JOURNALOFNEUROPHYSIOLQGY Vol. 68, No. 5, November 1992. Prinred

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Vestibular Inputs to Brain Stem Neurons That Participate in Motor Leaming in the Primate Vestibuloocular Reflex DIANNE M. BROUSSARD AND STEPHEN G. LISBERGER Department of Physiology, W. AL Keck Foundation Center for Integrative Neuroscience, and Neuroscience Graduate Program, University of California, San Francisco, Cal$ornia 94143 SUMMARY

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CONCLUSIONS

METHODS

I. Previous studies have described a subpopulation of interneurons in the vestibuloocular reflex (VOR) pathways that express large changes in their responses to head turns in conjunction with motor learning in the VOR. These neurons are called flocculus target neurons (FINS) because they are inhibited at monosynaptic latencies by stimulation of the flocculus and ventral paraflocculus. 2. Electrical stimulation of the vestibular labyrinth revealed that FTNs receive excitatory monosynaptic inputs from the ipsilatera1 vestibular labyrinth and longer-latency, excitatory inputs from the contralateral labyrinth. 3. Our data show that commissural inhibition, which has been thought to be an important feature of vestibular processing, does not provide the dominant inputs from the contralateral labyrinth to FTNs. Instead, the inputs from both labyrinths are excitatory and may be functionally antagonistic. Changes in the balance of excitatory inputs from the two horizontal canals to FINS could contribute to motor learning in the VOR.

INTRODUCTION

The vestibuloocular reflex (VOR) functions to stabilize the direction of gaze so that clear vision is possible during head movements. In rhesus monkeys, the VOR causes eye rotation that is equal in speed and opposite in direction to each head turn, even in the absence of any visual feedback (Miles and Eighmy 1980). The excellent performance of the VOR is ensured by an adaptive mechanism that uses visual inputs to adjust the amplitude of the eye movement evoked by a given head turn (Gonshor and Melvill Jones 1976). We view this adjustment, which occurs throughout life, as a simple form of motor learning. Physiological studies have revealed a group of neurons in the medial vestibular nucleus that express large changes in their responses to natural vestibular stimulation in association with learning. These neurons are called flocculus target neurons (FTNs) because they receive monosynaptic inhibition from the flocculus (Stahl 199 1) and ventral paraflocculus (Lisberger and Pavelko 1988) of the cerebellum. Lisberger ( 1988) has argued that vestibular synapses onto FTNs are a likely site for the physiological changes underlying learning. However, the synaptic organization of the vestibular inputs to FTNs is unknown. We have therefore used electrical stimulation of the vestibular labyrinth with single pulses to determine the vestibular inputs to FTNs in alert monkeys. Our data show that FTNs are excited at monosynaptic latencies from the ipsilateral labyrinth and at longer latencies from the contralateral labyrinth. 1906

Experiments were carried out on two young male rhesus monkeys. Our methods for training monkeys, for monitoring eye position, and for the surgical implantation of chronic head-holding devices have been described elsewhere (Broussard et al. 1992). A bipolar stainless steel stimulating electrode was implanted at a site in the ventral paraflocculus at which single 200 PA current pulses elicited ipsiversive eye movements. Platinum-iridium stimulating electrodes were implanted in the perilymph of one or both vestibular labyrinths with a transmastoid approach. Stimulation was referenced to a silver ball electrode in the middle ear. In monkey R, who yielded most of the data presented here, electrodes were implanted in the superior canals bilaterally; this placement provided excellent electrical activation of afferents from the horizontal canal without altering the mechanical function of that canal (Broussard et al. 1992; Bronte-Stewart and Lisberger 1990). In a second monkey, the stimulating electrode was placed in the perilymph of the vestibule. Similar results were obtained from both monkeys. For single-unit recording, a stainless steel cylinder was implanted stereotaxically and glass-insulated platinum-iridium microelectrodes were advanced into the brain stem in the region of the medial vestibular nucleus. Isolated neurons were first characterized behaviorally during smooth pursuit eye movements evoked by sinusoidal target motion at 0.4 Hz and & 10” amplitude. Neurons were next tested for inhibitory input from the lateral vestibulocerebellum by recording their responses to 200 individual current pulses (biphasic, 0.4-ms duration, 150-300 PA) delivered at 5 Hz through the stimulating electrode implanted in the ventral paraflocculus. Finally, the responses of FTNs were recorded while 200 individual current pulses (300-500 PA) were delivered at 5 Hz to each vestibular labyrinth. For the data shown here, we stimulated the vestibular apparatus at a current that evoked 50% of the maximum eye velocity evoked at any current by a single pulse. The discharge of single units was amplified and discriminated conventionally, and the time of occurrence of each unit spike was recorded to the nearest 10 ps by a computer. We also sampled the extracellular voltage trace at 100 kHz for 40 ms surrounding each stimulus and generated a template of the stimulus artifact and field potential by averaging traces in which there was clearly no evoked spike. Subtraction of the template from each raw data trace revealed any unit spikes that occurred immediately after the stimulus. RESULTS

The histogram in Fig. 1A shows a typical example of the suppression of firing that occurred in all FTNs after stimulation through electrodes in the ventral paraflocculus. Fig. 1B illustrates the average firing rate of the same FTN during pursuit of sinusoidal target motion along the horizontal me-

0022-3077192 $2.00 Copyright 0 1992 The American Physiological Society

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Figure 1C shows two responses of a typical FTN to electrical stimulation of the ipsilateral labyrinth. In trace I, the cell fired just before the stimulus artifact, and an evoked spike occurred superimposed on the field potential. In trace 2, the evoked spike did not fall on the field potential because the cell fired spontaneously just after the stimulus. For each cell, we estimated the cumulative probability that a given stimulus would evoke a spike. First, we measured the latency of the first spike after each of 150 stimuli. Then, we arranged the latencies in ascending order, assigned each measurement a probability value in an ascending series from 1 / 150 to 1, and plotted probability as a function of latency (Fig. 2A, trace labeled “stim”). To remove the contribution of spontaneous firing from these probability curves, we conducted the same analysis starting 20 or 30 ms before the stimulus (Fig. 2A, trace labeled “no stim”) and estimated the spontaneous probability at each latency by linear regression and interpolation (Fig. 2A, -). We then derived the cumulative probability that the stimulus pulse would evoke a spike (Fig. 2 B) by subtracting the spontaneous probability from the probability of firing after the stimulus. For the FTN whose data appear in Fig. 2, stimulation of the ipsilateral vestibular apparatus caused a sharp increase in the probability of firing that started 0.92 ms after the stimulus.

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1. Response properties of a typical FTN. A: peristimulus time histogram showing the inhibition of firing after the application of a single pulse to the ventral paraflocculus. Spikes were counted in 0.2-ms bins. The vertical dashed line shows the time of stimulation. B: average firing rate of the same FTN during pursuit of sinusoidal target motion. Upward deflections of the position traces indicate ipsiversive eye movement. A single cycle of the average has been repeated. For steady fixation straight ahead, this cell fired at 70 spikes/s. C: two records of extracellular voltage during electrical stimulation of the ipsilateral vestibular apparatus with a single pulse. The dashed lines bracket the intervals that contained the stimulus artifacts, which have been removed from the traces for clarity. The upward arrows indicate action potentials from the cell. FIG.

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ridian with the head stationary. Firing rate was strongly modulated and increased for eye motion away from the side of recording. Of the 33 FTNs we recorded during smooth pursuit eye movements in the horizontal and vertical planes, I8 increased their discharge rates during contraversive eye movements (E-contra), and 2 showed increased firing during ipsiversive eye movements (E-ipsi). The other 13 cells showed clear modulation of firing during both horizontal and vertical pursuit (E-oblique). We will focus on the 20 E-contra and E-ipsi FTNs. During fixation at straight ahead gaze, these FTNs were spontaneously active at rates that ranged from 43 to 134 spikes/s (mean 83 spikes/s).

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FIG. 2. Quantitative analysis of the responses of FTNs to stimulation of the ipsilateral labyrinth. A : cumulative probability of firing is plotted as a function of time. The trace labeled “stim” shows the probability immediately after application of a single pulse to the labyrinth and the trace labeled “no stim” shows the probability contributed by the resting rate when no stimulus was applied. B: probability of evoking a spike as a function of time. f, time of onset of the response, which was used to measure the response latency; f- and +, baseline and peak probabilities, respectively, that were used to analyze response amplitude. C: response latency is plotted as a function of the maximum probability of evoking a spike with the stimulus. Each point represents the responses of 1 cell; 13 and m, ITNs that had increased firing for contraversive and ipsiversive pursuit, respectively.

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tion cannot be attributed to the data analysis; because we subtracted the “no stim” probabilities from the measured probabilities, inhibitory responses would have appeared as regions of negative slope in plots like Fig. 3A.

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DISCUSSION

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3. Quantitative analysis of the responses of FTNs to stimulation oftbe contralateral labyrinth. A : cu mulative probability of firing that can be attributed to the stimulus as a function of time. B: latency to the response as a function of the maximum probability of evoking a spike. Each point represents the responses of 1 cell; D and l , FTNs that had increased firing for contraversive and ipsiversive pursuit, respectively. FIG.

For each FTN, we measured the total probability of evoking a spike as the peak (+ ) of the stimulus-related probability minus the baseline (+ ) (arrows in Fig. 2 B), and we defined the response latency as the time of onset of the rise in the stimulus-related probability of evoking a spike (t , Fig. 2B). Fig. 2C plots the latency as a function of the probability of evoking a spike for 16 E-contra FTNs and 2 E-ipsi FTNs. The probability of evoking a spike ranged from near 0 to 0.95 at the standard stimulus current. Almost all of the horizontal FTNs in our sample responded to stimulation of the ipsilateral labyrinth at short latencies (0.75-l .39 ms) . Three FINS were activated at longer latenties and had relatively small responses, and two FTNs were not activated and are not plotted in Fig. 2. Responses of FTNs to stimdation of the contralateral labyrinth Current pulses applied to the contralateral labyrinth increased the probability of firing in 16 of the 20 horizdntal FTNs that were tested; the remaining 4 showed no response. Figure 3A shows the probability of evoking a spike by stimulation of the contralateral labyrinth for a typical FTN. The time course of the rise in probability was slower for the contralateral (Fig. 3A) than for the ipsilateral inputs to FTNs (Fig. 2 B). Fig. 3 B plots response latency as a function of the probability of evoking a spike by stimulation of the contralateral labyrinth. For our sample of 16 horizontal FTNs, ‘the probability of evoking a spike ranged from 0.07 to 0.36 and the latency of the response ranged from 1.35 to 3.38 ms. Although we can report the responses of only two E-ipsi FTNs, it is interesting that their responses to electrical stimulation of the vestibular apparatus (u, Figs. 2C and 3B) could not be distinguished from those of E-contra FTNs (El, Figs. 2C and 3 B) . For the stimulation conditions we used, no FTN in our sample, including E-oblique FTNs, showed any evidence of a decrease in firing rate after stimulation of the contralateral labyrinth. The absence of inhibi-

Our data provide new information about the vestibular inputs to a group of functionally identified neurons that are known to play an important role in motor learning in the VOR. Previous studies conducted on decerebrate animals had demonstrated a reciprocal organization in the vestibular inputs from the bilateral labyrinths to neurons in the vestibular nuclei (Shimazu and Precht 1966; Shimazu 1968 ) . In the earlier studies, neurons that showed increased firing during ipsiversive head turns were excited monosynaptically by stimulation of the ipsilateral vestibular nerve and were inhibited by stimulation of the contralateral nerve. In addition, in both cats (Shimazu and Precht 1966) and monkeys (Goldberg et al. 1987) some neurons received an excitatory input from the contralateral labyrinth. In the present study, Fl’Ns increased their firing rates during the VOR evoked by ipsiversive head turns (Lisberger and Pavelko 1988 ) , and most received excitation from the ipsilatera1 labyrinth at latencies that are compatible with monosynaptic inputs. However, all FTNs received longer-latency excitatory input from the contralateral labyrinth and none showed any evidence of inhibitory inputs. Although this does not prove the absence of commissural inhibition to FTNs, it implies that commissural excitation dominates the responses of FTNs to stimulation of the contralateral labyrinth in the awake animal. Because FI’Ns are known to participate in the horizontal VOR, we assume that their synaptic inputs from both vestibular labyrinths arise from the horizontal canals. This assumption may not be valid because our stimulating electrodes activated afferents from receptors other than the horizontal canal. If the assumption is true, then the balance between antagonistic inputs from the two horizontal canals could be important in determining the responses of FTNs during the VOR. During an ipsiversive head turn, increased excitation from the ipsilateral labyrinth would be opposed by decreased excitation from the contralateral labyrinth. In E-contra FTNs, the input from the ipsilateral horizontal canal is normally stronger and firing rate increases during the VOR evoked by ipsiversive head turns. When the gain of the VOR is low, however, E-contra FI’Ns show a reversal in direction selectivity so that firing rate increases during the VOR evoked by contraversive head motion (Lisberger and Pavelko 1988 ). Antagonistic inputs from the two horizontal canals could be important in mediating this reversal in the responses of FTNs. When the gain of the VOR is low, either a more effective input from the contralateral canal or a less effective input from the ipsilateral canal would shift the balance of vestibular inputs to FTNs in favor of increased firing during contraversive head motion. We are grateful to Dr. Sascha du Lac for helpful comments on an earlier version of the manuscript, to Dr. David J. Perkel for suggesting the method of data analysis, and to J. Schwartz and P. Walton for technical assistance. The research was supported by National Eye Institute Grant R37-EY03878 and by a Development Award from the M&night Neuroscience Endowment Fund.

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INPUTS TO FLOCCULUS Address for reprint requests: D. M. Broussard, Box 0444, Dept. of Physiology, University of California, San Francisco, California 94 143. Received 1 July 1992; accepted in final form 20 August 1992. REFERENCES H. M. AND LISBERGER, S. G. Physiological properties of vestibular afferents participating in the plasticity of the vestibulo-ocular reflex. Sot. Neurosci. Abstr. 16: 733, 1990. BROUSSARD, D. M., BRONTE-STEWART, H. M., AND LISBERGER, S. G. Expression of motor learning in the response of the primate vestibuloocular reflex pathway to electrical stimulation. J. Neurophysiol. 67: 1493-1508, 1992. GOLDBERG, J. M., HIGHSTEIN, S. M., MOSCHOVAIUS, A. K., AND FERNANDEZ, C. Inputs from regularly and irregularly discharging vestibular nerve afferents to secondary neurons in the vestibular nuclei of the squirrel monkey, I. An electrophysiological analysis. J. Neurophysiol. 58: 700-718, 1987. GONSHOR, A. AND MELVILL JONES, G. Short-term adaptive changes in the BRONTE-STEWART,

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human vestibulo-ocular reflex arc. J. Physiol. Lmd. 256: 361-379, 1976. LISBERGER, S. G. The neural basis for learning of simple motor skills. Science Wash. DC 242: 728-735, 1988. LISBERGER, S. G. AND MILES, F. A. Role of primate medial vestibular nucleus in long-term adaptive plasticity of vestibuloocular reflex. J. Neurophysiol. 43: 1725- 1745, 1980. LISBERGER, S. G. AND PAVELKO, T. A. Brain stem neurons in modified pathways for motor learning in the primate vestibulo-ocular reflex. Science Wash. DC 242: 77 1-773, 1988. MILES, F. A. AND EIGHMY, B. B. Long-term adaptive changes in primate vestibuloocular reflex. I. Behavioral observations. J. Neurophysiol. 43: 1406-1425, 1980. SHIMAZU, H. Inhibitory commissural fibers interconnecting the bilateral vestibular nuclei. Brain Res. 8: 378-382, 1968. SHIMAZU, H. AND PRECHT, W. Inhibition of central vestibular neurons from the contralateral labyrinth and its mediating pathway. J. Neurophysiol. 29: 467-492, 1966. STAHL, J. S. Signal Processing in the Vestibulo-Ocular Reflex Circuitry of the Rabbit ( PhD dissertation). New York: New York University, 199 1.

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Vestibular inputs to brain stem neurons that participate in motor learning in the primate vestibuloocular reflex.

1. Previous studies have described a subpopulation of interneurons in the vestibuloocular reflex (VOR) pathways that express large changes in their re...
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