Convergent Properties of Vestibular-Related Brain Stem Neurons in the Gerbil

Galen D. Kaufman, Michael E. Shinder, and Adrian A. Perachio

Department of Otolaryngology, University of Texas Medical Branch, Galveston, Texas 77555-1063


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kaufman, Galen D., Michael E. Shinder, and Adrian A. Perachio. Convergent Properties of Vestibular-Related Brain Stem Neurons in the Gerbil. J. Neurophysiol. 83: 1958-1971, 2000. Three classes of vestibular-related neurons were found in and near the prepositus and medial vestibular nuclei of alert or decerebrate gerbils, those responding to: horizontal translational motion, horizontal head rotation, or both. Their distribution ratios were 1:2:2, respectively. Many cells responsive to translational motion exhibited spatiotemporal characteristics with both response gain and phase varying as a function of the stimulus vector angle. Rotationally sensitive neurons were distributed as Type I, II, or III responses (sensitive to ipsilateral, contralateral, or both directions, respectively) in the ratios of 4:6:1. Four tested factors shaped the response dynamics of the sampled neurons: canal-otolith convergence, oculomotor-related activity, rotational Type (I or II), and the phase of the maximum response. Type I nonconvergent cells displayed increasing gains with increasing rotational stimulus frequency (0.1-2.0 Hz, 60°/s), whereas Type II neurons with convergent inputs had response gains that markedly decreased with increasing translational stimulus frequency (0.25-2.0 Hz, ±0.1 g). Type I convergent and Type II nonconvergent neurons exhibited essentially flat gains across the stimulus frequency range. Oculomotor-related activity was noted in 30% of the cells across all functional types, appearing as burst/pause discharge patterns related to the fast phase of nystagmus during head rotation. Oculomotor-related activity was correlated with enhanced dynamic range compared with the same category that had no oculomotor-related response. Finally, responses that were in-phase with head velocity during rotation exhibited greater gains with stimulus frequency increments than neurons with out-of-phase responses. In contrast, for translational motion, neurons out of phase with head acceleration exhibited low-pass characteristics, whereas in-phase neurons did not. Data from decerebrate preparations revealed that although similar response types could be detected, the sampled cells generally had lower background discharge rates, on average one-third lower response gains, and convergent properties that differed from those found in the alert animals. On the basis of the dynamic response of identified cell types, we propose a pair of models in which inhibitory input from vestibular-related neurons converges on oculomotor neurons with excitatory inputs from the vestibular nuclei. Simple signal convergence and combinations of different types of vestibular labyrinth information can enrich the dynamic characteristics of the rotational and translational vestibuloocular responses.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Second-order neurons of the vestibular system (vestibular nuclei neurons) have been demonstrated to respond to inputs from multiple sensory organs of the labyrinth. This type of convergence has been proposed to explain a phenomenon originally named spatiotemporal convergence by Baker et al. (1984), whereby neuronal response gains and phases were found to vary systematically when head tilts were applied dynamically in each of several vertical head planes. Coactivation of semicircular canal and macular receptors (canal-otolith convergence) was proposed as the mechanism that accounted for the finding that responses varied as a function of head tilt angle.

In subsequent studies, in which pure translational acceleration was applied by horizontal translational motion of the head, a similar response was reported for second-order medial vestibular neurons in decerebrate rats (Angelaki et al. 1993; Bush et al. 1993). This form of spatiotemporal convergence was modeled by the probable convergence of inputs from otolith organ-related afferent neurons with different response dynamics, i.e., vector sensitivities or firing regularity (Angelaki 1993). Furthermore, medial vestibular neurons exhibited responses that appeared to encode different stimulus properties. For example, in some neurons the responses to stimulus vectors to which they were maximally sensitive remained in-phase with peak head acceleration and exhibited relatively constant gains across stimulus frequencies. In contrast, the responses to an orthogonal vector that resulted in minimal amplitude gain were characterized by a response phase lead of nearly 90°. However, this minimum response gain increased in magnitude ~10-fold over a decade increase in stimulus frequency. Those properties can be compared with the two forms of vestibuloocular responses to applied linear forces. The neuronal responses to the vector of maximum sensitivity best relate to the compensatory vestibuloocular response to static head tilt, which is dominated by low-pass characteristics (Tomko and Paige 1992). In contrast the vestibuloocular responses to dynamic translational acceleration have high-pass properties that reflect sensitivity to the rate of change of acceleration (jerk) during translational motion (Niven et al. 1966), similar to that found in neuronal responses to the vector of minimum sensitivity.

Among the other premotor inputs to oculomotor neurons are those from the perihypoglossal nuclei (including the prepositus, Rollers nucleus, and intercalatus), particularly the prepositus nucleus, which itself has reciprocal connections with the medial, inferior, and ventral lateral vestibular nuclei bilaterally (Belknap and McCrea 1988; McCrea and Baker 1985). The vestibular responses of perihypoglossal neurons have been examined to date primarily with regard to stimulation of horizontal semicircular canal receptors. The majority of the responses of those cells were classified as Type II activated by contraversive rotation (Blanks et al. 1977; McFarland and Fuchs 1992). Because the vast majority of medial vestibular nuclei cells receive convergent canal and otolith organ-related inputs (Bush et al. 1993), it is presumed that many of those cells project to the perihypoglossal area and thus would impart signals from both vestibular sources onto their target neurons. Perihypoglossal neurons also have been found to discharge during eye movements (McFarland and Fuchs 1992). Typically, they have been found to burst before the onset of saccades and/or exhibit sensitivity to ocular position (Escudero et al. 1996; McFarland and Fuchs 1992). A subset of those cells also responds to head rotation.

Perihypoglossal neurons projecting to the abducens nuclei have been characterized immunocytochemically as GABAergic (Lahjouji et al. 1997) and glycinergic (Spencer et al. 1989). Vestibular-related output of those cells therefore would be sign reversed compared with that of converging vestibular nuclei inputs to oculomotor neurons. We hypothesize that signal cancellation might occur at the oculomotor neuron unless the perihypoglossal neurons' dynamic responses are different from those of the vestibular nuclei. More complete characterization of the vestibular properties of perihypoglossal cells would provide potential insight as to the role of those cells in regulation of vestibuloocular function.

One of the objectives of the current study was to examine vestibular- and oculomotor-related activity in and near the perihypoglossal neurons during the application of either horizontal rotational or translational head acceleration. We then compared the results to previous findings in similar studies of medial vestibular nucleus neurons to assess how both sets of premotor neurons might influence and regulate the rotational and translational vestibuloocular responses.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

For measurement of extracellular unit activity and the vestibuloocular reflex (VOR), we developed an alert gerbil preparation in which orthodromic vestibular labyrinth stimulating electrodes, a recording access cap, and a head restraint bolt were implanted surgically several days before the first recording session. Under general anesthesia [pentobarbital sodium (Nembutal) and ketamine, each 20 mg/kg], a postauricular approach through the thin bulla was used uni- or bilaterally to place silver monopolar stimulating electrodes near the oval window. Self-tapping stainless steel screws and inverted T-bolts were placed into the skull at several points as anchors for the acrylic cap, which fixed the hardware together. After the surgery and for the extent of its use, each animal was housed individually to protect the implant. These and all other procedures were reviewed and approved by the University of Texas Medical Branch Institutional Animal Care and Utilization Committee.

On a recording day, the animal was anesthetized briefly with isoflurane gas (3% induction, 1% maintenance) while an eye coil (see following text) was secured to the locally anesthetized cornea of one eye. The animal was allowed to recover from the isoflurane while restrained. The head was fixed by holding the skull-cap restraint stud in a plane that brings the horizontal semicircular canals to an earth horizontal position (20° nose down). The body was confined loosely in a plexiglass tube the diameter of which slightly exceeded the animal's body. Within 5 to 10 min the gerbil became alert. We have determined that by 30 min after isoflurane anesthesia, the gerbil VOR recovers and remains stable. Therefore at 30 min we began to obtain a record of horizontal and translational VOR at several frequencies and began simultaneous extracellular unit recordings in the perihypoglossal region. Horizontal translational acceleration angles were defined with the right-hand rule so that positive values represent an acceleration vector pointing out the animal's left ear.

Using these techniques it was possible to perform multiple recording sessions with individual animals approximately biweekly spanning several weeks. At the end of the terminal experiment, a glass pipette containing saturated Fast Green dye in 2 M KCl was advanced into the perihypoglossal region. Cell latency was determined by applying 100-µs pulses to the oval window electrode. Both polarities were tested for the maximum effect, and the current was decreased to determine a 50% threshold where the cell fired after about half of the pulses. The threshold current was typically 25-50 µA. The reference electrode was subcutaneous in the neck region. After a representative unit was found and characterized, cathodal (positive) DC (+10 µA for 10-15 min) was applied to the recording electrode to eject the Fast Green dye. After the experiments, the animal was anesthetized deeply using 17% intraperitoneal chloral hydrate and perfused transcardially with 4% paraformaldahyde. The brain stem was removed and cut in 40-µm frozen sections for electrode track and dye spot recovery. The Fast Green requires no further processing and can be seen clearly in unstained or lightly counterstained tissue (see Fig. 2).



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Fig. 1. Camera lucida of the transverse estimated stereotaxic position of the recorded cells in the alert gerbil preparation. Sampled population includes neurons encountered between 4.0 and 6.5 mm down from the surface of the cerebellum using a dorsal approach. Group was bounded caudally by the area postrema and inferior olivary nucleus (top, -3 mm AP), and rostrally by the genu of the seventh nerve (g7, bottom, 0 mm AP or interaural). Three middle sections represent estimated recording locations in 1-mm-thick slices flattened for presentation. Areas where 1 cell occurred at the same stereotaxic position have larger dots according to the scale shown. Note that the bulk of the recordings took place in the rostral half of the perihypoglossal area.



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Fig. 2. Photomicrograph of a unit located in the right prepositus nucleus verified by Fast Green dye spot (left-arrow ). Sulcus limitans---defining the border between the MVe and PrH---is shown on each side of the 4th venticle with arrowheads. - - -, extent of the prepositus nucleus. This cell was characterized as translational-only, with a maximum translational gain of 168 S/s/g at 0.5 Hz, 0° polar angle. Scale bar = 500 µm.

Using aseptic technique, the acrylic skull caps used in our experiments for restraint and brain stem extracellular recording access were stable for several weeks without antibiotics or other preventative treatments. One animal was maintained with a skull cap for 6 wk with no behavioral pathology. However, after perfusion, the tissue over the recording site was thickened and fibrous, and the surface of the cerebellum was concave where repeated electrode entry was performed. Significant cerebellar compression and pathology were present. Therefore an optimum period for experimental manipulation using these methods is ~2 wk with no more than three to four separate recording days. Baseline data were collected during the first recording sessions, while anatomic and/or adaptive experiments could be performed during the terminal recording day, with subsequent tissue collection.

Some early data were acquired in the decerebrate preparation for comparison. The technique in the gerbil has been described previously (Newlands and Perachio 1990a,b). There was a clear difference in unit activity between the decerebrate and alert preparations. This generally was characterized by lower background discharge rates and response gains across frequencies in the decerebrate animal (P = 0.0001, ANOVA). In addition, variations from the alert sample were common. At 1.0 Hz, for example, rotational convergent neurons in the decerebrate preparation significantly phase-led their counterparts as observed in alert preparations (P = 0.034). Nonconvergent, eye-independent Type I decerebrate cells had an especially strong phase shift with increasing stimulus frequency.

SEARCH-COIL TECHNIQUE. We adopted the technique of Hess and Dieringer (1991) to record two-dimensional eye movements in the gerbil. Briefly, an eye coil (Sokymat, Switzerland; 1.8 mm, 80 turns) was glued onto the cornea using cyanoacrylate adhesive while the gerbil was maintained under isoflurane anesthesia. The eye was held open with a tungsten wire speculum and irrigated with a viscous topical anesthetic (lidocaine, 2%) before allowing the animal to become alert. Calibration of the eye coil was accomplished by physically moving the attached coil through rotational horizontal and vertical steps of the eye against a small protractor while the animal was still under anesthesia or by horizontally rotating the head of the anesthetized animal. After the recording session, excess topical anesthetic was applied to the eye, and the coil was removed with a gentle shearing motion. No significant damage occurred to the cornea from this procedure, which could be repeated in the same eye after 2-3 days. The signal derived by the calibration procedure was linear over a range of ±30° for horizontal displacement and ±20° for vertical displacement. Vision in the eye to which the coil was attached probably was reduced.

In another eye coil variation, 10 turns of 0.001-in-diam H-poly nylon-coated copper wire was placed around the lateral margins of the globe after temporal canthectomies for exposure. After gluing the coil in place with cyanoacrylate, a loop of wire was exteriorized for strain relief to allow unhindered eye movement, followed by generous lubrication of the eye with topical anesthetic (lidocaine 1%). For this procedure, calibration was accomplished by systematically moving the head position of a lightly anesthetized animal relative to the field coils (after Godaux and Cheron 1996).

Natural vestibular stimulation was provided using a 23 lb-ft DC torque motor for rotation about an earth vertical axis, and an earth horizontal granite air bearing driven by a 40 lb-ft torque motor via a cable/capstan arrangement for horizontal translational motion. For rotational horizontal stimuli, sinusoidal oscillations between 30 and 120°/s peak velocity were applied between 0.1 and 2.0 cycles/s. Horizontal translational stimuli was varied between 0.25- and 2.0-Hz sinusoids. In addition, the head orientation was varied in 30° incremental steps through polar angles from 0 (nasooccipital head axis aligned with the acceleration vector) to ±180° in the horizontal plane. Polar angle data were used to determine the maximum response vector (Smax) of each tested neuron. The magnitude of the acceleration was 0.1 g.

The translational air bearing sled provided an exceptionally stable platform on which to test neurons for rotational and translational motion. In the alert preparation, spontaneous motor activity of the animal became the critical factor determining testing time for each unit. There was no clear benefit in training the gerbil to become accustomed to the restraint. Even in active animals, however, it was not unusual to achieve long periods of time (minutes) between motor activity during which stable recordings were obtained.

Each cell encountered was tested with a sequential protocol of rotational and translational accelerations at several frequencies in the dark. Stereotaxic position was referenced to a mark set into the skull cap during the implantation surgery and verified by lesion or dye spot. The data are likely skewed to reflect sampling bias created by the type of electrodes used (1-10 MOmega ) and therefore the size of neurons detected. There was also an unbalanced frequency sampling due to the protocol order during recordings interrupted by losing a unit signal. The entire bilateral prepositus nuclei are 1 mm wide and 2-3 mm long in the gerbil. The dataset was selected based on a combination of microdriver coordinates recorded during the experiment (depth from the surface was 4.0-6.5 mm), with histological verification of electrode tracts and dye spots (see Figs. 1 and 2). These coordinates were centered on the prepositus region.

Most of the data were collected from electrode placement ~1-2 mm caudal to the interaural axis, roughly the rostrocaudal extent of the prepositus and Roller's nuclei (see also Fig. 1). Approaching the prepositus nucleus dorsally, medial vestibular (medial MVe) neurons could be encountered at more lateral penetrations, therefore we could not rule out a subset of neurons from this nucleus (or the equivalent of the so-called marginal zone in monkeys) (McFarland and Fuchs 1992) among our data. We saw no histological evidence of electrodes bending in their vertical track to reach the target area.

Half of the alert animals had an eye coil that allowed us to correlate eye movement to unit activity. In earlier recordings without an eye coil present, if a unit responded to rotational motion with bursting activity that matched the frequency of the fast phase of nystagmus in the gerbil and varied tonically with the stimulus frequency, it was classified operationally as an oculomotor-related cell even though we did not collect an eye-position signal to correlate with that unit. Such data represent approximately half of the cells classified as "oculomotor related." All such bursting cells in animals with an eye coil were related to eye movement.

Unit data were band-pass filtered (20 Hz to 5 kHz), digitized, and stored for off-line analysis. Some mixed spike population recordings were separated based on a waveform fitting algorithm using a multiple spike detector (AlphaOmega Engineering, Nazarith, Israel). However, all the data included in the analysis was single-unit data. Nondiscriminable "multiunit" recordings were not analyzed. Cycle histograms of single unit data were constructed. The neuron's firing rate was fit to the stimulus profile with a sinusoidal function using a least-squares algorithm (Bush et al. 1992). For a detailed discussion of translational spatiotemporal fitting algorithms for the calculation of Smax and Smin, see Angelaki et al. (1992). Gain and phase responses were referenced to the maximum rotational velocity or maximum translational acceleration with both parameters held constant across stimulus frequency. Gains were calculated by dividing the mean peak response amplitude averaged over several cycles by the stimulus peak amplitude (rotational: spikes/s per maximum rotational velocity in °/s; translational: spikes/s per g, where 1 g = 9.8 m · s-1 · s-1).

In some cells that stopped firing completely during the inhibitory response phase of the cycle, gains were based on full-cycle fits of the positive response portion (so that the mean firing rate was near 0). In other cells that exhibited bursting activity during half of the cycle, a fit was performed only on the other half of the cycle where activity was more or less tonically modulated with the stimulus. The bursting activity was clearly related to the fast phase of nystagmus in animals with an eye coil (Fig. 6). Translational eye gains were calculated using the ratio of measured eye rotation in degrees over the translational displacement of the track in centimeters (°/cm).

We tested unit responses to pure rotational or translational motion in the horizontal plane in the dark. We used several general categories to separate neuron types for comparisons and analysis. These groupings were rotational or translational responses (including canal-otolith and spatiotemporal convergence); the rotational vestibular type (e.g., I or II); the frequency of the stimulus applied; the presence of oculomotor-related unit activity (nystagmus related burst and pause); a Phase response classification (where "in-phase" < ±45° with respect to rotational velocity or translational acceleration, and "out-of-phase" > ±45°); and the horizontal polar angle (for determining Smax/Smin) at which a translation was applied.

The data and figures presented in these categories are from the alert preparation only; the decerebrate data are not included except in Table 1. For the majority of our data the maximum magnitude of the sinusoidal rotational stimulus was 60°/s, and for the translational stimulus the peak acceleration was ±0.1 g (0.98 m · s-1 · s-1).


                              
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Table 1. Summary data of unit recordings

The nature of our recording sessions meant that partial characterizations of unit activity were occasionally collected; that is, some units were observed only at one or a few frequencies and/or polar angles. In other cases, a unit was characterized briefly at one rotational or one translational frequency only, and the cell was lost before it was tested for canal-otolith convergence. These cells were excluded from the convergent ratio analysis. Most neurons were held for characterization over a period of 1-15 min. After a brief summary, the results will be presented by the classifications outlined in the preceding text.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General trends and distributions

The combined data represent 223 neurons in and near the perihypoglossal region of alert (n = 13) and decerebrate (n = 15) gerbils (total n = 28). A summary of response types is presented in Table 1. The distribution is based on units recorded in that region and separated by canal-otolith convergence and horizontal vestibular rotational Type. Type I neurons are defined as responding to ipsilateral rotations, Type II to contralateral rotation, Type III to both, and Type IV to neither (Duensing and Schaeffer 1958). Note that a much higher cell yield was obtained in the alert preparation. More units were recorded on each day (the animal was more stable) and over days several recording sessions were possible.

Stereotaxic distribution

The approximate stereotaxic distribution of the presented cells is shown in Fig. 1. The region includes the medial MVe, the prepositus nucleus, the intercalatus and Roller's nucleus caudally, the medial longitudinal fasciculus, and dorsal cells of the reticular medullary region. Stereotaxic data were verified histologically with electrode tracts and dye spots in sectioned brain stem tissue (Fig. 2). No significant unit response differences based on anatomic location were found, although we did not systematically vary stereotaxic position. Because no significant relationships were found between physiological and anatomic properties, we focused on the significant variables and interactions described in the following text. The majority of recorded cells were located in the rostral half of the prepositus region.

Latency from labyrinth

Galvanic labyrinth stimulation was applied to test the responses of some of the cells to determine their latency and synaptic relationship to the vestibular periphery. Neurons in the prepositus nucleus, the location of which was verified later with Fast Green dye spots, had latencies ranging from 1.5-4 ms. These latencies indicate di- and polysynaptic connections to either or both the ipsilateral and contralateral labyrinth (Newlands and Perachio 1990a). This supports the functional position of these cells as primarily third order from the labyrinth.

Finally the sampled population was cells with irregular firing characteristics (coefficient of firing variation, CV > 0.3). Rotational-only neurons had a significantly higher CV than their canal-otolith convergent counterparts (1.4 ± 0.85 vs. 1.1 ± 0.55, means ± SD; P = 0.023, F = 5.345, ANOVA). The vast majority of the cells in this region had a response to passive head motion in some form.

Results by category

We categorized the cells by examining several of their functional attributes and found the tested population to be highly heterogenous. No clear grouping of cell types with several similar characteristics was observed. Rather we encountered a mixture of cells with a variety of individual patterns of these attributes.

Rotational responses were tested across frequencies and classified according to the following attributes: rotational response Type (I or II), the presence of otolith convergence (canal-otolith convergence), the presence of an oculomotor-related response (nystagmus related activity), and finally a classification of the response phase. We arbitrarily defined a cell to be "in phase" or "out of phase" if the cycle phase angle of that cell's maximum firing response occurred within or outside of ±45° relative to the peak stimulus (re: peak acceleration for translational stimuli or re: peak velocity for rotational stimuli).

The attributes of the translational response in canal-otolith convergent cells, and in purely translational cells, also were categorized. Translational response parameters included the following: whether the cell was canal-otolith convergent and if so, its rotational vestibular Type (I or II), the presence of an oculomotor-related response (nystagmus or translational VOR related activity), the classification of the response phase (in or out re: acceleration), and finally whether the cell had additional spatiotemporal convergent properties (i.e., a non-null minimum response or Smin and shifting gain and phase with stimulus vector orientation). This latter determination could be made by testing across different polar angles of stimulus vector orientation. All of the translational vectors were parallel to the Earth's horizontal plane.

Clearly all of these unique attributes could combine in many patterns. The majority of the unit data were canal-otolith convergent and revealed in-phase responses. Translational-only neurons more likely had responses that were out of phase, and those cells generally had a higher response gain than in-phase translational-only neurons. Each category included cells for which individual patterns of attributes differed, and a particular attribute could be observed across many categories. For example, oculomotor-related neurons occurred in all of the categorical divisions. The response gain also varied greatly within one category.

The number of factors tested meant that the actual number of recorded neurons that could be placed into any one particular combination of attributes was sometimes low. However, by employing variance analysis methods we gained an appreciation of which factors and interactions of factors were most important in shaping the response of neurons in this region. Multivariate ANOVA (MANOVA) was performed on the rotational and translational gain and phase data separately. The results from this analysis will be used to support classification response differences in the figures to follow. Pair-wise comparisons (contrasts) also are reported as P values throughout RESULTS to support specific observations. These data were a subset of the factors shown to be significant by the overall MANOVA.

Data illustrated in Figs. 4 and 5 were derived from a subset of the total data summarized in Table 1, omitting decerebrate, Type III, and any data obtained in tests in which the peak stimulus was not either ±0.1 g or 60°/s. One hundred and sixty-four cells remained for analysis of data from the alert preparation.

Ocular VOR responses

The presence of an earth-stationary visual surround slightly increased the rotational response gain of the VOR above that observed in the dark. Horizontal rotational VOR gains were generally <0.6 in the light and increased with stimulus frequency. Figure 3 shows an example of calculated rotational and translational VOR gain and phase at several frequencies in one animal.



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Fig. 3. Gerbil vestibuloocular reflex (VOR). An example of rotational (A-D; °/°) and translational (E and F; °/cm) gerbil VOR data. Several stimulus magnitudes are shown for the rotational data (30, 60, and 90°/s with standard deviation). Rotational response in the dark was slightly lower, especially at the middle frequency.

The translational horizontal VOR (tVOR) maximum ocular response gain varied with the stimulus vector polar angle. In general, the horizontal tVOR ocular gain was maximal when the vector of horizontal acceleration was oriented orthogonally to the optic axis (~50° from the nasooccipital axis). Vertical eye movements were maximal near horizontal translations near the optic axis. Torsional eye movements were not recorded. Individual animal responses always revealed a sinusoidal pattern the amplitude of which varied with stimulus polar angle. The translational VOR was characterized by a gain increase and advancing phase with increasing stimulus frequency (Fig. 3).

Rotational unit responses

ROTATIONAL TYPE AND CANAL-OTOLITH CONVERGENCE. All of the four rotational response types as defined by Duensing and Schaeffer (1958) were found in cellular responses encoded in the perihypoglossal region. Fifty-five percent of the cells with a response to horizontal rotation (Types I, II, or III) were vestibular Type II (sensitive to contralateral rotation). Type I (ipsilateral sensitivity) and III (bidirectional sensitivity) cells constituted 35 and 9%, respectively, of the responsive neurons (Table 1). This distribution remained fairly stable across classifications.

Figures 4 and 5 show Bode gain and phase plots of the rotational and translational data, respectively, using several different groupings of the tested attributes. These different views of the data help to illustrate the factor interactions. In Fig. 4-1, the first view separates Type I and II neurons by their canal-otolith convergence or lack thereof.




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Fig. 4. Rotational responses. Part 1: rotational type by canal-otolith convergence. Alert rotational response gain and phase (re: velocity) across stimulus frequency for convergent (- - -) and rotational-only [---, non-convergent (NC)] neurons, and separated by Type I (A and B) and II (C and D) rotational responses. Cell number for each group is shown next to its trace and reflects the maximum sample number at 1 of the frequencies. SE bars are shown. Part 2: oculomotor sensitivity by rotational type. Alert rotational response gain and phase (re: velocity) across stimulus frequency for Type I (- - -) and II (---) neurons, separated by eye-movement sensitivity (A and B: vestibular only; C and D: oculomotor-related). Part 3: rotational type by phase-related response. Alert rotational response gain and phase (re: velocity) across stimulus frequency for rotational Type I (A and B) and II (C and D) neurons, separated by the phase response classification ("in phase" = less than ±45°, ---; "out of phase" = more than ±45°, - - -).




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Fig. 5. Translational responses. Part 1: rotational type by canal-otolith convergence. Alert translational response gain and phase (re: acceleration) across stimulus frequency for Type I convergent (dashed line), Type II convergent (solid line) and translational-only (NC, nonconvergent, thin solid line) neurons (A and B). Data includes all horizontal translational polar angles (Smax plus other recorded response angles). Part 2: oculomotor sensitivity by rotational type. Alert translational response gain and phase (re: acceleration) across stimulus frequency (and for all polar angles) for Type I (dashed line), II (solid line), and nonconvergent (NC, translational-only; thin solid line) neurons, separated by eye-movement sensitivity (A and B: vestibular only; C and D: oculomotor-related). There were insufficient oculomotor-related vestibular-only neurons identified to plot in C and D. Part 3: rotational type by phase-related response. Alert translational response gain and phase (re: acceleration) across stimulus frequency for rotational Type I (A and B), II (C and D), or nonconvergent (E and F) neurons, separated by the phase response classification (in phase = less than ±45°, solid line; out of phase = more than ±45°, dashed line).

Neurons with Type I rotational vestibular responses typically had a higher rotational gain than Type II neurons at higher stimulus frequencies (>1 Hz, P = 0.002, Fig. 4, 1 and 2). Furthermore, these Type I neurons were typically rotational-only cells, i.e., not canal-otolith convergent (Fig. 4-1). Type II neurons and canal-otolith convergent Type I neurons had a relatively flat gain across stimulus frequency.

The rotational phase responses with respect to angular head velocity lagged with increased stimulus frequency for all vestibular types with a slight peak stimulus velocity lead at 0.2 Hz, and a slight phase lag at 2.0 Hz.

OCULOMOTOR-RELATED RESPONSES. Eye movements during applied motion were correlated with simultaneous unit recordings. Within the tested neurons a continuum of response dynamics was observed, including pure vestibular responses (no oculomotor-related activity), and cells with both head-motion and oculomotor-related sensitivity. The paucity of spontaneous eye movement in the gerbil, along with the lack of behaviorally trained visuomotor responses (e.g., fixation or saccades to visual targets), precluded detailed evaluation of oculomotor related activity or ocular-position sensitivity. Nevertheless the presence or absence of an oculomotor-related signal associated with the vestibuloocular fast phases was one of several significant factors accounting for differences in response gain across many cell categories.

The second view of the rotational data (Fig. 4-2) splits neurons by their response or lack of response to oculomotor activity during the horizontal VOR. Neurons shown to burst or pause in correlation with the fast phase of nystagmus were classified as oculomotor-related, and separated further by Type I and II responses.

Thirty percent (30%) of the rotational vestibular neurons had oculomotor-related activity. Of these, 72% (n = 42) had Type II rotational responses. However, most of the high-pass rotational frequency response was attributable to rotational-only Type I, not Type II, neurons (Fig. 4, 1 and 2).

Neurons with oculomotor-related sensitivity had higher rotational gains than neurons in the same rotational response category that did not exhibit oculomotor-related activity (Fig. 4-2, P = 0.03).

At least one pattern of cell attributes was commonly observed. In 16% of vestibular-related neurons in the alert preparation, the cells responded to contralateral (Type II) head rotation, burst with ipsilateral ocular fast phases and paused with fast phases in the opposite direction. That is, bursting correlated with ipsilateral fast phases during the portion of the stimulus cycle in which the vestibular canal input diminished, whereas brief pauses of unit activity occurred during the rotational vestibular response to contraversive head rotation associated with fast ocular motion in the direction opposite to the recording side (see Fig. 6). Bursting typically preceded the onset of the fast phase eye movement by ~10 ms.



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Fig. 6. Eye and unit recording. Response of a Type II oculomotor-related neuron to horizontal head rotation at 0.2 Hz, 60°/s maximum velocity. ---, least squares fit of the head velocity signal. Horizontal eye velocity was calculated by differentiation of the eye position signal. Bottom: unit discharge; bursting correlated with fast phases occurred during ipsilateral head movement, and pauses correlated with fast phases occurred during the tonic response to contralateral head movements.

PHASE CLASSIFICATION. The third view of the rotational data (Fig. 4-3) separates Type I and II neurons by their phase response classification (in phase = less than ±45° vs. out of phase = more than ±45°). There was significant variation in the cyclic phase relationship between the maximum neuronal response gain and the maximum sinusoidal stimulus amplitude during rotational stimuli. Using the two categories of response phase classification, a distinct difference could be observed in the response gain (MANOVA, P = 0.016, power = 0.67, Fig. 4-3). Neurons in which rotational response gains increased with increasing stimulus frequency were characterized by in-phase responses. In addition, their activity was often also oculomotor-related, and their vestibular responses were statistically mostly nonconvergent, Type I responses to head rotation (see Fig. 4, 1-3).

Translational unit responses

CANAL-OTOLITH CONVERGENCE AND ROTATIONAL TYPE. We observed neurons with translational-only, rotational-only, and canal-otolith convergent responses in a 1:2:2 ratio (see Table 1). The response gains during translational motion decreased with increasing stimulus frequency (low-pass characteristics, Fig. 5), but only for some categories.

The first view of the translational data (Fig. 5-1) shows the translational response gain across stimulus frequency of canal-otolith convergent neurons (both Type I and Type II), with translational-only neurons. Type II canal-otolith convergent neurons had higher response gains at 0.25 Hz than Type I and nonconvergent (translational-only) cells (P = 0.0095). The translational response gain of nonconvergent cells had only slight low-pass characteristics.

This low-pass characteristic often was observed in convergent Type II neurons. Compared with the rotational response of Type I neurons, the vestibular rotational types, I and II, appear to complement each other in their frequency sensitivity to semicircular canal or otolith organ stimulation.

The second view (Fig. 5-2) separates the translational responses of Type I and II canal-otolith convergent neurons and translational-only neurons by the presence of an oculomotor-related signal. The oculomotor-related classification was assigned to neurons with rotation-induced, nystagmus-related bursting activity. However, we did observe unit responses during translational motion that correlated with fast phases of the translational response. These neurons also exhibited bursting behavior during the rotational VOR. Figure 5-2 shows that it was primarily the oculomotor-related Type II neurons that were responsible for the low-pass characteristic. Oculomotor-related translational neurons had a significantly greater response gain than their vestibular-only counterparts (MANOVA P = 0.03, power = 0.58)

Note that both the rotational and translational VOR responses reveal high pass activity (Fig. 3), yet a significant subset of perihypoglossal neurons encode low-pass activity. This apparent discrepancy is incorporated in a model presented in the discussion.

PHASE CLASSIFICATION AND SPATIOTEMPORAL CONVERGENCE. Convergent input from both regularly and irregularly firing vestibular afferent neurons (Goldberg et al. 1987) arguably results in what has been referred to as two-dimensional responses to stimulation of the otolith organs (Angelaki 1993b; Bush et al. 1993). More than half of the neurons we encountered that responded to pure translational acceleration displayed this type of spatiotemporal convergence, i.e., a nonzero minimum response gain (Smin) and a maximum response phase that varied as a function of the stimulus vector angle of horizontal translational motion (see Fig. 7). The phase relationship usually inverts at a polar angle near the Smin but varies systematically on either side. The resulting response gain appears elliptical-, hourglass-, or dumbbell-shaped when plotted over 360° in the stimulus plane. The width of the ellipse varies greatly depending on the ratio of the maximum and minimum response angle (Smax/Smin).



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Fig. 7. Translational spatiotemporal convergent properties in a prepositus unit. Center: unit gain (S/s/G) at 5 horizontal polar angles of translational horizontal sinusoidal stimulation. Cycle unit histograms and the maximum response phase relative to the track position stimulus are shown near each polar angle radial. Cell's maximum response (Smax) was between 0 and 330° (near the naso-occipital axis) and the Smin was at 60°, as shown by the dip in the radial gain polygon. Note the phase angle shifting across polar angle vectors and that there remains some modulation even at 60°. (cell 347-5, 5.3 DV)

The third view of the translational data (Fig. 5-3) splits convergent Type I and II neurons, and translational-only neurons, by their phase response classification (in phase vs. out of phase). Again, it is the convergent Type II neurons, but specifically the out-of-phase Type II neurons, that reveal a large low-pass attribute. These neurons have a phase response more aligned with either jerk or velocity than with acceleration.

Cells found to have low-pass characteristics (which were typically out of phase) in response to translational acceleration often also were characterized as having oculomotor-related activity and receiving canal-otolith convergence (Fig. 5).

The Smax/Smin ratio of out-of-phase translational responses was higher than the ratio of in-phase neurons at 0.25 Hz (5.3 ± 1.1 vs. 2.9 ± 0.3, means ± SE; P = 0.07). There was a significant interaction between vestibular rotational type and response phase classification in the translational gain data (MANOVA P = 0.008, power = 0.80). Type II convergent neurons had higher Smax/Smin ratios, representing a polar response that was spatially more narrowly tuned.

Analysis of the horizontal translational response vectors of neurons tested at a minimum of four polar vector angles (collected at a single stimulus frequency, 0.5 Hz, n = 51) revealed no grouping of horizontal directional sensitivities across the sampled cells. Taken together, the Smax response vectors for the sample were distributed over 360° within the horizontal head plane.


    DISCUSSION
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Our results indicate that neurons in the perihypoglossal region of the gerbil receive highly convergent (canal-otolith, spatiotemporal, and oculomotor) inputs in variable patterns. The convergence of otolith and canal signals was coincident with lower high-frequency horizontal rotational gains and higher low-frequency translational gains (see Figs. 4 and 5). The translational low-pass characteristic of canal-otolith convergent units was carried often on Type II neurons (Fig. 5). Nonconvergent neurons with a Type I rotational response displayed significantly higher rotational gains at frequencies above 1 Hz compared with convergent Type I and all Type II neurons.

Cells with an oculomotor-related signal displayed higher rotational gains over all frequencies tested, especially at higher frequencies, and had higher translational gains at lower frequencies, than their vestibular-only counterparts. Oculomotor-related neuronal response gains also varied as a function of stimulus frequency to a greater extent than vestibular-only units.

However, these patterns had many exceptions. It would be an oversimplification to conclude that there were two primary neuron behaviors. The data indicate a diffuse relationship among neuron categories, with individual units possessing nontypical patterns of response dynamics. Only by examination of the entire sample of cells and applying MANOVA did the observed trends emerge.

Finally we confirmed that the reduced decerebrate preparation is not an accurate representation of normal function. These findings support the notion that the perihypoglossal and surrounding regions are an anatomic and functional extension of the MVe, combining multiple signals on many neuron types. The data suggest that this region could serve diverse multimodal transformations in the CNS.

Comparison with MVe neurons

MVe neurons are known to display spatiotemporal convergence properties that are characterized by response gains and phases that vary continuously throughout a range of stimulus vectors. During horizontal head rotations, both Type I and II neurons in the MVe display a phase relationship with head velocity that leads by 20-30° at 0.15 Hz and becomes virtually in-phase at higher frequencies.

Burst-position neurons in the primate marginal zone [on the border of prepositus hypoglossi (PrH) and MVe] have been shown to be one source of a horizontal eye position signal (McFarland and Fuchs 1992). Our findings reveal that translational vestibular-related responses, although similar to MVe neurons, possess orthogonal response phase properties that could control eye movement during specific types of head motion. We suggest that prepositus neurons, by virtue of their responses to vestibular input, could compete with the excitatory inputs from vestibular neurons on abducens motoneurons. The convergence of prepositus and medial vestibular inputs on abducens neurons might summate to provide both peak acceleration and jerk-related signals in response to translational acceleration (Fig. 8A). We assume that MVe input across the midline is excitatory (Scudder and Fuchs 1992) and that the PrH is inhibitory (Escudero et al. 1992). The MVe input to the abducens nucleus is characterized by a relatively flat gain in the signal that is phase-related to peak head acceleration. The signal that represents the same cells' responses to an orthogonal vector is phase related to the rate of change of acceleration (jerk) and exhibits a gain increase with stimulus frequency (Angelaki et al. 1993). Conversely, our present findings suggest that Type II PrH neurons can provide a signal to the contralateral abducens that is phase-related to jerk and exhibits a falling gain as a function of stimulus frequency (Fig. 5). The postsynaptic effect of these two inputs, converging an abducens motoneuron (as depicted in Fig. 8A), would result in a greater sensitivity to head acceleration at lower frequencies and an increasing sensitivity to jerk as the stimulus frequency increases. These combined characteristics are reflected in the gerbil tVOR (Fig. 3).



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Fig. 8. VOR pathways model. A model of horizontal VOR pathways using convergence of medial vestibular (MVe) and prepositus hypoglossi (PrH) convergent neurons and supported by the data. See the text for details. A: translational inputs. B: rotational inputs.

Interpretation of the functional significance of the rotational response of vestibular-related neurons requires certain underlying assumptions. We assume that the postsynaptic responses of cells receiving Type I inputs are excitatory, whereas Type II inputs from the PrH produce inhibitory responses. Because the PrH projections to the abducens nuclei are largely crossed (Belknap and McCrea 1988; McCrea 1988; McCrea and Baker 1985), we propose that those connections involve PrH Type II cells the axon terminals of which converge with those of contralaterally projecting MVe Type I neurons. By logical extension, the PrH projection to the ipsilateral medial rectus division of the oculomotor nucleus also would be carried by axons from PrH Type II cells. Because the MVe and PrH are known to be reciprocally connected bilaterally (McCrea 1988), we propose that PrH Type I neurons provide excitatory feedback to the vestibular nuclei, specifically through contralateral projections to the MVe Type II inhibitory interneurons that also receive inputs, via the vestibular commissural connections, from contralateral MVe Type I cells.

The gerbil horizontal rotational VOR exhibits a frequency dependent gain increase (Fig. 3). This is consistent with the response gain increase found in PrH Type I neurons that likely converge and summate with MVe crossed inputs to the abducens nucleus. The additional proposed inhibitory input from the PrH Type II cells should have a lesser effect on the rotational VOR because their gains are relatively low and flat across the tested frequency range. The models are predicated on circuitry that largely is based on anatomic findings. In our previous work (Angelaki et al. 1993) and in the current study, none of the recorded neurons was identified in terms of their direct synaptic projections to the oculomotor nuclei. Therefore the models serve to suggest potential connections that could be examined in future studies. For the present, we can only draw comparisons between the response properties of the various populations of neurons and those of the VOR. Adding the anatomic evidence of the connectivity within the VOR pathway, the most parsimonious arrangement would be one in which the projection of the PrH to the abducens and the oculomotor nuclei are those of Type II cells as depicted in Fig. 8, A and B. Based on our present findings this is a conceivable set of connections.

Comparison with other species

A comparison of unit data from several species (rat: Lannou et al. 1984; cat: Blanks et al. 1977; Escudero et al. 1996; Godaux and Cheron 1996; Kitama et al. 1995; Lopez-Barneo et al. 1982; and monkey: Cullen et al. 1993; McFarland and Fuchs 1992) reveals similar findings with regard to rotational gains and cell types. Although previous studies focused on different stimuli and response dynamics, like others (Lopez-Barneo et al. 1982; McFarland and Fuchs 1992), we observed vestibular-only and Type II vestibular cells with ipsilateral eye-movement sensitivity. However, we noticed a higher percentage of canal-otolith convergent cells than a previous study that employed static head tilt as an otolith stimulus (92/223 vs. 2/13) (Blanks et al. 1977). Other studies (immobilized rat: Lannou et al. 1984; and anesthetized cat: Blanks et al. 1977; Godaux and Cheron 1996), in agreement with this report, also showed an ~2:1 ratio of Type II to Type I neurons encountered in the perihypoglossal region.

The horizontal translational VOR response in the gerbil was similar to that reported in the rat (Hess and Dieringer 1990, 1991). Gerbils normally possess a monocular temporal-nasal optokinetic reflex (Israelian gerbils) (Sontheimer and Hoffmann 1987). The lack of foveal vision in this and other rodent species prevents tasks that can separate sensory from oculomotor signals on individual units.

The rich and diverse nature of the neurons found in the vestibular-related brain stem could provide dynamically appropriate output responses through simple combinations of those neuronal response properties. Given subsets of neurons may dominate the region's impact on vestibular function for a common range of motion stimuli, but the responses of other neural groups could provide dynamic signals for performance outside of that range to maintain proper behavioral performance. The increasing level of complexity in the understanding of vestibular function necessitates higher resolution descriptions of individual neural groups in the vestibular pathway. This study provides evidence to indicate a manner in which a neural population could accomplish such dynamic modulation by simple signal convergence.


    ACKNOWLEDGMENTS

This work was supported in part by National Institute on Deafness and Other Communication Disorders Grant DC-00385, National Aeronautic and Space Administration Grant 5-6009, and National Research Council Associate Grant 9602390.


    FOOTNOTES

Address for reprint requests: A. A. Perachio, Dept. of Otolaryngology, University of Texas Medical Branch, 7.102 MRB, Galveston, TX 77555-1063.

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

Received 29 June 1999; accepted in final form 22 December 1999.


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