization as a control. B: saccade-related membrane potential inverted to depolarization after Cl- ion injection into the same cell as in A. C: extracellular potential recorded in the vicinity of the cell. Hor and Ver, horizontal and vertical eye positions, respectively.

For some saccades, the hyperpolarization was followed by a slight depolarization and then returned to the presaccadic membrane potential level. The late depolarization, probably caused by the excitatory postsynaptic potentials, was generally small and will not be considered further.

Temporal characteristics of the saccade-related hyperpolarization

Figure 3 illustrates an example of a continuous record of the membrane potential in an OPN (top) together with saccadic eye movements (bottom), showing that a clear hyperpolarization was associated with each saccade. The time course and amplitude of the hyperpolarization varied from saccade to saccade. Because the hyperpolarization occurred for saccades in all directions, the radial eye velocity (middle) is shown for comparison. There is a gross similarity between the profiles of the hyperpolarization and the radial eye velocity. Temporal characteristics of the hyperpolarization such as the lead time, the duration, and the time course were analyzed quantitatively in relation to those of the radial eye velocity.



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Fig. 3. Continuous record of the membrane potential of an OPN and eye movements. Top: membrane hyperpolarization associated with saccades. Middle: radial eye velocity calculated from horizontal and vertical component velocities. Bottom: Hor and Ver, horizontal and vertical eye positions, respectively.

LEAD TIME. The onset of hyperpolarization in OPNs was found to precede the onset of saccades. The interval from the onset of hyperpolarization to the onset of eye velocity change (lead time) showed some variability even in a single OPN, as exemplified in the histograms for three OPNs in Fig. 4, A-C. In these neurons, the mean lead time was 19.7 ± 3.4, 16.0 ± 4.3, and 16.3 ± 4.0 (SD) ms, respectively. The mean lead times for 23 OPNs (average number of saccades, 16.6) ranged from 7.5 to 23.9 ms, with an overall mean of 15.9 ± 3.8 ms (Fig. 4D).



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Fig. 4. Lead time of the onset of steep hyperpolarization of OPNs relative to the onset of saccades. A-C: histograms of the lead time for 3 OPNs. D: distribution of the mean lead time for 23 OPNs.

The lead time of the saccade-related hyperpolarization was compared with that of the saccade-related depolarizing potential obtained after Cl- injection in two OPNs. There was no significant difference in lead time between these two conditions; the mean lead time for one OPN was 20.6 ± 1.2 ms (n = 3) for the hyperpolarizing responses before Cl- injection and 19.7 ± 4.9 ms (n = 21) for the reversed depolarizing IPSPs after Cl- injection and that for another OPN was 12.0 ± 2.9 ms (n = 5) for hyperpolarizing IPSPs and 14.5 ± 3.6 ms (n = 13) for reversed IPSPs. Thus the onset of saccade-related hyperpolarization represented the onset of IPSP production.

DURATION. As described in the preceding text, the duration of the hyperpolarization appeared to be almost the same as the saccade duration. To confirm this observation, we examined quantitatively the relation between the two parameters. Because the end of the hyperpolarization was usually very gradual and it was difficult to determine the precise time of its termination, the interval measured at the potential level of 20% of the maximum hyperpolarization was taken as an indicator of the duration. Correspondingly, the duration of saccades was also measured at the velocity level of 20% of its peak. Figure 5 shows an example of the relationship between thus measured duration of the hyperpolarization and saccade duration for an OPN. Regression analysis indicated that the correlation between these two variables was highly significant (r = 0.98, P < 0.001, n = 39). The regression line intersected the y axis at -1.06 ms with its slope of 1.03. The correlation coefficient for 17 OPNs (average number of saccades, 15.4), including the cell shown in Fig. 5, ranged from 0.85 to 0.99 with a mean of 0.95 ± 0.04 (P < 0.001). The regression lines intersected the y axis at 4.21 ± 14.51 ms with a mean slope of 0.98 ± 0.15. In the remaining six OPNs, the number of saccades was not sufficient for statistical analysis. However, when the data for these six neurons were pooled, a significant correlation also was found (r = 0.93, P < 0.001, n = 22). The regression line intersected the y axis at 11.87 ms with its slope of 0.91. The results indicate that the duration of hyperpolarization was almost equal to the duration of concomitant saccade.



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Fig. 5. Relationship between the duration of the hyperpolarization and the duration of saccades in an OPN. Duration of the hyperpolarization (y) was measured at the potential level of 20% of the maximum amplitude, and the duration of saccades (x) was also measured at the velocity level of 20% of its peak. Regression line follows y = 1.03x - 1.06 (r = 0.98, P < 0.001).

TIME COURSE. Figure 6 shows three examples of saccade-related hyperpolarization (bottom) in an OPN exhibiting different time courses together with radial eye velocity profiles (top). The three records are arranged from short (A) to long duration (C) of saccades. Eye velocity traces are inverted to facilitate comparison with hyperpolarization traces. There is a close resemblance between the time course of the falling phase of eye velocity and the time course of the decay phase of the hyperpolarization. The similarity between the hyperpolarization and the eye velocity profiles was found for all saccades in all of 23 OPNs. However, there was a disparity between the hyperpolarization and the eye velocity profiles at their initial phase. The disparity was apparent especially when the velocity had a slow rise and prolonged time to peak. For example, in Fig. 6C, the slope of the initial phase of the hyperpolarization was much steeper than that of the eye velocity.



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Fig. 6. Membrane hyperpolarization in an OPN associated with single step, stereotyped saccades exhibiting different durations and time courses of eye velocity. A-C, top: radial eye velocity; bottom: saccade-related hyperpolarization. Eye velocity trace is inverted to facilitate comparison with hyperpolarization. Vertical broken line indicates the onset of steep hyperpolarization.

To quantify the relation between the time course of the hyperpolarization and that of the eye velocity, the instantaneous amplitude of the hyperpolarization was compared with the instantaneous eye velocity. In Fig. 7, the analysis was performed on the averaged data for several saccades with approximately equal peak eye velocity. Both hyperpolarization and eye velocity for individual saccades were aligned on the onset of hyperpolarization for averaging, and then the averaged velocity trace was corrected for the lead time. Figure 7A shows the averages for relatively slow saccades (averaged peak velocity; 90°/s, n = 4) and Fig. 7B for relatively fast saccades (averaged peak velocity; 157°/s, n = 5). Thick and thin lines in top and middle indicate the mean ± SE of eye velocity and hyperpolarization, respectively. In bottom, the averaged velocity is superimposed on the averaged hyperpolarization for ease of comparison. In Fig. 7, C and D, the instantaneous amplitude of the hyperpolarization is plotted against the instantaneous eye velocity, where open and closed circles indicate the relationship before and after the peak of eye velocity. Because the hyperpolarization attained its maximum earlier than the velocity did (Fig. 7, A and B, bottom), the trajectory followed a clockwise loop. Regression analysis for the falling phase of eye velocity showed that the correlation between the instantaneous amplitude of hyperpolarization and the instantaneous eye velocity was highly significant (r = 0.98 for C and 0.99 for D, P < 0.001). Thus the time course of the decay phase of hyperpolarization and that of the falling phase of the eye velocity were almost identical. This close relationship between the hyperpolarization and the falling phase of eye velocity was found quantitatively for all of 23 OPNs (average number of saccades used, 4.5). The correlation coefficients ranged from 0.88 to 0.99 with a mean of 0.96 ± 0.03 (P < 0.001).



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Fig. 7. Relationship between saccade-related hyperpolarization of OPNs and radial eye velocity. A and B: average membrane potential and the average eye velocity for saccades of approximately equal peak velocities, 4 relatively slow saccades (A) and 5 relatively fast saccades (B). Top and middle: eye velocity and membrane potential, respectively. Thick and thin lines indicate the mean ± SE. Shown at the bottom is the averaged velocity superimposed on the averaged membrane potential. Eye velocity traces are corrected for the lead time and inverted. C and D: relationship between the instantaneous amplitude of the hyperpolarization and the instantaneous eye velocity (C for A and D for B). Open circles, data plots during the increasing phase of eye velocity (rightward oblique arrows); filled circles, those during the declining phase of eye velocity (leftward oblique arrows). During the declining phase, the two variables are correlated linearly (r = 0.98 for C and 0.99 for D, P < 0.001).

The similarity between the time courses of the hyperpolarization and the eye velocity, except for the initial phase, was found not only for simple, single step saccades (Figs. 6 and 7) but also for saccades interrupted in midflight (Fig. 8A) or with a peculiar velocity profile (Fig. 8B). In Fig. 8A, the velocity profile exhibits two peaks, indicating an interrupted saccade, and there are also two peaks in the hyperpolarization trace. After correcting the velocity trace for the lead time, the timing of the second peak of hyperpolarization (thick line in Fig. 8A) was found to be almost synchronous with the second peak of the eye velocity (thin line in Fig. 8A). The amplitude of hyperpolarization around the second peak also varied in parallel with eye velocity. In Fig. 8B, the eye velocity is relatively low during the earlier half of the saccade, greatly increases during the later half, and quickly declines to zero at the end of saccade. Correspondingly, the hyperpolarization attains its peak in the later half of the saccade and quickly returns to the presaccadic level at the end of saccade. A disparity between the hyperpolarization and the eye velocity at the initial phase is also seen in Fig. 8: the slope of the initial hyperpolarizing deflection is much steeper than the slope of the eye velocity curve.



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Fig. 8. Parallelism between the profile of saccade-related hyperpolarization of an OPN and that of radial eye velocity of nonstereotyped saccades. A: interrupted saccade. B: saccade with early slow and late steep increase in velocity. Top: eye velocity; bottom: hyperpolarization (thick line) superimposed on eye velocity (thin line), the latter being shifted by the lead time and inverted. Vertical broken line indicates the onset of the hyperpolarization. Note that the initial part of hyperpolarization is much steeper than that of eye velocity in both A and B.

The initial part of the saccade-related hyperpolarization was similar in shape regardless of the later potential profile that paralleled the eye velocity. To characterize this initial change in the membrane potential, the time to peak of the hyperpolarization was measured for each saccade and plotted against that of eye velocity. In cases where there were two or more peaks in the membrane potential, the time to the initial peak was taken for measurement. As shown in Fig. 9 for two OPNs, the time to peak of the hyperpolarization distributed in a narrow range in spite of a wide distribution of the time to peak of eye velocity. There was no significant correlation between the two parameters. The mean times to peak of hyperpolarization in the two OPNs were 19.1 ± 4.0 (n = 33) and 23.0 ± 4.5 ms (n = 32). Overall mean for 23 OPNs (average number of saccades, 16.6) was 20.3 ± 2.6 ms. The distribution of data points in Fig. 9 agrees with the above-described finding that the disparity between the hyperpolarization and the eye velocity at the initial phase was clearly found when the velocity curve had a slow rise and prolonged time to peak.



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Fig. 9. Relationship between the time to peak of the saccade-related hyperpolarization and the time to peak of the eye velocity.  and open circle , data plots for 2 OPNs. Line (slope = 1.0) would apply if the times to peak of hyperpolarization and eye velocity were equal.

In some OPNs, a small slow hyperpolarizing potential appeared to precede the steep hyperpolarization. Because such a slow potential change was not consistently found for each saccade, an averaging of the membrane potential was made to evaluate its significance. Figure 10 shows an example of averaged data obtained from records aligned on the onset of steep hyperpolarization for 26 saccades in an OPN. The averaged membrane potential showed a presaccadic slow change that began ~20-30 ms before the steep hyperpolarization, but it was extremely small in amplitude compared with the amplitude of the steep hyperpolarization. The significance of the presaccadic slow change was examined by comparing the means of the potential during the period from 1 to 30 ms and from 51 to 100 ms (measured every 1.0 ms) before the steep hyperpolarization. The difference between the two means was statistically significant (P < 0.01). Similar analysis was performed on a total of 14 OPNs by averaging the data for 11-55 saccades (average, 22.0). A statistically significant slow potential was found for 7 of the 14 OPNs and not for the remainder. The amplitude of the slow potential measured at the onset of the steep hyperpolarization for the 14 OPNs was, on the average, 6% of the peak hyperpolarization.



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Fig. 10. Average of saccade-related hyperpolarizations in an OPN for 26 single-step, stereotyped saccades. Eye velocity (top) and the membrane potential (bottom) were aligned on the onset of the steep hyperpolarization (vertical broken line). Horizontal line indicates the averaged membrane potential level during the intersaccadic period. An arrow indicates a slow potential change that precedes the steep hyperpolarization.

Relationship of the hyperpolarization to the peak eye velocity

The amplitude of the eye velocity-related hyperpolarization was compared with the peak radial eye velocity for each saccade. The amplitude of the hyperpolarization was measured at the time corresponding to the peak of eye velocity corrected for the lead time (Fig. 11A, up-down-arrow ). Because the amplitude of IPSPs should be affected by the membrane potential level, the correlation was calculated for the records in which the membrane potential was maintained at a relatively constant level (range of variation less than ±4 mV) during intersaccadic intervals. Figure 11B shows an example of the relationship between the peak eye velocity and the amplitude of hyperpolarization. The amplitude of the hyperpolarization increased with the amplitude of the peak eye velocity. Linear regression analysis indicated that the correlation between the two variables was statistically significant (r = 0.73, P < 0.01). Similar correlations were found for all of eight OPNs examined (average number of saccades, 21.1; P < 0.01). The correlation coefficients ranged from 0.63 to 0.85 with a mean of 0.75 ± 0.07. In the remaining OPNs, the number of saccades or the range of saccade velocity at a constant level of the membrane potential was not sufficient for this analysis.



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Fig. 11. Relationship between the amplitude of saccade-related hyperpolarization and the peak eye velocity. A: example of record showing the method of amplitude measurement. Amplitude of hyperpolarization (up-down-arrow  in bottom) was measured at the time corresponding to the peak of eye velocity (up-down-arrow  in top) after correcting for the lead time. B: relationship between the 2 variables for an OPN (r = 0.73, P < 0.01).

If the OPN hyperpolarization is produced by a linear summation of IPSPs attributable to afferents which convey each of horizontal and vertical component velocity signals and converge on an OPN, one might argue that the amplitude of hyperpolarization should be compared with a simple sum of horizontal and vertical components of eye velocity. Therefore the correlation between the amplitude of hyperpolarization and the sum of the absolute values of horizontal and vertical component velocities was calculated. The correlation coefficients for the eight OPNs ranged from 0.66 to 0.84 with a mean of 0.75 ± 0.07 and were almost identical to the values for the correlation with the radial eye velocity. Thus these regression analyses did not distinguish whether the saccade-related hyperpolarization in OPNs better reflected the radial eye velocity or the sum of horizontal and vertical component velocities.

Relationship of the hyperpolarization to the amplitude of saccades

An attempt was made to quantify the total inhibitory afferent input impinging on an OPN during each saccade. The membrane potential level at the onset of each hyperpolarization was taken as a baseline, and the area enclosed by this baseline and the hyperpolarization curve was calculated. Because the precise termination of the hyperpolarization was difficult to determine, the hyperpolarization curve was integrated from its onset for the period equal to the saccade duration. Figure 12 shows an example of the relationship between the area of the hyperpolarization and the radial amplitude of saccades. Regression analysis indicated that the two parameters were significantly correlated (r = 0.73, P < 0.01). Significant correlations were found for the eight OPNs (P < 0.01) described in the preceding section. The correlation coefficients ranged from 0.64 to 0.89 with a mean of 0.78 ± 0.08. 



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Fig. 12. Relationship between the area (time integral) of the saccade-related hyperpolarization and the radial amplitude of saccades for an OPN (r = 0.73, P < 0.01). Same cell as in Fig. 11B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed that a saccade-related pause of OPN spikes was caused by a hyperpolarization consisting of IPSPs, the time course of the hyperpolarization was in parallel with that of instantaneous saccadic eye velocity except for the initial steep part of hyperpolarization, the initial steep hyperpolarization occurred ~16 ms before the beginning of saccades and attained its peak in ~20 ms regardless of eye velocity profile, and the total duration of the hyperpolarization was equal to the duration of the saccade. We think that the saccade-related hyperpolarization reflects the time course and the density of total afferent discharge because it consists of IPSPs induced by afferent impulses. Discharge patterns and possible origins of afferents producing the saccade-related IPSPs will be considered on the basis of the present findings. Before that, we will first compare the present results with previous extracellular studies on the discharge patterns of OPNs, thereby trying to explain their pause based on the membrane potential changes.

Comparison with extracellular studies on timing parameters of the pause

Evinger et al. (1982) reported that the mean lead time of the OPN pause was 22.5 ms in cats. As pointed out by these authors, these values tended to overestimate the actual lead time by half the average interspike interval (~5 ms). The mean lead time of the steep hyperpolarization (15.9 ms) is in good agreement with that of the pause. This indicates that the steep hyperpolarization exerts, at its very onset, a powerful inhibitory effect on spike generation.

The pause duration of OPNs is somewhat shorter than the saccade duration in cats (Evinger et al. 1982). This finding is well explained by the present intracellular data. After the hyperpolarizing deflection reached its peak and completely suppressed spikes, the membrane potential reached threshold for spike generation during the gradual return to the presaccadic level. The firing began before the end of the saccade, and the rate gradually increased in parallel with the membrane potential recovery (Fig. 1B). The gradual resumption of firing rate after a pause is seen in extracellularly recorded OPN spikes in cats (cf. Fig. 1 of Evinger et al. 1982). In monkey OPNs, the duration of the pause is almost equal to the duration of the saccade (Keller 1974; Luschei and Fuchs 1972), and only the initial interspike interval after the pause is slightly longer than succeeding intervals (Cohen and Henn 1972; Fig. 10B of Everling et al. 1998). The difference between cat and monkey OPNs in the relation of pause duration to saccade duration is presumably related to the difference in velocity profile of saccade. Because the postpeak fall of saccade velocity in monkeys is much steeper than that in cats, we assume that the velocity-related hyperpolarization may return more rapidly to the presaccadic level in monkeys than in cats, making pause duration almost equal to duration of hyperpolarization.

Role of brain stem MLBNs in production of OPN pause

The present results show a close correlation between the duration of OPN hyperpolarization and saccade duration, between the amplitude of hyperpolarization and saccade velocity, and between the time integral of hyperpolarization and saccade amplitude. These relationships between the parameters of OPN hyperpolarization and saccadic eye movements are very similar to the relationships between the parameters of MLBN firing and saccadic eye movements: i.e., there is also a close correlation between the burst duration and saccade duration (King and Fuchs 1979; Luschei and Fuchs 1972), between the intraburst firing rate and saccade velocity (Kaneko et al. 1981; Keller 1974; King and Fuchs 1979; Yoshida et al. 1982), and between the number of spikes and saccade amplitude (Kaneko and Fuchs 1981; Kaneko et al. 1981; Keller 1974; King and Fuchs 1979; van Gisbergen et al. 1981; Yoshida et al. 1982). The similarities between patterns of MLBN firing and OPN IPSPs suggest that MLBNs provide OPNs with eye velocity-related signals during saccades.

The time course of OPN hyperpolarization resembles that of MLBN firing not only for the eye velocity-related component but also for the initial steep changes. The instantaneous firing rate of MLBNs rises more steeply than eye velocity does (Keller 1974), and the trajectories of the relationship between these two parameters generally make a loop (van Gisbergen et al. 1981; Yoshida et al. 1982), like the relationship between the instantaneous amplitude of OPN hyperpolarization and the instantaneous eye velocity (Fig. 7). This raises the question whether the abrupt increase in MLBN firing influences the onset of OPN hyperpolarization. To clarify this point, it is essential to know the temporal relationship between the onset of OPN inhibition and the onset of MLBN bursts. The mean lead time of MLBN bursts in previous studies ranged from 7.1 to 12.7 ms (Hepp et al. 1989 for review). These values are shorter than the mean lead time of the steep hyperpolarization in OPNs (15.9 ms). The methods of measurement to determine the onset time of saccadic eye movements might vary somewhat with each laboratory. Therefore we reexamined the lead time of MLBN bursts with the same techniques used for determination of the lead time of OPN hyperpolarization (see METHODS). The lead time of bursts in horizontal MLBNs was found to be 8.3 ± 2.0 ms (n = 10), which was within the range of the values found in the previous studies. It follows that bursts of MLBNs are induced after the onset of steep hyperpolarizations in OPNs by 7.6 ms on the average. Thus the onset of steep hyperpolarization appears to be determined by afferent discharge that precedes MLBN bursts. However, because the time to peak of the initial steep hyperpolarization in OPNs is ~20 ms, its profile may be influenced by the MLBN discharge.

Because OPNs are inhibited for saccades in all directions, they would have to receive converging inputs from MLBNs with different ON directions. It is possible that afferents from horizontal and vertical IBNs may converge directly on OPNs. Alternatively, it is also possible that afferents from horizontal and vertical EBNs may converge on common inhibitory interneurons that project to OPNs. Anatomic studies have shown that the IBN region contains neurons that project to the OPN region (Langer and Kaneko 1984, 1990). Morphophysiological studies with intraaxonal horseradish peroxidase staining of IBNs identified as projecting to the contralalateral abducens nucleus could not reveal their termination in the OPN area in cats (Yoshida et al. 1982) and monkeys (Strassman et al. 1986b). Strassman et al. have argued that the inhibitory input to OPNs during the saccade arises from a different population of burst neurons than immediate premotor IBNs. Although the eye velocity-related inhibitory signals to OPNs most likely originate from the burst generator in the brain stem, the location of the inhibitory neurons that make direct connection with OPNs remains to be studied.

Role of the SC in production of OPN pause

Likely candidates for the origin of the input that generates the earliest IPSPs in OPNs appear to be the SC and the frontal eye field. Because firing characteristics of SC cells have been studied extensively with reference to saccade metrics, we will discuss the relation between the OPN IPSPs and SC activity.

INHIBITORY INPUT FROM SC BURST CELLS. Ablation of both the SC and the frontal eye field abolishes generation of visually evoked saccades (Schiller et al. 1980), and focal electrical stimulation of the intermediate and deep layers of the SC induces saccades in monkeys (Robinson 1972; Schiller and Stryker 1972) and cats (Straschill and Rieger 1973). Wurtz and Goldberg (1971, 1972) first described neurons in the SC that discharge prior to saccades. Saccade-related bursts begin ~20 ms (ranging from 16.0 to 24.8 ms) before saccade onset in monkeys (Sparks 1978). Similar lead times (means ranged from 14 to 26 ms) have been found for saccade-related burst cells in the cat SC that have spatial and temporal properties similar to primate burst cells (Peck 1987). Some of the SC cells that project to the brain stem have been shown to exhibit saccade-related discharge (Berthoz et al. 1986; Munoz et al. 1991). Morphophysiological studies have demonstrated that saccadic signals are conveyed by a class of SC cells, called T cells (Moschovakis et al. 1988a,b), which project to various structures including the OPN region (Scudder et al. 1996a). These findings agree with the notion that the SC plays a crucial role in the initiation of saccades (see Sparks 1986; Sparks and Mays 1990 for review). Because stimulation of the SC produces disynaptic IPSPs in OPNs (Iwamoto et al. 1997; Yoshida et al. 1996), it seems reasonable to suggest that SC burst cells provide, via inhibitory interneurons, signals that determine the onset of saccade-related IPSPs. It should be noted here that the initial steep hyperpolarization does not necessarily represent the profile of the "trigger signal" per se (Robinson 1975; van Gisbergen et al. 1981), because it may contain IPSPs caused by afferents from MLBNs as described in the preceding text.

Burst cells in the SC frequently exhibit a low-frequency prelude of spikes before the intense burst in monkeys (Schiller and Koerner 1971; Sparks 1978; Wurtz and Goldberg 1971, 1972) and cats (Munoz et al. 1991). Keller (1981) has suggested the possibility that a group of pontine long-lead burst neurons is intercalated between the SC and OPNs. Axons exhibiting long-lead bursts have been shown to terminate in the OPN region (Scudder et al. 1996a,b). A small slow hyperpolarization before the steep IPSPs observed in some OPNs (Fig. 10) may correspond to the low frequency prelude of inhibitory input (cf. Kamogawa et al. 1996). Because the slow hyperpolarization was extremely small in amplitude compared with the steep IPSPs and was not consistently observed, it appears that the intense burst in the SC and long-lead burst neurons plays a more important role in saccade initiation than the low-frequency prelude does.

Several studies have shown a close relation between the temporal discharge pattern of burst cells in the SC and saccadic eye velocity. The instantaneous firing rate of some tecto-reticulo-spinal neurons is correlated with the instantaneous eye velocity in cats (Berthoz et al. 1986). There are SC burst cells whose mean firing rates are correlated with the peak eye velocity in cats (Munoz et al. 1991) and in monkeys (Rohrer et al. 1987). When saccades are interrupted in midflight by brief stimulation of the OPN region (Keller and Edelman 1994) or the rostral pole of the SC (Munoz et al. 1996), the SC burst discharge is suppressed during the interruption and increases in association with a resumed saccade. A similar suppression and resumption of spikes of SC burst cells also is observed during spontaneous interruptions of saccades (Munoz et al. 1996). Correspondingly, the amplitude of OPN hyperpolarization decreased during interruptions of saccades and increased during reacceleration (Fig. 8A). These similarities between the discharge pattern of SC burst cells and the IPSP pattern of OPNs suggest the possibility that burst cells in the SC also may contribute to the eye velocity-related OPN hyperpolarization. However, quantitative analysis of SC burst discharge during postinterruption saccades does not support velocity coding of SC burst cells (Keller and Edelman 1994), whereas the time course and the amplitude of the OPN hyperpolarization were in parallel with eye velocity for any saccadic profile including postinterruption saccades. It also should be recalled that feline tecto-reticulo-spinal neurons do not seem to carry simple eye movement signals (Berthoz et al. 1986; Munoz and Guitton 1986; Munoz et al. 1991) and that saccades with identical parameters can be associated with SC burst discharges that differ considerably in intensity in cats (Peck 1987). In the latter study, saccade duration is only weakly correlated with the duration of SC bursts. In contrast, the present study showed a tight relation between the profile of OPN hyperpolarization (except its initial phase) and saccade parameters (duration, velocity, and amplitude). Thus it cannot be assumed that the temporal patterns of IPSPs in OPNs during saccades directly reflect the activity of SC burst cells. It is more likely that the temporal discharge patterns of afferents responsible for the eye velocity-related IPSPs in OPNs are generated in the burst generator in the brain stem.

DISFACILITATION RELATED TO SC FIXATION CELLS. A recent study (Everling et al. 1998) has shown that fixation cells located in the rostral SC (Munoz and Guitton 1991; Munoz and Wurtz 1992, 1993; Munoz et al. 1991; Paré and Guitton 1994) pause earlier than OPNs. On the basis of their presumed excitatory connections with OPNs, disfacilitation caused by the pause of fixation cells may assist decreasing excitability of OPNs during saccades. However, this disfacilitation effect seems to be too weak to act as a trigger signal for the OPN pause. It has been shown that the onset of pause in fixation cells is more gradual and less synchronized to saccade onset than the onset of pause in OPNs (Everling et al. 1998). Tonic activity of OPNs probably is maintained not only by the SC but also other sources of excitatory input (Raybourn and Keller 1977). The present results indicate that an abrupt onset of OPN pause is caused by the steep IPSPs. Because IPSPs are accompanied with an increase in the membrane conductance (Eccles 1964), they would exert a strong inhibitory action on spike generation even if some tonic excitatory inputs are still existent.

In summary, the data indicate that the onset of the OPN pause is determined by an abrupt inhibitory input manifest as the initial steep hyperpolarization and probably due to the saccade-related discharge of SC neurons. They further show that the duration of the saccade is controlled by OPNs that are, in turn, controlled by an eye velocity-related inhibitory input most likely from MLBNs. These data constitute the first substantive findings regarding both the previously postulated trigger and latch signals (Robinson 1975; van Gisbergen et al. 1981).


    ACKNOWLEDGMENTS

The authors thank A. Ohgami for technical assistance.

This study was supported by Grants-in-Aid for Scientific Research (Grants 10164210 and 11145209 to K. Yoshida) from the Ministry of Education, Science, and Culture of Japan and by Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation. S. Chimoto was supported by the Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.


    FOOTNOTES

Address reprint requests to K. Yoshida.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 September 1998; accepted in final form 20 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society