Nystagmus Induced by Pharmacological Inactivation of the Brainstem Ocular Motor Integrator in Monkey

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Vision Research 39 (1999) 4286–4295 www.elsevier.com/locate/visres

Nystagmus induced by pharmacological inactivation of the brainstem ocular motor integrator in monkey

Donald B. Arnold a,1, David A. Robinson a, R. John Leigh b,c,*

a Department of Ophthalmology and Biomedical Engineering, The Johns Hopkins School of Medicine, Baltimore, MD, USA b Departments of Neurology, Neuroscience and Biomedical Engineering, Uni6ersity Hospitals, 11100 Euclid A6e., Cle6eland, OH 44106-5000, USA c Veterans Affairs Medical Center, Case Western Reser6e Uni6ersity, Cle6eland, OH, USA

Received 29 July 1998; received in revised form 27 January 1999

Abstract

A common cause of pathological nystagmus is malfunction of the mechanism by which the brain integrates eye velocity signals to produce eye position commands. For horizontal gaze, neurons in the nucleus prepositus hypoglossi-medial vestibular nucleus region (NPH-MVN) play a vital role in this neural integrator function. We studied the effects on gaze stability of pharmacological intervention in the NPH-MVN of monkeys by microinjections of eight drugs. Agents with agonist or antagonist actions at gamma-aminobutyric acid (GABA), glutamate, and kainate receptors all caused gaze-evoked nystagmus with centripetal eye

drifts; glycine and strychnine had no effect. When the GABAA-agonist muscimol was injected near the center of MVN, the eyes drifted away from the central position with increasing-velocity waveforms, implying an unstable neural integrator. The observed effects of these drugs on gaze stability may be related to inactivation either of neurons within NPH-MVN or the cerebellar projections to them that control the ﬁdelity of neural integration. Drugs that inﬂuence GABA or glutamine transmission may have a role in the treatment of nystagmus due to an abnormal neural integrator. Published by Elsevier Science Ltd.

Keywords: Nystagmus; GABA; NMDA; Cerebellum; Neural integrator

1. Introduction discharge rate with eye position — and also with velocity during movements (Fuchs, Scudder & Kaneko, For a clear and stable view of the world, images must 1988). However, premotor neurons that send vestibular be held steadily on the retina, and the fovea must be (Waespe & Henn, 1977), saccadic (Van Gisbergen, pointed at the object of interest (Carpenter, 1991). In Robinson & Gielen, 1981), and pursuit signals (Miles & order to view clearly an object located off to one side, Fuller, 1975) to the ocular motoneurons modulate their each eye must be held at an eccentric position in the discharge with eye velocity, not position. This differ- orbit. The latter requires a tonic contraction of the ence implies that an integration of velocity-coded to extraocular muscles to oppose the elastic forces im- position-coded signals is achieved by the nervous sys- posed by the orbital tissues, which act to return the eye tem (Robinson, 1975; Arnold & Robinson, 1997). It is to a central position (Leigh & Zee, 1999). To achieve now established that, for horizontal eye movements, this sustained muscle contraction, the ocular motoneu- this neural integration depends heavily upon the nu- rons must encode commands that have an eye position cleus prepositus hypoglossi-medial vestibular nucleus component to them. Electrophysiological studies region (NPH-MVN) in the caudal pons and medulla conﬁrm that the ocular motoneurons do modulate their (Cannon & Robinson, 1987; Cheron & Godaux, 1987); for vertical eye movements, the interstitial nucleus of * Corresponding author. Tel.: +1-216-421-3224; fax: +1-216-421- Cajal plays a key role (Helmchen, Rambold, Fuhry & 3040. Bu¨ttner, 1998). In addition, the cerebellum, especially E-mail address: [email protected] (R.J. Leigh) 1 Present address: Enders Research Unit, Children’s Hospital, the ﬂocculus, makes an important contribution (Car- Boston, MA, USA. penter, 1972; Robinson, 1974; Zee, Yamazaki, Butler &

0042-6989/99/$ - see front matter Published by Elsevier Science Ltd. PII: S0042-6989(99)00142-X D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295 4287

Gu¨cer, 1981). Thus, lesions of any of these structures position midway between the abducens nuclei (Smith, impair the ability to hold the eyes in eccentric gaze; Kastella & Randall, 1972). they drift back towards the center of the orbits, and corrective quick phases cause gaze-evoked nystagmus 2.2. Measurement of eye mo6ements, unit recording — perhaps the commonest form of pathological nys- and stimulation tagmus (Leigh & Zee, 1999). Recently, attempts have been made to understand Eye movements were recorded using the eye-coil/ what neurotransmitters may be involved in the process magnetic-field system (Robinson, 1963). The monkey of neural integration, using the technique of pharmaco- sat in a primate chair with its head fixed in the center of logical inactivation. Such knowledge is directly relevant the field coils. The noise level of the system was approx- to understanding and treating pathological nystagmus imately 15 min of arc, and bandwidth was 0–1 kHz. An which interferes with clear vision. In the cat, microin- ultra-violet mirror-galvanometer (Honeywell Visicorder jection of the GABAA-agonist muscimol into NPH- 1108) and an FM tape recorder (Vetter Model B), MVN abolishes neural integration for horizontal simultaneously recorded horizontal and vertical move- movements, so that the centripetal drift of the eyes after ments of one eye, and target position, when relevant. a saccade is determined by the elastic pull of the orbit The goal of recording from single neurons was to tissues (Mettens, Godaux, Cheron & Galiana, 1994a). locate the abducens nuclei and identify sites for injec- Similar results are reported after muscimol injections tion. Monopolar tungsten micro-electrodes were used into the MVN of monkeys (Straube, Kurzan & Bu¨ttner, for single-unit extracellular recording and stimulation. 1991). However, other neurotransmitters are reported The tips were insulated with epoxylite varnish until to contribute to normal function of the neural integra- their impedance (at 1 kHz) was between 1 and 3 MV tor (Mettens, Cheron & Godaux, 1994b). One goal of for recording electrodes and about 500 kV for stimulat- the present study was to compare the effects of agonist ing electrodes. The electrode was placed in a guide tube and antagonist agents acting at GABAA, glutamate, consisting of 22 gauge tubing cut to a length between kainate, and glycine receptors. 68 and 72 mm depending on the size of the monkey’s head. This assembly was then inserted into the brain so that the tip of the guide tube lay approximately 6 mm 2. Methods above the floor of the fourth ventricle. The electrode was then advanced out of the guide tube, through the 2.1. Preparation of animals cerebellum and fourth ventricle, and into the brainstem using a Trent Wells hydraulic microdrive (3-0590). All animal experiments were conducted in strict ac- Recordings were made at various locations to allow a cordance with principles of laboratory animal care map of brainstem landmarks to be made. The two (NIH publication No. 86-23, revised 1985). Three juve- abducens nuclei were identified by the characteristic nile rhesus monkeys, Macaca mulatta, weighing be- ‘singing’ sound produced on the audio monitor. In tween 2.5 and 4 kg, were used in this study. Each addition, the cells were very densely packed and all monkey underwent two operations performed under fired at a rate proportional to ipsilateral eye position pentobarbital anesthesia and aseptic conditions. In the and eye velocity. The rostro-caudal and medio-lateral first, a coil of wire was implanted in one eye, as borders of these areas were delineated to make sure previously described (Arnold & Robinson, 1997), to that they were the abducens nuclei. A volume was then measure eye movements. One week later the animal was explored 1 mm to 2 mm caudal to the caudal border of trained to fixate dots back-projected on a screen by a the abducens nuclei and 1–4 mm lateral, where many laser under the control of an x–y mirror galvanometer. sites were found in which firing rates of cells varied The monkeys were deprived of water for 1 day prior to with eye movements, and stimulation produced eye each training session and during the session the monkey movements. This was presumably the relevant region was given a liquid reward for correct fixation. After within the NPH-MVN and would especially include the several sessions the monkey learned to fixate the light at marginal zone (McFarland & Fuchs, 1992). Chemicals various positions and the eye coil was calibrated. The were injected throughout these regions. second surgery involved trephining a hole 2 cm in diameter in the midline of the occipital area of the 2.3. Injections monkey’s skull. A stainless steel recording chamber (Trent Wells 3-0558) was then implanted over the hole. Chemicals were injected into the NPH-MVN region Its axis lay in the midsagittal plane, tilted back from the via a 29 gauge metal cannula that was advanced vertical by 20° and was positioned so that it would pass through a guide tube in the same manner as the elec- through a point on the midline 4 mm posterior to and trodes. Injections were made with a 10 ml Hamilton 2 mm above the inter-aural axis. This corresponds to a Luer tip microsyringe controlled by a stepper motor. 4288 D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295

The volume of all injections was 1 ml; all were unilat- (0.2–20 s) an attempt at greater accuracy in measuring eral in each monkey. The chemicals were mixed in a t would not have contributed to any better interpreta- solution of saline and Hepes buffer with a pH of 7.4. tion. Consequently, time constants were calculated by The following chemicals were used: muscimol (Sigma), drawing a line tangent to the eye movement trace at a 10 mM; bicuculline (Sigma), 10 mM; strychnine point immediately after a saccade and taking the dif- (Sigma), 100 mM; glycine (Sigma), 1 mM; kainic acid ference between that time and the time at which the (Sigma), 50 mM; DNQX (6,7-Dinitroquinoxoline-2,3- tangent line crossed the null position. The null posi- dione) (Research Biochemicals), 10 mM; NMDA (N- tion was measured, in each case, as the position (usu- m methyl-D-aspartic acid) (Sigma), 50 M; APV ally near zero) at which the postsaccadic eye velocity (2-amino-5- reversed its sign. Each time constant was calculated at phosphonovaleric acid) (Sigma), 1 mM. Before and at five different points on a given recording and the various times after the injections the monkey’s random average was used as the nominal value; the standard saccades, and post-saccadic drifts in the dark, were deviation reflected variability. Injections at some sites recorded to assess the function of the integrator. Dur- caused constant-velocity slow phases of nystagmus; in ing these times the monkeys were kept alert by audi- these cases, the velocity when the nystagmus was most tory stimulation. Generally, the monkey’s integrator, if intense, V , was measured at five different points affected, recovered by 2 h, so that its time constant max was at least 50% of the pre-injection value, when mon- and averaged. Sometimes, injections caused increasing- itoring was stopped. velocity drifts (reflecting an unstable integrator); in these cases, negative time constants were measured in 2.4. Lesions and identification of injection sites the same manner as positive time constants, except that the tangent line intercepted the null position in Electrolytic midline lesions were made in two mon- the negative direction on the time axis. keys by passing 100 mA of anodic DC current into the brainstem for 1 min. Eye movement data were col- lected before and after the lesions. Each monkey was 3. Results then allowed to recover for 4–5 weeks at which point its spontaneous eye movements in the dark were 3.1. Location of injections recorded again to determine what recovery had taken place; these studies have been previously reported Injections were made at points between 1 and 2 mm (Arnold & Robinson, 1997). Shortly after the last caudal to the abducens nuclei and 1 and 4 mm lateral recordings the monkeys were perfused under deep pen- to the midline. The positions of injections were deter- tobarbital anesthesia with a cardiac injection of 10% mined by stereotaxic extrapolation from the physiolog- neutral formalin through the left ventricle. The brains ically identified loci of the abducens nuclei and were were blocked and 50 mm frozen sections were cut corroborated by histology. Fig. 1 illustrates some of from them in a transverse plane containing the elec- the injection sites for muscimol (A), bicuculline (B), trode tracks. Alternate sections were then stained with and other chemicals (C). This figure in conjunction cresyl violet. These sections allowed localization of with the coordinates given in Table 1, permits the sites of injections and lesions. approximate location of all injection sites. With 41 injections in three monkeys, many overlapping, all 6 2.5. Analysis of eye mo ements cannula tracks could not be identified individually, although all were confined to the region illustrated. Eye movements were analyzed from the Visicorder Moreover, 1 ml injections will probably spread over an output by hand. We analyzed gaze-holding in darkness effective distance of at least 90.5 mm. Although dif- after a saccade brought the eye to a new orbital posi- ferences were seen between different injection sites (see tion; smooth-pursuit and vestibular eye movements were not studied. The most common effect of the below), the techniques used here were not intended to injections was to impair gaze holding so that the eyes elucidate micocircuitry but only to test more general drifted, with decreasing velocity, toward the center. In pharmacological localization of the neural integrator. these cases, the time constant of the drift was mea- No overt non-oculomotor effects were noticed, ex- sured to provide an estimate of neural integrator func- cept in the three muscimol injections that made the tion. It is a property of an exponential of time integrator unstable with negative time constants (indi- ~ constant , that a tangent to it at a time t0 will cated by asterisk in Table 1). In each of these cases ~ intersect the abscissa at time t0 + , thus providing a the monkey put the ear ipsilateral to the injection side simple graphic way to estimate ~. Since the variations down on the floor of its cage for about 1 h following in ~ created by these experiments covered two decades the end of the experiment. D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295 4289

3.2. Muscimol (GABA agonist) injections course of the injection diffusing away from the site. Smaller time constants were associated with longer Ten unilateral, 1 ml injections of 10 mM muscimol recovery periods. After an injection, the null point were made in the caudal pons, 1–2 mm caudal to the (estimated by noting the eye position at which post-sac- abducens nuclei and 1–3 mm lateral to the midline cadic drift reversed direction) shifted, even within 1 (Table 1). Five of these injections, between 1 and 2.5 min, up to 10° either ipsilaterally or contralaterally, mm lateral, resulted in a marked reduction in the time with no consistent trend. It tended to decrease toward ~ constant of the integrator to the minimum value min zero, within an hour. shown. A typical example of such injections causing the In two cases, injection of muscimol created simple integrator to become leaky is shown in Fig. 2. Prior to nystagmus (Vmax, Table 1). The slow phases were injection (Fig. 2A), the monkey could hold eccentric straight indicating a normal integrator — otherwise gaze steadily in darkness. The normal time constant of they would have been curved. This is indicative of a centripetal eye drift for humans is approximately 20 s vestibular imbalance similar to a peripheral vestibular (Leigh & Zee, 1999), and monkeys are comparable. The lesion. Both sites were at the lateral limit (3 mm) mild downbeat nystagmus in Fig. 2A is normal in the investigated. The constant-velocity slow phases had a rhesus monkey. After the injection (Fig. 2B), the inte- peak velocity of about 10 °/s, ipsilateral to the injection. grator has become very leaky, causing gaze-evoked Finally, injection at three sites (indicated by asterisks nystagmus. Note, however, that there was some vari- in Table 1) created a novel finding — they made the ability in the centripetal drift, from beat to beat; that neural integrator unstable. As illustrated in Fig. 4A, the some individual slow phases appeared to show more eyes drifted away from the null point with a waveform than one rate of drift; and some were almost linear (see resembling a growing exponential, which can be de- Section 4). After each of these five injections, the inte- scribed by a negative time constant. In each case the ~ grator time constant, min was reduced to between 0.2 monkey only made eye movements on one side of the and 1.5 s (mean 0.9 s) within 5–10 min of the injection. null point, the side ipsilateral to the injection. Duringa2hperiod following each injection, the The dependence of the results on the location of the time constant slowly increased to greater than 5 s. The injection is reported in Table 1. Injections between 1.0 time constant eventually returned to normal within a and 2.7 mm produced a reduction in integrator time day or two. Fig. 3 illustrates typical time courses of the constant. Injections around 2.7–3.0 mm made the inte- integrator time constants following these injections. The grator unstable (*) while more lateral injections at 3.0 changes in time constants followed similar trajectories mm produced nystagmus with linear slow phases. From after each injection, and probably reflected the time Fig. 1, the boundary between the production of a leaky

Fig. 1. The location of the region of the injection sites. PH, nucleus prepositus hypoglossi; MVN, medial vestibular nucleus; DVN, descending vestibular nucleus; LVN, lateral vestibular nucleus; Y, y-group of the vestibular nucleus; SCP, superior cerebellar peduncle. The symbols represent examples of the loci where injections were made that resulted in the following effects: ﬁlled triangle, leaky integrator (see Fig. 2B for examples); star, unstable integrator (see Fig. 4A and B for examples); c, either an unstable integrator or constant-velocity nystagmus without affecting the integrator; ﬁlled circles, nystagmus without affecting the integrator (see Fig. 4C), ×, no effect. All slices are through a transverse section plane tilted caudally from stereotactic vertical by 20° and 1.5 mm caudal to the abducens nuclei. (A) muscimol sites; (B) bicuculline sites; (C) sites where NMDA, APV, kainic acid, DNQX, glycine, strychnine, or saline were injected. For clarity, not all sites are shown; many sites overlapped; Table 1 provides the lateral location of every injection site. 4290 D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295

Table 1 were made in roughly the same area of the caudal pons a Results of injections of chemical agents on gaze stability as the muscimol injections (Fig. 1B and Table 1). The Agent Location Monkey Result three most medial injections, all between 1.0 and 1.5 mm and probably conﬁned to the NPH, made the Muscimol (GABAA receptor agonist), 10 mM integrator leaky. The average time constant was 2.8 s. ~ 9 1.0 mm L 2 min =1.0 0.1 s ~ 9 The time constants generally were reduced less and 1.5 mm L 1 min =0.2 0.02 s ~ 9 recovered faster than after muscimol injections (Fig. 3). 1.7 mm L 3 min =1.1 0.1 s ~ 9 2.0 mm R 1 min =1.5 0.2 s The null points stayed within 5° of the center of gaze ~ 9 2.5 mm R 2 min =0.8 0.1 s and the direction of the shift was inconsistent. Four ~ 9 2.7 mm L 3 min =−0.8 0.2 s* ~ 9 other injections from 1.5 to 2.7 mm lateral produced a 2.7 mm L 3 min =−1.1 0.2 s* ~ 9 nystagmus characterized by slow phases of constant 3.0 mm R 2 min =−1.2 0.2 s* 9 3.0 mm R 2 Vmax =13 1°/s velocity (around 15 °/s, Table 1) as illustrated in Fig. 9 3.0 mm R 2 Vmax =8 1°/s 4B. These injections were mainly in the medial half of

Bicuculline (GABAA receptor antagonist), 10 mM the MVN. Unlike the muscimol injections, the direction ~ 9 1.0 mm L 2 min =4.8 0.6 s of the slow phases of the nystagmus could be ipsilateral ~ 9 1.5 mm L 1 min =1.9 0.2 s ~ 9 (1 case) or contralateral (3 cases) to the side of the 1.5 mm L 1 min =1.7 0.1 s 9 injection. Two injections at 4 mm, undoubtedly in the 1.5 mm R 2 Vmax =18 1°/s 9 2.0 mm L 3 Vmax =11 1°/s DVN (Fig. 1), had no effect. 9 2.7 mm L 3 Vmax =15 1°/s 2.7 mm L 3 V 1491°s max = / 3.4. NMDA and APV (NMDA receptor antagonist) 4.0 mm R 2 No effect 4.0 mm R2 No effect injections NMDA, 50 mM ~ 9 Three 1 ml, 50 mM unilateral injections of NMDA 1.5 mm L 1 min =0.8 0.2 s ~ 9 3.0 mm L 3 min =1.5 0.3 s were made at locations within 3 mm of the midline ~ 9 3.0 mm R 2 min =0.9 0.1 s (Table 1). All three injections made the integrator ~ APV (NMDA antagonist), 1 mM leaky. The mean min was 1.1 s. The time constants ~ 9 1.5 mm L 1 min =3.0 0.3 s exhibited a time course similar to muscimol, but with a 1.5 mm L 1 ~ 3.690.3 s min = slightly faster recovery (Fig. 3). Four unilateral 1 ml 3.0 mm L 3 No effect ~ 9 injections of 1 mM APV were made. As can be seen in 3.0 mm R 2 min =2.8 0.3 s Table 1, three of the four resulted in a leaky integrator, Kainic acid, 50 mM ~ 9 while the fourth produced no effect. The minimum time 1.5 mm L 1 min =0.7 0.1 s ~ 9 2.7 mm L 3 min =0.5 0.1 s constant (mean 3.1 s) as well as the time course of ~ 9 3.0 mm R 2 min =1.3 0.1 s recovery were virtually the same as in the case of DNQX (kainate receptor antagonist), 10 mM injections of bicuculline (Fig. 3). These injections ~ 9 1.5 mm L 1 min =2.0 0.2 s ranged from the marginal zone (NPH-MVN boundary) 3.0 mm R 2 ~ 1.090.2 s min = to lateral MVN but laterality seemed not to make a 2.7 mm L 3 No effect difference. a ~ min refers to the minimum time constant reached by the integrator. Negative time constants, indicated by *, indicate an unstable 3.5. Kainic acid and DNQX (kainate receptor integrator where the eyes moved away from the center, as in Fig. 4. antagonist) injections Vmax is the velocity of the slow phases of constant-velocity nystagmus, when it was most intense. In both cases following muscimol m m injection, the slow phases were ipsilateral. For the four cases follow- Three 1 l injections of 50 M kainic acid were made ing bicuculline injection, the slow phases of Vmax were ipsilateral in within 3 mm of the midline (Table 1). All three injec- one case and contralateral in the other three. Recall from Fig. 1 that tions produced a leaky integrator. The mean time con- the locations less than 1.7 mm are probably in the NPH; from 1.7 to stant was 0.8 s, the lowest of all averages. Three 3.5 in the MVN; and beyond 3.5 in the DVN. All injections were on m the left (L) in Monkeys 1 and 3 and the right (R) in Monkey 2. unilateral, 1 l injections of 10 mM DNQX were made Variability is shown by 91 S.D. All injections were 1 ml. within 3 mm of the midline (Table 1). Two out of the three injections produced a leaky integrator, while the and an unstable integrator is near the medial-lateral third had no effect. center of the MVN at this level. 3.6. Glycine and strychnine (glycine antagonist) 3.3. Bicuculline (GABA antagonist) injections Three unilateral, 1 ml injections of 1 mM glycine (one Nine unilateral 1 ml injections of 10 mM bicuculline in each monkey) were made within 1.5–3 mm of the D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295 4291 midline. In each case there was no discernible effect on 4. Discussion eye movements. Three unilateral, 1 ml injections of 100 mM strychnine were made within 1.5–3 mm of the The main ﬁnding of this study is that injection of midline (at the locations similar to those in Fig. 1C). No chemicals with either agonist or antagonist effects on changes in eye movements were apparent after these GABA, glutamate, or kainate receptors in the NPH- injections. These results suggest that glycine is not a MVN region interferes with the ability to hold the eyes major factor in integrator connections. steadily at an eccentric position in the orbits. Glyciner- gic agents had no effect. The commonest effect of 3.7. Controls pharmacological inactivation was to make the neural integrator for eye movements ‘leaky’, causing cen- In each of the three monkeys a 1 ml injection of tripetal drifts of the eyes and gaze-evoked nystagmus. normal saline was made within 3 mm of the midline. At some injection sites, either bicuculline or muscimol These produced no discernible change in eye caused nystagmus with linear slow phases. Several of movements. these effects have been previously reported for indiv-

Fig. 2. An example of eye movements associated with an injection that caused the neural integrator to become leaky. (A) Prior toa1mlinjection of 10 mM muscimol. (B) Following the injection, at the time of maximum effect. In (A), the trained monkey, in darkness, is searching for the targets normally visible at 0 and 20° right. Note the normal horizontal gaze-holding. The mild down-beating nystagmus is normal for the rhesus monkey. In (B) the integrator has become very leaky, creating gaze-evoked nystagmus. See text for discussion. In this and other ﬁgures, U, D, R, and L indicate up, down, right and left, respectively. 4292 D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295

Fig. 3. Representative time courses of the neural integrator time constant following injections of each of the chemical agents employed in this study. Muscimol, NMDA, DNQX, and kainic acid all decreased the time constant below 2 s, whereas Bicuculline and APV had milder effects. Smaller time constants were associated with longer recovery periods. For minimum values of the time constant measured for all injections, see Table 1. idual agents (Cannon & Robinson, 1987; Cheron & of neurons; in the horizontal plane, the NPH-MVN Godaux, 1987; Straube et al., 1991; Mettens et al., region is critical (Arnold & Robinson, 1997). Pharma- 1994a, b). A novel ﬁnding was that muscimol injections cological agents may prevent the constituent neurons of into the center of the MVN sometimes caused the eyes this network from modulating their discharge in re- to drift away from the central position with increasing- sponse to inputs from other neurons; this would cause velocity waveforms, suggesting that the neural integra- the integrator to become leaky. This effect would be tor had become unstable. A similar effects has been expected both for agents such as muscimol, which previously reported by Straube et al. (1991), after injec- hyperpolarizes cells (and drives them into cutoff) and tion of bicuculline into the vestibular nuclei. How can drugs such as bicuculline, which depolarize cells (and these results — in which agonist or antagonist of drives their discharge rates into saturation). speciﬁc transmitter receptors had similar effects — be A suggested mechanism whereby the neural integra- explained? tor could become unstable is that its constituent neurons remain functional, but that the feedback control of them — by the cerebellum — be disrupted (Kamath & 4.1. Interpretation of the effects of pharmacological Keller, 1976; Zee, Leigh & Mathieu-Millaire, 1980). inacti6ation Experimentally, such instability is reported following

injection of either the GABAA agonist, muscimol (this

Present evidence suggests that the neural integration study) or the GABAA antagonist bicuculline (Straube et of oculomotor signals depends on a distributed network al., 1991) into that portion of the MVN which receives D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295 4293

ﬂoccular afferents and lies lateral to the marginal zone to be almost linear. This phenomenon has been previ- (Bu¨ttner-Ennever, 1992). Floccular Purkinje cells are ously documented (see, for example, Fig. 6 from the know to be GABAergic (Mugnaini & Oertel, 1985) and study by Straube et al., 1991). It has been suggested so it seems possible that inactivating their projections to (prompted by clinical observations) that centripetal neurons in MVN might be responsible for instability of drifts with more than one time constant could be due to the neural integrator. Perhaps of relevance to this issue non-linearities within the neural integrator (Abel, Del- is that acquired nystagmus with increasing velocity l’Osso & Daroff, 1978); this seems likely in the present waveforms is reported in patients with cerebellar lesions study, since microinjections only inactivated part of the (Zee et al., 1980; Barton & Sharpe, 1993). network of cells contributing to normal gaze-holding. Finally, a ﬁnding that deserves some explanation is In those cases when slow phases of nystagmus were that, after some injections with muscimol (for example, uniformly linear (especially following bicuculline injec- Fig. 2B), slow phases of nystagmus appeared to show tion as in Fig. 4C), a vestibular imbalance has been more than one time constant of drift, some appearing postulated (Straube et al., 1991).

Fig. 4. Examples of injections that caused nystagmus with either increasing-velocity (A, B) or linear (C) slow phases (note different scales on each panel). (A) Effects of 1 ml injection of 10 mM muscimol in Monkey 3 injected 2.7 mm lateral to midline. (B) Effects of 1 ml injection of 10 mM muscimol in Monkey 2 injected 3 mm lateral to midline. Note that the slow-phase drift away from the null point approximates an increasing exponential, indicating an unstable integrator. (C) Example of an injection causing nystagmus without integrator failure. The effect was produced by 1 ml injection of 10 mM bicuculline in Monkey 3. The record shows spontaneous nystagmus, in darkness, that has linear slow phases with a velocity of 15 °/s. 4294 D.B. Arnold et al. / Vision Research 39 (1999) 4286–4295

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