Response of medial medullary reticular neurons to otolith stimulation during bidirectional off-vertical axis rotation of the cat

BRAIN RESEARCH ELSEVIER

Brain Research 732 (1996) 159-168

Research report

Response of medial medullary reticular neurons to otolith stimulation during bidirectional off-vertical axis rotation of the cat Y.S. Chan *, C.W. Chen, C.H. Lai Department of Physiology, Faculty of Medicine, The University of Hong Kong, 5 Sassoon Road, Hong Kong Accepted 23 April 1996

Abstract In decerebrate cats, the extracellular activities of neurons in the medial medullary reticular fortnation were studied during constant velocity off-vertical axis rotations (OVAR)in the clockwise(CW) and counterclockwise(CCW) directions(at 10”tilt). Spontaneously

active neurons demonstratedsinusoidalposition-dependentdischargemodulationsto OVAR which selectivelystimulatesthe otoliths. Two features of neuronalresponsesto bidirectionalOVAR were identified.Within the velocity spectrumtested (1.75–150/s), some neuronsshowedsymmetricbidirectionalresponsesensitivity(8 value)to CW and CCWrotations.The spreadof the 3 valuesof each of these neurons with velocity was small. This group of reticular neurons were described as exhibitingsymmetricand velocity-stable bidirectionalresponse sensitivity.The mathematicallyderived gain tuning ratios of these neuronswere within the range of narrowly spatiotemporal-tuned neurons.Anothergroupof reticularneurons,however,showedasymmetricbidirectionalresponsesensitivityto CW and CCWrotations;a few of theseneuronswereresponsiveonlyto OVARof one directionbut not to both.For each of this secondgroup of neurons,the spread of the 8 values with velocitywas large. These reticularneuronswere describedas exhibitingasymmetricand velocity-variablebidirectionalresponsesensitivity.The gain tuningratios of these latter neuronswere foundto be within the range of broadlyspatiotemporal-tunedneurons.Singleneuronsof both groupsdisplayedorientationaltuning.Both the best responseorientations of neuronsthat showedsymmetricand velocity-stablebidirectionalresponsesensitivityand the preferredorientationsof neuronsthat showedasymmetricand velocity-variablebidirectionalresponsesensitivitywere foundto point in all directionson the rotaryplane. The responsedynamicsof the former groupof neuronswas also examined.All showedflat responsegain across the entire velocityrange. Someshoweda flat responselead whileothersshoweda progressiveshiftfromsmallresponselead at low velocityto phasecloseto zero at highervelocities.The functionalsignificanceof these medialmedullmyreticularneuronsto the directionand orientationof head tilt is discussed. Keywords: Otolith; Medial medullary reticular neuron; Off-vertical axis rotation; Bidirectional response sensitivity; Spatial property; Response dynamic; Cat

1. Introduction Neurons in the pontomedullary reticular formation have been shown to encode head movements close to the horizontal plane [6,17,33,39,45]. Responses reflecting different spatiotemporrd inputs are evoked in reticular neurons during sinusoidal rotations that consisted of both linear and angular head accelerations [6,17]. Complex spatiotemporal patterns were also observed in the vestibular nuclei [30,50] and the vestibulospinal reflex [49] during similar sinu-

‘ Corresponding author. E-mail: [email protected]

soidal vertical rotations. The elicitation of these patterns has been attributed to the spatiotemporal convergence of inputs arising from the vertical semicircular canals and the otolith organs [5,30]. No attempt has hitherto been made to investigate the spatiotemporal properties of reticular neurons in response to pure otolith stimulation. Despite a dearth of pure otolith-related data on the reticular formation, neurons in the vestibular nucleus of rats were shown to exhibit narrowly tuned (or one-dimensional) spatiotemporal sensitivity and broadly tuned (or two-dimensional) spatiotemporal sensitivity with the use of linear accelerations that stimulate only the macular receptors [4,8]. Different spatiotemporal response sensitivities were also observed in tilt-sensitive vestibular nuclear neu-

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rons during off-vertical axis rotation, OVAR [32], that activates only the otolith afferents [13]. Constant velocity OVAR, which simulates natural head rotation about a tilted axis, is an appropriate method to test otolith function [21]. This mode of vestibular stimulation is also of interest because natural head rotations are often about axes that deviate from the vertical. In monkeys, central vestibular neurons mediating the sequential change in the rotating gravity vector acting on the otolith receptors during OVAR [40] are shown to be involved in the generation of maculo-ocular reflex [13,27]. Besides the vestibular nucleus [50], processing of otolith signals in the reticular formation [6,33] is implicated in eliciting reflexes of the muscles in the neck and limbs [43,49]. The OVAR provides a good stimulus paradigm for investigations of the role of otolith inputs in spatiotemporal processing within neurons of the medullary reticular formation. In this paper, neurons in the medullary reticular formation were first grouped into two classes according to their response sensitivity to CW and CCW OVAR at different velocities. The otolithic evoked behaviors of these two groups of neurons were then studied with an aim to understand their functional features in the coding of head orientations near the horizontal plane.

2. Material and methods 2.1. Surgical procedures Experiments were performed on 23 adult cats (2.5-3.8 kg). Surgical procedures were performed under halothanenitrous oxide anesthesia. The right femoral artery and vein were cannulated for monitoring of arterial blood pressure and injection of fluid respectively. The head of the animal, mounted on a Kopf stereotaxic frame, was maintained in the earth’s horizontal plane. The cervical column of the cat was oriented parallel to gravity [cf. [4811with the surgically exposed spinous process of the C2 vertebra clamped to the stereotaxic frame. Relative movement of the neck, shoulder and forelimbs was minimized by a freshly prepared plaster cast placed on these areas of the body. The trunk was placed in a tailor-made plastic tray which was attached to the tilt table while the extended limbs of the animal were secured rigidly to the tilt table. These served to minimize any somesthetic stimulation during subsequent natural vestibular stimulation. A small portion of the left occipital cortex overlying the colliculi was aspirated and the animal was precollicularly decerebrated. The cranial cavity was gently packed with cotton wool to minimize mechanical movement of the brain during subsequent vestibular stimulation. Craniotomy of 5 mm in diameter was made on the skull overlying the left vermal cerebellum between 0.5 and 1 mm from the midline. The dura of the cerebellum was removed to allow placement of the recording microelectrode. The exposed brain surface to-

gether with the exposed site of decerebration were covered with 3Y0 agar in physiological saline. Anesthesia was discontinued after these surgical maneuvers. In all animals, pressure points and wounds were infiltrated with 2$Z0 lidocaine (Astra). Body temperature was maintained at 37°C with a thermostatically-controlled heating pad. The animal respired spontaneously and the endtidal carbon dioxide, monitored with a carbon dioxide monitor (Datex), was within the range of 3.8–4.2% throughout the experiment. Blood pressure was maintained between 100-120 mmHg with intravenous drip of 80 Kg/ml Aramine (Merck, Sharp & Dohme), if blood pressure appeared to fall. 2.2. Vestibular stimulation and unit recording The whole animal was fixed on the rotating platform and the whole assembly was tilted to a fixed angle (10°) deviated from the earth’s vertical. Constant velocity rotations (1.75, 4.7, 9 or 150/s) were then applied about the tilted axis to the whole experimental set-up in either the clockwise (CW) or the counterclockwise (CCW) direction. The rotary axis passed through the center of the animal’s head. This form of natural vestibular stimulation was described as off-vertical axis rotation (OVAR). The motion of the table was controlled by a stepping motor device (Daedal Inc.). During rotation, the instantaneous position of the table was monitored via the output of a potentiometer placed on the rotatory axis of the table. A tungsten microelectrode (tip diameter 1 p,m, 10 Mi2, Frederick Haer) inclined at 15° from the vertical was inserted dorsoventrally through the intact vermal cerebellum to the reticular formation in the left medulla. Extracellular recording was made from the medullary reticular formation at 2 h after cessation of anesthesia. The firing rate of each spontaneously active neuron in the medullary reticular formation was continuously monitored at two static head positions (first at horizontal and then at 10° head-down) for 10 min. Only those units with stable spontaneous firing rate were studied. At 10° tilt, the responses of medial medullary reticular units to 2–4 successive complete 360° constant velocity (1.75, 4.7, 9 and 150/s) OVAR in the CW and CCW directions, or vice versa, were studied. In some cases, the response patterns were also studied with OVAR at 5°, 15° and/or 20° tilt. The waveform of each action potential was closely monitored on a slave storage oscilloscope (displayed with a fast time base). Neurons which varied in the spike shape or amplitude during successive rotations were discarded. The neuronal activities were conventionally amplified, filtered (0.3-3 kHz) and fed to a window discriminator (Frederick Haer). The unitary activities and the signals indicating the table positions during OVAR were directed through a data acquisition device (Cambridge Electronics Design, 1401 plus) and subsequently stored in a PC486-computer for off-line analysis. For on-line inspection of the neuronal

Y.S. Chan et al./Brain Research 732 (1996) 159-168

responses, the unitary firing rate, derived from a rate counter (Frederick Haer), and the signal of the table position were displayed on a 2-channel chart recorder. 2.3. Data analysis The unitary firing rate during each set of OVAR was averaged into a cycle histogram of 256 bins/cycle. The periodic modulation of neuronal responses were then subjected to best sine curve fit by a least-squares procedure. The harmonic distortion (ratio of the Fourier amplitude of the second harmonic to that of the fundamental) and signal-to-noise ratio (ratio of the Fourier amplitude of the fundamental harmonic to the root mean square amplitude of the harmonics above the second one) of each unit were also determined. Unitary responses in which the harmonic distortion was <3590 or the signal-to-noise ratio was >1.5 were considered significant [cf. [11,44]]. The positions of the CW and CCW discharge maxima as well as the response gain were evaluated from the curve. The response gain (impulses/s/degree) was defined as half peak-to-peak change of firing rate per degree of tilt from the earth’s vertical. The response magnitudes during OVAR in the CW and CCW directions were expressed in terms of the bidirec[see tional response sensitivity ratio, 8 = GainCw\GainCCw [1]]. This ratio has been shown to be related to the gain tuning ratio o [I] by the equation: u =(8– 1)/(8+ 1). A gain tuning ratio of 0.1 was used in recent linear acceleration studies [4,8] to separate narrowly spatiotemporal-tuned unitary response (i.e. one-dimensional neuron) from broadly spatiotemporal-tuned response (i.e. two-dimensional neuron). Application to OVAR requires u values of +0.1 to define this separation; this u range was equivalent to 8 values of 0.82–1.22, meaning that the amplitude of discharge modulation to CW rotation is within 82–12270 of that obtained during CCW rotation. With this range as reference, 8 values of 0.75–1 .25 was adopted in the present study to segregate the narrowly spatiotemporaltuned neurons from the broadly tuned ones. Thus, neurons showing CW discharge modulation within 75–125% of the CCW discharge modulation should correspond to narrowly tuned neurons and those showing CW discharge modulation either <7570 or > 1259Z0of the CCW discharge modulation should correspond to broadly tuned ones (see [32]). The former pattern was described as exhibiting symmetric response sensitivity to CW and CCW rotations while the latter pattern was described as exhibiting asymmetric response sensitivity to CW and CCW rotations. To assess the spread of the 8 values of each neuron within the velocity spectrum tested, the difference between the maximum and minimum 8 values of each neuron at different velocities was also calculated. For each unit which demonstrated symmetric bidirectional response sensitivity, the orientation of the best response on the plane of rotation was determined from the

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mean of the orientations of the CW and CCW discharge maxima delineating the small sector on the plane of rotation [see also [1,12]]. The spatial difference (in degrees) between the orientation of the best response and the CW or CCW discharge maximum was taken as the response phase of each unit on the rotatory locus: a response lag was observed when the CW maximum, compared with the best response orientation shifted in the CW direction and a response lead was observed when the CW maximum, compared with the best response orientation shifted in the CCW direction [see also [1]]. For unitary responses which displayed asymmetric bidirectional response sensitivity, the orientations of the CW and CCW discharge maxima were presented as a pair so as to illustrate the directional preference. The response phase of these units, however, cannot be determined using the present experimental paradigm. 2.4, Histological confirmation The site of the last recorded unit and the deepest point of electrode penetration were marked by lesion with electrical current (0.25 mA for 8 s) at the end of the recording session. The animal was then given pentobarbital injection (60 mg/kg, i.p.) before transcardiac perfusion with 1 1 physiological saline followed by 11 10Yoformaline-saline. The brain was removed and stored in 10?ZOformaline. Before histological processing, the brain was stored for 48 h in a solution of 20910sucrose in formaline-saline. Frozen parasagittal sections (60 p,m thickness) of the brainstem were cut with a sliding microtome. Histological location of the recorded units were reconstructed from cresyl violet stained serial sections with reference to the lesions and depth of the microelectrode penetrations.

3. Results A total of 55 spontaneously active medial medullary reticular neurons responded to 10° OVAR (1.75–150/s) in the CW and CCW directions with sinusoidal position-dependent discharge modulation. For each neuron, the variation in the magnitude of the discharge modulation (i.e. peak-to-peak firing rate) during successive rotations in the same direction was < 10% of the mean. Also, the DC firing rate (the rate about which the response was modulated) during successive rotations remained unchanged for each neuron. The different neuronal response behaviors reported in this study were sampled throughout the recording period and there was no noticeable bias in their frequency of occurrence during the course of each experiment. Neurons recorded were histologically confirmed to be in the left medullary reticular formation. The recorded area was found to be between 0.5–1 mm from the midline, extending rostrocaudally from the posterior border of the left abducens nucleus to 2 mm further caudal, and span-

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ning dorsoventrally between a region just ventral to the abducens nucleus and dorsal to the inferior olive. 3.1. Bidirectional response sensitivity Neurons showed two patterns of bidirectional response sensitivities, 8, to OVAR. One group of neurons (19 units were tested with 3–4 stimulus velocities; 10 units were tested with 2 velocities, viz. 1,75 and 90/s) exhibited

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symmetric response sensitivities to each set of CW and CCW rotations (i.e. the CW gain was between 75–125% of the CCW gain) within the velocity spectrum tested (Fig. 1A). In this group of neurons, the spread in 8 values of each neuron within the velocity spectrum tested ranged between 0.06–0.3. Such a range of difference in 8 values with velocity was taken as stable. Neurons in this group are thus classified as exhibiting both symmetric response sensitivity to each set of CW and CCW rotations as well as velocity-stable bidirectional response sensitivities. Furthermore, the gain tuning ratio, u, an index used in linear acceleration studies [4,8] to compare the magnitude of the minimum and maximum sensitivity vectors thereby separating narrowly spatiotemporal-tuned neurons from broadly spatiotemporal-tuned neurons, was also calculated using the equation proposed by Angelaki [1]. The u values of the 29 neurons in this group were within > –0.1 and <0.1. Their u values also remained stable with increase in the velocity of OVAR. These characteristics of the u values were comparable to those of the narrowly spatiotemporal-tuned one-dimensional neurons [cf. [8]]. Thus, the present group of neurons with symmetric and velocitystable 8 should correspond to narrowly spatiotemporaltuned neurons. The other group of neurons (15 units were tested with 3–4 stimulus velocities; 6 units were tested with 2 velocities, viz. 1.75 and 90/s; 5 units were tested with only 1 velocity, viz. 90/s) exhibited asymmetric bidirectional response sensitivities (i.e. the CW gain was either < 75?Z0or > 125% of the CCW gain) in at least one of the velocities tested (Fig. IB). All except 4 neurons showed discharge modulation to both CW and CCW rotations at various velocities tested. These 4 neurons were tested with 3–4 velocities and responded to OVAR in only one direction (8 value of O or ~) at some velocities (3 only to CW rotation and 1 only to CCW rotation) while remained bidirectionally responsive in other velocities. A large spread in 8 values with velocity change was observed in each of these 4 neurons as their 8 values spanned from -1 to O or ~. Of the remaining 17 neurons (tested with >2 velocities) that were bidirectionally responsive to OVAR, the spread in 8 values of each neuron within the velocity spectrum ranged between 0.35–1 .48, significantly higher than that of neurons exhibiting velocity-stable 8 values. This range of difference in 8 values with velocity was taken as velocity-variable. Thus, all neurons in this population were described as exhibiting asymmetric response sensitivity to each set of CW and CCW rotation as well as velocity-variable bidirectional response sensitivities. Besides, the gain tuning ratios, u, of these neurons were < –0.1 and >0.1 in at least one of the velocities tested. These m values were characteristic of the broadly spatiotemporal-tuned two-dimensional neurons [cf. [8]]. Thus, this population of neurons exhibiting asymmetric and velocity-variable 8 should correspond to the broadly spatiotemporal-tuned neurons.

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Velocity (O/s) Fig. 1. The response pattern of reticular neurons that showed symmetric (A) and asymmetric (B) bidirectional response sensitivity (8 ratios) with changes in the velocity of OVAR. In A and B, the set boundaries that separate symmetric and asymmetric bidirectional response sensitivities (8 ratios of 0.75 and 1.25) were denoted by dotted lines. A: the 8 ratios were within 0.78 and 1.19. The spread in 8 values of each neuron within the velocity spectrum tested was 0.06–0.3; all neurons showed velocitystable 8 vatues. + indicates neuron with a spread of the 8 ratios <0.15 within the velocity spectrum tested. B: most neurons showed symmetric 8 ratios at low velocity and asymmetric 8 ratios at high velocity; 2 neurons (labeled with +) showed asymmetric 8 ratios at all velocities tested. In this group, the spread in 8 values of each neuron within the velocity spectrum tested was 0.35–1.48: all neurons were classified as exhibiting velocity-variable 8 values.

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3.2. Spatial response to OVAR In neurons which showed symmetric and velocity-stable bidirectional response sensitivity, the best response orientation of each neuron remained stable at different velocities of rotation. The variation in the best response orientations of each of these neurons at different velocities ranged from 3.6° to 33.9°. The distribution of the best response orientations of these neurons was also examined on the rotary plane that was tilted at 10° from the earth’s horizontal (Fig. 2A). The best response orientations of these neurons pointed in all directions on the plane of rotation. For descriptive purpose, the plane of rotation was arbitrarily divided into 4 quadrants. The head-down and head-up quadrants were denoted as the pitch direction while the left-side-down and the right-side-down quadrants were denoted as the roll direction. For neurons with their best response orientations near roll, 10 were excited by rightside-down tilt (type ~) and 4 were excited by left-side-down tilt (type ~). For those neurons with their best response orientations near pitch, 12 were excited by head-up tilt (type 0 md 3 were excited by head-down tilt (type 2). NO significant difference was found between the number of

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roll and pitch neurons. For neurons with their best response orientations along the roll direction, the mean response gain in the right-side-down quadrant was 0.87 + 0.15 (S.E.M., n = 10) imp\s/O and that in the left-sidedown quadrant was 0.55 + 0.16 (S.E.M., n =4) imp/s/O. For those neurons with their best response orientations along the pitch direction, the mean response gain in the head-up quadrant was 0.70 + 0.11 (S.E.M., n = 12) imp/s/O and that in the head-down quadrant was 0.79 + 0.28 (S.E.M., n =3) imp/s/O. For the neurons which showed asymmetric and velocity-variable bidirectional response sensitivities, the relative behavior of each pair of asymmetric CW and CCW unitary responses to OVAR was plotted in Fig. 2B. The gain and orientation of the discharge maximum of each neuron for OVAR in the CW direction was paired with that in the CCW direction. The majority of the response pairs was found on one-half of the polar diagram, i.e. in the half-circle of the polar diagram bound by 180°–2700–00.To facilitate the analysis, only the distribution of the preferred orientations (i.e. orientation of the rotational direction in which a . . wa~ GIWILGUJ U,ese neurons was exammea greater galu ‘-’- ‘---“’-’’-J’ U. ‘r ‘L(filled symbols in Fig. 2B). Eight and 2 were in the

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Fig. 2. Polar diagrams showing on the plane of rotation (at 10° tilt) the gain and location of the discharge maxima of reticular neurons to bidirectional of the diagram represents either the mean g~n (imp/s/”) of the Cw’ ~d CCW response (A) or the OVAR. The distance of each symbol fromthecenter’ CW gain and CCW gain (B) of each neuron. Neurons with the response gain greater than the radius are drawn along the circumference and their response h ( left-side-down (LSD), h gain values are shown in brackets. Dotted lines arbitrarily divide the plane of rotation into 4 q 4 st~dmd head positions me referred to as 0°, 90°, 180”and 270”, respectively. A: spatial distribution of the best (Hu)andr%M-Side-dOWrr @sDl ‘rhes’e response orientations of neurons at 1.750/s. O represents neurons that showed symmetric and velocity-stable 8; * represents neurons that showed symmetric 3 at 1.750/s but asymmetric 8 at > 4.70/s (circle pairs, square pairs and star pairs in panel B); X represents neurons that showed symmetric 8 at 1.750/s but responsive to only one direction of rotation at higher velocities. B: relation of CW and CCW responses in neurons that showed asymmetric and velocity-variable b vahres. Responses of the same unit to CW and CCW rotations are connected by a straight line. Neurons showing a greater gain with CW rotation were paired by solid line and those with CCW rotation were paired by dashed line. Filled symbols indicate the preferred orientations. Triangle (n = 2) and rhomboid (n = 1) pairs represent neurons showing asymmetric ~ at 1.750/s; triangle P~rs showed asymmetric ~ at all velocities tested. Other symbol pairs represent neurons showing asymmetric 8 at 4.70/s (circle pairs, n = 2), 9“/s (square pairs,n = 9) or 150/s (st~ pairs,n = 3); afl showed symmetric 8 at 1.750/s (illustrated as * in panel A).

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For a majority of the reticular neurons that showed asymmetric and velocity-variable bidirectional response sensitivities to OVAR, a greater gain was always evoked at a particular rotating direction at velocities that elicited asymmetric response sensitivity. Neurons can thus be classified as CW or CCW neurons. Thirteen were CW neurons and 6 were CCW neurons. Two neurons, however, cannot be so grouped as at some velocity a greater gain was observed at CW direction while at other velocity a greater gain was observed at CCW direction. 3.3. Dynamic response of neurons with symmetric and velocity-stable bidirectional response sensitivi~

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Velocity (“/s) Fig, 3. The effect of velocity on the response gain (upper panel) and response phase (lower panel) of medullary reticula’ neurons that showed symmetric and velocity-stable bidirectional response sensitivity to OVAR in the CW and CCW directions. All neurons showed a stable gain with velocity (upper panel). Lower panel: positive response phase values indicate response lead, negative values indicate response lags, and zero value indicates the overlap of CW and CCW maxima. Two dynamic response behaviors were evident. One group showed stable response lead of - 30° and the other showed a progressive shift from small response lead to response phase hovering about zero. Circle and bar represent mean and S.E.M., respectively. The number of neurons for each set of data was indicated in parentheses.

right-side-down and left-side-down quadrants, respectively while 5 and 2 were in the head-up and head-down quadrants, respectively. Such a pattern of distribution was comparable to that of the best response orientations of the aforementioned neurons that showed symmetric and velocity-stable bidirectional response sensitivities. Amongst the reticular neurons that showed asymmetric and velocity-variable bidirectional response sensitivities, only 2 exhibited asymmetric response gains throughout the velocities tested (triangle pairs in Fig. 2B). The remaining 19 neurons exhibited asymmetric response gains only at some of the velocities tested. Most (n = 14) of these latter neurons showed asymmetric gains to CW and CCW rotations at velocities > 4.70/s (circle, square and star pairs in Fig. 2B) and showed symmetric response gains at 1.750/s (10° tilt). The best response orientations of these 14 neurons derived from stimulus eliciting symmetric gains (1.750/s; 10° tilt) were also included in Fig. 2A (asterisks): 7 were roll neurons and 7 were pitch neurons. For the 4 neurons that responded to OVAR in only one direction at some of the velocities tested (with 8 value of Oor ~), their response gains were symmetric at 1.750/s (10° tilt) and their best response orientations so derived were also illustrated in Fig. 2A (crosses).

In reticular neurons that showed symmetric and velocity-stable bidirectional response sensitivities, the response gain was stable as a function of velocity (Fig. 3 upper panel). The mean gain ranged from 0.79 to 0.85 imp/s/O. The response phase of these neurons to change in the velocity of OVAR was also studied (Fig. 3 lower panel). Some units exhibited stable response lead, between 24.9° and 34.5°. Most units showed a progressive shift in response phase with velocity, i.e. from response lead of 14.6° (at 1.750/s) to response lag of – 8.9° (at 150/s). The response phase of these latter units hovered around zero for stimulus between 4.7 and 150/s.

4. Discussion The present study describes the response characteristics of spontaneously active medullary reticular neurons during constant velocity OVAR which selectively stimulates the otolith afferents [13]. During OVAR, presumably the utricle as compared with the saccule is more effectively activated based on the directional sensitivity of its afferents [19,46]. We demonstrate that medial reticular neurons in response to OVAR exhibited either symmetric and velocity-stable bidirectional response sensitivity or asymmetric and velocity-variable bidirectional response sensitivity. Single neurons of both groups displayed orientational tuning, indicating their capability in the coding of head position during natural otolith stimulation. 4.1. Spatiotemporal-tuned neurons and otolith stimulation Constant velocity OVAR provides a good paradigm with which the spatiotemporal tuning property of otolith neurons can be characterized by assessing the response sensitivity of individual neurons to CW and CCW rotations. Such a characterization is based on the working hypothesis that this bidirectional response sensitivity can be correlated with the gain tuning ratio [1], an index employed in horizontal linear acceleration studies to separate narrowly spatiotemporal-tuned neurons from broadly tuned ones [8]. With OVAR, vestibular nuclear neurons

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have been shown to exhibit symmetric or asymmetric bidirectional response sensitivity to bidirectional rotation [11,32]. With the application of our working hypothesis, those vestibular nuclear neurons that exhibited symmetric bidirectional response sensitivities were found to show gain tuning ratios similar to the range of narrowly spatiotemporal-tuned neurons [32]. Likewise, those neurons exhibiting asymmetric bidirectional response sensitivities were found to have gain tuning ratios of broadly tuned neurons [32]. Nevertheless, it is essential to comment that our gain tuning ratio calculated from the equation of Angelaki [1] has yet to be experimentally proven as equivalent to the actual gain tuning ratio (S~iJS~,X) determined from measured responses to pure linear accelerations [8]. In the present study, neurons with different response sensitivities to bidirectional OVAR were found to coexist in the medial medullary reticular formation. Based on the implicit correlation between bidirectional response sensitivity and gain tuning ratio, one might speculate that the neuronal behaviors presently observed can be attributable to spatiotemporal convergence of otolith inputs. In other studies on the reticular formation, although neurons with behaviors of spatiotemporal convergence were observed [6,17], the vertical rotations employed activated both the vertical canals and otoliths. The possible contribution of the vertical canals became especially eminent in the rabbit study which used a sinusoidal frequency of 0.2–0.6 Hz [17]. Thus, the spatiotemporal features of reticular neurons evoked by pure otolith inputs cannot be revealed with these data. In the present study, reticular neurons with different asymmetric response patterns have been identified with bidirectional OVAR which selectively stimulates the otoliths. Using our working hypothesis, 42% of the reticular neurons that showed asymmetric and velocityvariable response patterns were found to have gain tuning ratios similar to those of broadly spatiotemporal-tuned neurons [8]. A much lower proportion (27%) of vestibular nuclear neurons displaying comparable response asymmetry (’spatiotemporal property’) was found in the rat [32]. Besides, the extent of response asymmetry to bidirectional OVAR was much greater in medial medullary reticular neurons than in vestibular nuclear neurons. This is reflected in the spread of 8 values of individual neurons showing asymmetric and velocity-variable response patterns: 0.35–1.48 for bidirectionally responsive reticular neurons and 0.35–0.67 for bidirectionally responsive vestibular nuclear neurons [32]. Furthermore, a few reticular neurons responded to OVAR in only one stimulus direction, showing a 8 value of O or CO,an observation that was not found amongst OVAR-responsive neurons in the vestibular nucleus of rats [32] and cats [10]. These phenomena suggest a difference in the processing of otolith information between central vestibular and medullary reticular neurons. That reticular neurons receive otolith inputs presumably via the vestibular nucleus [31,35] tempts one

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to speculate that the observed strong asymmetric response behavior of reticular neurons is partly attributable to additional processing of the spatiotemporal signals in the neural circuitry involved. The behaviors of the broadly tuned neurons in the vestibular nucleus have been suggested [3] to result from the convergence of regular and irregular otolith afferents [20,23]. In this context, it is noteworthy that some secondary vestibular neurons receive a mixed regular and irregular input, though it was indistinguishable whether the input arose from canal or otolith [7]. Besides, that afferent terminals of single axons have been shown to innervate otolith hair cells of different morphological polarization vectors [24,41] infers that spatially and temporally mismatched neural information may reach the afferent terminals. Thus, otolith afferents displaying different spatiotemporal-tuned properties [16] may provide the necessary information in the central construction of the directionality of response. The asymmetric and velocity-variable neuronal response patterns observed in our experiments may also emerge through other possible origins. Although no directional difference in the response gain to OVAR was observed amongst a small sample of otolith afferents [22], the generation of response asymmetry in central neurons may quantitatively involve the inputs of a family of afferents that display a spectrum of spatial and temporal signals. Contribution from the contralateral otoliths is also implicated as the proportion of vestibular nuclear neurons exhibiting asymmetric response sensitivity to OVAR was significantly higher in hemilabyrinthectomized cats than in control cats [9]. 4.2. Properties of neurons with symmetric and ueloci&stable bidirectional response sensitivi~ The best response orientations of reticular neurons that showed velocity-stable and symmetric bidirectional response sensitivity were found to point in all directions on the plane of rotation, with an equal number in the roll and pitch directions. Such a pattern of distribution is comparable to that of rat vestibular nuclear neurons which displayed similar spatiotemporal behaviors to OVAR [32]. This pattern is also comparable to that of the functional polarization vectors in otolith afferents [16,19,46]. The present findings indicate that head orientations close to the horizontal plane are being encoded in medullary reticular neurons that showed velocity-stable and symmetric bidirectional response sensitivity. A slightly different distribution was observed in other studies. With the use of sinusoidal vertical rotations, the optimal response plane orientations of most medial reticular neurons of rabbits were within &75° of the interaural axis, with only a few in the pitch direction [17]. It should be noted that though these neurons discharged almost in phase with position between 0.1–0.5 Hz, presumably receiving otolithic inputs, twothird of these neurons receive converging input from the

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canal as demonstrated by exponential step stimulation [17]. In another preparation using wobble rotations [6], the best vectors of pontomedullary reticulospinal neurons of cats were predominantly in the roll direction, i.e. within &45° of the contralateral-side-down position; less than 10Yowas in the pitch direction. Within the low frequency range employed in the OVAR (0.005–0.042 Hz) and the wobble rotation (0.05 Hz) studies, it is conceivable that essentially the same stimulus is delivered to the otolith organs. Thus, the difference in the fine tuning of the best response orientations between the experimental paradigms remains inexplicable. For the best response orientations along the roll direction, there was a predominance of type @ response (i.e. units that were excited in the right-side down quadrant) over those with type a response (i.e. units that were excited in the left-side down quadrant). In addition, the mean response gain was higher in units showing type ~ response. When these features of narrowly tuned neurons were compared with reticular neurons whose spatiotemporal properties were not characterized [6], similar asymmetric pattern of distribution between type a and type ~ responses was found amongst neurons that showed otolithic dynamics [6]. Comparable asymmetric distribution in the roll direction was also observed in another study on reticular neurons though it is evident that some of these neurons received canal inputs as well [33]. The medial reticular formation receives bilateral inputs from the vestibular nucleus [31,35] and labyrinth [36,37]. It is of interest to note that the ratio of a to ~ neurons is reversed between the narrowly tuned reticular neurons and narrowly tuned vestibular nuclear neurons on the same side of the brainstem [32]. This reciprocal behavior suggests that macular signals encoding different head orientations along the roll direction exert a differential (complementary) influence on the medullary reticular formation and the vestibular nucleus. Asymmetry in the proportion of the best response orientations in the head-up (type 1) and head-down (type 2) quadrants along the pitch direction was also observed during OVAR. A greater proportion was found in the head-up than in the head-down quadrant. Similar distribution was observed in vestibular nuclear neurons of cats [12] though the spatiotemporal properties of these neurons are not characterized. In alert cats, the asymmetric upward and downward nystagmus beats observed during lowfrequency otolithic stimulation ([47]; see also [14,34]) was hypothesized to compensate for the difference in the asymmetric inertia load of the head during upward and downward head movements [47]. The asymmetric distribution of central vestibular and reticular neurons along the pitch direction may reflect the underlying asymmetric features of the neural circuitry that controls vertical head and eye movements. The OVAR-evoked dynamic response presented in this study was not elicited in the same manner as those evoked

in other studies which used sinusoidal rotations in vertical planes [6,33]. Nevertheless, the OVAR-evoked neuronal responses are also generated as a result of sinusoidal stimulation delivered to the macular hair cells though the stimulus plane was inclined at a small angle from the horizontal. In the present study (0.005-0.042 Hz), medial medulkuy reticular neurons with symmetric and velocitystable bidirectional response sensitivity to OVAR showed either a flat response lead of N 30° or a response phase close to zero. The gain of these neurons also remained stable with velocity. These dynamic properties are similar to those elicited in a subgroup of reticular neurons during sinusoidal roll tilt between 0.008–0.051 Hz [33]. It may also be pertinent to note that the response phase of the presently sampled reticular neurons showed a more positional characteristics than that of vestibular nuclear neurons exhibiting similar spatiotemporal behaviors [32]. 4.3. Preferred orientation: neurons with asymmetric and velocity-variable bidirectional response sensitivity Results of the present study provide evidence that head orientations close to the horizontal plane are also encoded in medullary reticular neurons that showed velocity-variable and asymmetric bidirectional response sensitivity. Amongst the broadly spatiotemporal-tuned reticular neurons, the preferred orientations were predominantly found on the head-up/contralateral-side-down quadrants. This pattern was comparable to the asymmetric distribution of the best response orientations of reticular neurons that showed velocity-stable and symmetric bidirectional response sensitivity. For those reticular neurons which responded to OVAR in only one direction, the coding of head orientations on the plane of rotation is only attained in the responsive rotatory direction as characterized by the prefen-ed orientations. The functional implication of this behavior remains to be elucidated. Nevertheless, the CWCCW asymmetry observed provides directional information for individual neurons of this group. Besides, the present data suggest that the cat reticular formation maintains a spatial frame of reference in the central processing of broadly spatiotemporal-tuned information from the otoliths. 4.4. Functional consideration Neurons in the medial pontomedullary reticular formation are known to be involved in the control of eye, head or gaze movements [25,28,29,37,38,42]. In alert head-free cats, neurons in the medial pontomedullary reticular formation showed preferred response direction with respect to orienting movements [28]. Medullary reticular neurons with preferences for movement were also found in head-fixed cats [15,26]. That the histological distribution of the spatiotemporally characterized medial medullary reticular neurons sampled in the present study overlaps with that of

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neurons which are responsible for the control of head and eye movements [15,25,28,29,37] infers that our neurons would comprise part of the neural substrates that are involved in head and eye movements. Characteristic unidirectional continuous nystagmus with slow phase velocity that is compensatory to head angular velocity is generated during OVAR through selective stimulation of the otolith organs [13]. It was hypothesized in a neural model that this steady-state ocular response is related to the broadly spatiotemporal-tuned vestibular nuclear neurons which would exhibit directionally specific behaviors during OVAR [2,18]. Since the OVAR-evoked directional asymmetry observed in central vestibular [32] and medullary reticular neurons fits those predicted for broadly tuned neurons [1], it seems reasonable that the presently sampled reticular neurons that displayed velocity-variable S and CW–CCW asymmetry are also good candidates to be involved in the central transformation of head velocity signals into oculomotor responses during maculo-ocular reflex [[2], cf. [27]]. In a recent study on rabbits, it was proposed that reticular neurons with variable phase may shift muscle function from art agonist to an antagonist as the direction of head tilt changes [17]. Since broadly spatiotemporal-tuned vestibular nuclear neurons showed variable phase as a function of the stimulus direction [8], it is pertinent to consider that our directionally asymmetric reticular neurons are capable of behaving like these central vestibular neurons in the adjustment of muscle activity based on the direction and orientation of head tilt.

Acknowledgements This research was supported by research grants from the Hong Kong Research Grants Council and the University of Hong Kong (Committee on Research and Conference Grants and Lee Wing Tat Medical Research Fund) to YSC. The author would also like to thank Mr. Simon S.M. Chan for excellent technical assistance.

References [1] Angelaki, D.E., Two-dimensional coding of linear acceleration and the angular velocity sensitivity of the otolith system, Biol. Cybern., 67 (1992) 511-521. [2] Angelaki, D.E., Detection of rotating gravity signals, Mol. Cybern., 67 (1992) 523-533. [3] Angekdci,DE., Vestibular neurons encoding two-dimensional linear acceleration assist in the estimation of rotational velocity during off-vertical axis rotation, Ann. NYAcad. Set., 656 (1992) 910–913. [4] Angel&i, D.E., Bush, G.A. and Perachio, A.A., Two-dimensional spatiotemporal coding of linear acceleration in vestibular nuclei neurons, J. Neurosci., 13 (1993) 1403–1414. [5] Baker, J., Goldberg, J., Hermann, G. and Peterson, B., Spatiat and

167

temporal response properties of secondary neurons that receive convergent input in vestibular nuclei of alert cats, Brain Res., 294 (1984) 138-143. [6] Bolton, P.S., Goto, T., Schor, R.H., Wilson, V.J., Yamagata, Y. and Yates, B.J., Response of pontomedullary reticulospirud neurons to vestibular stimuli in vertical planes. Role in vertical vestibulospinal reflexes of the decerebrate cat, J. Neurophysiol., 67 (1992) 639-647. [7] Boyle, R., Goldberg, J.M. and Highstein, S.M., Inputs from regularly and irregulady discharging vestibuktr nerve afferents to secondary neurons in the vestibuku nuclei of the squirrel monkey. III. Correlation with vestibulospinal and vestibuloocular output pathways, J. Neurophysiol,, 68 (1992) 471–484. [8] Bush, G.A., Perachio, A.A. and Angelafri, D.E,, Encoding of head acceleration in vestibular neurons. I. Spatiotemporal response properties to linear acceleration, J. AJeurophysiol.,69 (1993) 2039–2055. [9] Chan, Y.S. and Cheung, Y.M., Response of otolith-related neurons in bilateral vestibular nucleus of acute hemilabyrinthectomized cats to off-vertical axis rotations, Ann. NY Acad, Sci., 656 (1992) 755-765. [10] Chan, Y.S., Cheung, Y.M. and Hwang, J.C., Effect of tilt on the response of neuronal activity within the cat vestibular nuclei during slow and constant velocity rotation, Brain Res,, 345 (1985) 271-278. [11] Chan, Y,S., Cheung, Y.M. and Hwang, J.C., Response characteristics of neurons in the cat vestibular nuclei during slow and constant velocity off — vertical axes rotations in the clockwise and counterclockwise directions, Brain Res., 406 (1987) 294–301. [12] Chan, Y.S., Cheung, Y.M. and Hwang, J.C., Unit responses to bidirectional off-vertical axes rotations in central vestibular and cerebelkw fastigial nuclei. In O. Pompeirmo and J.H.J. Alhrm (Eds.), Vestibulospinal Control of Posture and Locomotion. Progress in Brain Research, Vol. 76, Elsevier, Amsterdam, 1988, pp. 67-75. [13] Cohen, B., Suzuki, J.-I, and Raphan, T., Role of the otolith orgam in generation of horizontal nystagmus: effects of selective Iabynnthine lesions, Brain Res., 276 (1983) 159–164. [14] Darlot, C., Lopez-Borneo, J, and Tracey, D., Asymme~ of vertical vestibular nystagmus in the cat, Exp. Brain Res., 41 (1981) 420–426. [15] Delgado-Gracis, J.M., Vidal, P.P., Gomez, C. and Berthoz, A., Vertical eye movement related signals in antidromically identified medulkuy reticular formation neurons in the alert cat, Exp. Brain Res., 70 (1988) 585-589. [16] Dickman, J.D., Angelaki, D.E. and Correia, M.J., Response properties of gerbil otoliti afferents to small angle pitch and roll tilts, Brain Res., 556 (1991) 303-310. [17] Fagerson, M.H, and Barmack, N.H., Responses to vertical vestibular stimulation of neurons in the nucleus reticularis gigantocellularis in rabbits, J, Neurophysiol., 73 (1995) 2378–2391. [18] Fanelli, R., Raphan, T, and SchnaboIk, C., Neural network modelling of eye compensation during off-vertical-axis rotation, Neural Networks, 3 (1990) 265-276. [19] Femandez, C. and Goldberg, J.M., Physiology of peripheral neurons innervating otolith organs of the squirrel monkey. II. Directional sensitivity and force-response relations, J. Neurophysiol., 39 (1976) 985-995. [20] Fernandez, C. and Goldberg, J.M., Physiology of peripheral neurons innervating otolith organs of the squirrel monkey. III. Response dynamics, J. Neurophysiol., 39 (1976) 996-1008. [21] Furman, J.M., Schor, R.H. and Schumann, T.L., Off-vertical axis rotation: a test of the otolith-ocular reflex, Ann. Otol. RhinoL L.uryngol., 101 (1992) 643-650. [22] Goldberg, J.M. and Femandez, C., Eye movements and vestibulmnerve responses produced in the squirrel monkey by rotations about an earth-horizontal axis, Exp. Brain Res., 46 (1982) 393–402. [23] Goldberg, J,M., Desmadryl, G., Baird, R. and Femandez, C., The vestibular nerve of the chinchilla. IV. Discharge properties of utricular afferents, J. NeurophysioL, 63 (1990) 781-790. [24] Goldberg, J.M., Desmadryl, G., Baird, R. and Femandez, C., The vestibular nerve of the chinchilla. V. Relation between afferent

168

Y.S. Chan et al. /Brain Research 732 (1996) 159-168

discharge properties and peripheral innervation patterns in the utricular macula, J. Neurophysiol., 63 (1990) 791-804. [25] Grantyn, A. and Berthoz, A., Reticulo-spinal neurons participating in the control of synergic eye and head movements during orienting in the cat. L Behavioral properties, Exp. Brain Res., 66 (1987) 339-354. [26] Gmntyn, A., Berthoz, A., Hardy, O. and Gourdon, A., Contribution of reticulospinal neurons to the dynamic control of head movements: presumed neck bursters. In A. Berthoz, P.P. Vidal and W. Graf (Eds.), The Head-Neck Sensov Motor System, Oxford University Press, New York, 1992, pp. 318-329. [27] Hess, B.J.M. and Diennger, N., Spatial organization of the maculoocular reflex of the rat: responses during off-vertical axis rotation, Eur. 1 Neurosci., 2 (1990) 909–919. [28] Isa, T. and Naito, K., Activity of neurons in the medial pontomedullary reticular formation during orienting movements in afert head-free cats, J. Neurophysiol., 74 (1995) 73-95. [29] Iwamoto, Y., Sasaki, S. and Suzuki, L, Input-output organization of reticulospinal neurons, with special reference to connexions with dorsal neck motoneurones in the cat, Exp. Brain Res., 80 (1990) 260-276. [30] Kasper, J., Schor, R.H. and Wilson, V.J., Response of vestibular neurons to head rotations in vertical planes. I. Response to vestibular stimulation, J. Neurophysiol., 60 (1988) 1753–1764. [31] Ladpli, R. and Brodal, A., Experimental studies of commissural and reticular formation projections from the vestibular nuclei in the cat, Brain Res., 8 (1968) 65-96. [32] Lai, C.H. and Chan, Y.S., Properties of otolith-related vestibular nuclear neurons in response to bidirectional off-vertical axis rotation of the rat, Brain Res., 693 (1995) 39–50. [33] Manzoni, D., Pompeiano, O., Stampacchia, G. and Srivastava, U.C., Responses of medullary reticulospinal neurons to sinusoidal stimulation of labyrinth receptors in decerebrate cat, J. Neurophysiol., 50 (1983) 1059-1079. [34] Matsuo, V. and Cohen, B., Vertical optokinetic nystagmus and vestibular nystagmus in the monkey: up-down asymmetry and effects of gravity, Exp. Brain Res., 53 (1984) 197–216. [35] Peterson, B.W. and Abzug, C., Properties of projections from vestibular nuclei to medial reticular formation in the cat, J. Neurophysiol., 38 (1975) 1421-1435. [36] Peterson, B.W., Filion, M., Felpel, L.P. and Abzug, C., Responses of medial reticular neurons to stimulation of the vestibulra nerve, Exp. Brain Res., 22 (1975) 335-350.

[37] Peterson, B,W., Fukushima, K., Hirai, N., Schor, R.H. and Wilson, V,J., Responses of vestibulospinal and reticulospinal neurons to sinusoidal vestibular stimulation, J. Neurophysiol,, 43 (1980) 1236– 1250. [38] Peterson, B.W., Pitts, N.G., Fukushima, K. and Mackel, R., Reticulospinal excitation and inhibition of neck motoneurons, Exp. Brain Res., 32 (1978) 471-489. [39] Pompeiano, O., Manzoni, D., Srivastava, U.C. and Stampacchia, G., Convergence and interaction of neck and macular vestibular inputs on reticulospinal neurons, Neuroscience, 12 (1984) 111–128. [40] Reisine, H, and Raphan, T., Neural basis for eye velocity generation in the vestibular nuclei of alert monkeys during off-vertical axis rotation, Exp. Brain Res., 92 (1992) 209–226, [41] Ross, M.D., Morphological evidence for parallel processing of information in rat macula, Acta Otolatyngol,, 106 (1988) 213–218. [42] Sasaki, S. and Shimazu, H,, Reticulovestibular organization participating in generation of horizontal fast eye movement, Ann. NY Acad. Sci., 374 (1981) 130-143. [43] Schor, R.H. and Miller, A.D., Vestibular reflexes in neck and forelimb muscles evoked by roll tilt, J. Neurophysiol., 46 (1981) 167-178. [44] Schor, R.H., Miller, A.D. and Tomko, D.L., Responses to head tilt in cat central vestibular neurons. I. Direction of maximum sensitivity, J. NeurophysioL, 51 (1984) 136–146. [45] Schor, R.H. and Yates, B.J., Horizontal rotation responses of medulliwy reticular neurons in the decerebrate cat, J. Vest. Res., 5 (1995) 223-228. [46] Tomko, D.L., Peterka, R,J. and Schor, R.H., Responses to head tilt in cat eighth nerve afferents, Exp. Brain Res., 41 (1981) 216–221. [47] Tomko, D.L., Wall, C, III, Robinson, F.R. and Staab, J.P., Influence of gravity on cat vertical vestibule-ocular reflex, Exp. Brain Res., 69 (1988) 307-314. [48] Vidal, P.P., Graf, W. and Berthoz, A., The orientation of the cervical vertebral column in unrestrained awake animals. L Resting position, Exp. Brain Res,, 61 (1986) 541-559. [49] Wilson, V.J., Schor, R.H., Suzuki, I. and Park, B.R., Spatial organization of neck and vestibular reflexes acting on the forelimbs of the decerebrate cat, 1 Neurophysiol., 55 (1986) 514-526. [50] Wilson, V.J., Yamagata, Y., Yates, B.J., Schor, R.H. and Nonaka, S., Response of vestibular neurons to head rotations in vertical planes. III. Response of vestibulocollic neurons to vestibular and neck stimulation, J. Neurophysiol., 64 (1990) 1695–1703.