 |
INTRODUCTION |
The cerebellum receives information from two distinct afferent pathways. One of these pathways consists of mossy fibers that originate from many different regions in the brain stem and synapse on granule cells. The other pathway consists of climbing fibers that originate exclusively from the contralateral inferior olive and synapse directly on Purkinje cells (Eccles et al. 1966a
, 1967
).
Cerebellar Purkinje cells are characterized by two distinctly different action potentials. One kind of action potential, the "simple spike" has a spontaneous rate of 30-60 imp/s and generally is attributed to the activation of Purkinje cells by parallel fibers, the cell bodies (the granule cells) of which receive excitatory inputs from mossy fibers. Simple spikes (SSs) also are influenced by the dynamic interplay of inhibitory interneurons within the cerebellum (Eccles et al. 1967
). By contrast, a second kind of action potential, the "complex spike" or "climbing fiber response" (CFR) has a clear and unambiguous proximate cause (Granit and Phillips 1956
). This broad, multiple-peaked action potential is due to the unique climbing fiber synapse on cerebellar Purkinje cells (Eccles et al. 1966a
). The climbing fiber synapse is responsible for a large excitatory postsynaptic potential that opens both voltage-sensitive calcium channels and calcium-sensitive potassium channels in Purkinje cell dendrites (Konnerth et al. 1990
; Llinás and Sugimori 1980
; Miyakawa et al. 1992
; Regehr and Mintz 1994
). CFRs have a low spontaneous frequency of 1-5 imp/s (Granit and Phillips 1956
).
After the occurrence of a CFR, there is a decreased probability of a SS. The short-duration "inactivation response" lasts on the order of several milliseconds (Granit and Phillips 1956
). However, longer duration "climbing fiber pauses" last 15-300 ms (Bell and Grimm 1969
; Granit and Phillips 1956
; McDevitt et al. 1982
; Sato et al. 1992
). The longer duration pauses are likely to reflect the action of climbing fibers on other cerebellar interneurons. Such pauses can be induced by stimulation of the inferior olive at stimulus currents that are just below threshold for evoking a CFR in the Purkinje cell from which the SSs are recorded (Bloedel and Roberts 1971
). Both decreases and increases in SSs after CFRs have been observed in different regions of the cerebellum during spontaneous discharge (Bell and Grimm 1969
; Kano et al. 1991
; McDevitt et al. 1982
; Sato et al. 1992
) or under conditions in which the CFR and/or SS were evoked by sensory stimulation or movement (Ebner and Bloedel 1981
; Thach 1970
).
In the uvula-nodulus, both vestibular and optokinetic stimulation can be used to evoke climbing fiber activity. These stimuli activate particular parasagittal zones related to projection patterns from the inferior olive (Barmack and Shojaku 1995
; Wylie et al. 1994
, 1995
). Vestibular climbing fiber projections originate from both the
-nucleus and dorsomedial cell column of the contralateral inferior olive (Barmack et al. 1993b
; Gerrits et al. 1985
; Sato and Barmack 1985
; N. H. Barmack, B. J. Fredette, and E. Mugnaini, unpublished data). The topography of these vestibular climbing fiber projections has been characterized previously at the level of the inferior olive (Barmack 1996
; Barmack et al. 1993b
) and at the uvula-nodulus (Barmack and Shojaku 1995
). For example, in the uvula-nodulus, a parasagittal zone, related to stimulation in the plane of the ipsilateral posterior semicircular canal and contralateral anterior semicircular canal (LPC-RAC), is located near the midline. Further laterally, a parasagittal zone related to stimulation of the ipsilateral anterior semicircular canal and contralateral posterior semicircular canal (LAC-RPC) exists (Barmack and Shojaku 1995
). By activating a climbing fiber input to one of these zones, it is possible to study interactions between CFRs and SSs in Purkinje cells within the same and adjacent zones.
In this experiment, we have sought answers to three questions: 1) is the climbing fiber-associated decrease in SSs restricted to the sagittal zone in which the CFR is recorded? Or, conversely, can climbing fiber activity in an adjacent sagittal zone modulate the simple spike activity in a particular zone? 2) Does the zonal organization, observed in the nodulus and lower layer of the uvula (9d), regions that receive a rich primary (Barmack et al. 1993a
) and secondary vestibular afferent input (Barmack et al. 1992
; Brodal and Hoivik 1964
; Epema et al. 1985
, 1990
; Magras and Voogd 1985
; Miles et al. 1980
; Sato et al. 1989
; Tan et al. 1995
; Thunnissen et al. 1989
), as well as a vestibular climbing fiber input, extend to the other folia of the uvula where such primary and secondary vestibular afferent inputs are absent? 3) Is there uniform modulation of both CFRs and SSs within a particular zone or is there any evidence of a spatial variation in modulation within a zone? We answered these questions by using the technique of extracellular microelectrode recording from the entire uvula-nodulus of anesthetized rabbits.
 |
METHODS |
Anesthesia
Twenty pigmented rabbits (weight 0.8-1.7 kg) were anesthetized intravenously with
-chloralose (50 mg/kg) and urethan (500 mg/kg). In addition, prednisolone (5 mg/kg) was administered intramuscularly to reduce inflammation during recording sessions lasting 5-10 h. Rectal temperature was monitored and maintained at 37°C. The adequacy of anesthesia was evaluated using the corneal reflex as an indicator.
Vestibular stimulation
The head of the rabbit was attached by implanted head bolts to a restraining bar. This bar held the head rigidly in the center of rotation of a three-axis vestibular rate table with the plane of the horizontal semicircular canals maintained in the earth horizontal plane. The body of the rabbit was encased in foam rubber and fixed with elastic straps to a plastic tube aligned with the longitudinal axis of the rate table. The rate table was oscillated sinusoidally about its vertical axis (yaw), about its longitudinal axis (roll), or about its interaural axis (pitch) (±10°, 0.005-0.800 Hz). During vestibular stimulation, the vision of the rabbit was occluded completely.
Static vestibular sensitivity
A static roll test was used to decide whether the discharge of a Purkinje cell was related to otolithic stimulation. In this test, the rabbit was tilted 5-10° about the longitudinal axis. After an adaptation period of 20-30 s, the average discharge frequency was measured for the next 20-30 s. The rabbit then was tilted in the opposite direction. A difference in mean discharge frequency of >20%, evoked for roll tilts in the two opposite directions, indicated sensitivity to linear acceleration. If there were any doubts about the otolithic responsiveness of a Purkinje cell, the test was repeated. Based on the predominant mediolateral polarization vector of hair cells within the utricular maculae as opposed to the predominant ventraldorsal polarization vectors for hair cells within the saccular maculae (Fernandez and Goldberg 1976
), we presume that a static roll stimulus evoked activity originating primarily from utricular hair cells.
Vestibular null plane measurement
A null technique was used to characterize the peripheral origin of vestibularly modulated Purkinje cell activity in the uvula-nodulus. While the rabbit was rotated about the longitudinal axis of the rate table, the angle of the rabbit's head about the vertical axis was changed systematically until a minimum in the vestibularly-modulated neuronal activity was detected (null plane). On either side of this null plane, the phase of the vestibularly modulated activity was shifted with respect to the sinusoidal vestibular forcing function by 180°. A change in head position of <4° was sufficient to cause this 180° phase shift. For each tested neuron, the null plane characterized the polarization vector of the hair cells of particular end organs that contributed to the modulated response. The optimal plane, the plane of maximal CFR modulation, was assumed to be orthogonal to the null plane. In four Purkinje neurons, this assumption was tested directly. The optimal plane was within 90 ± 8° (mean ± SD) of the null plane. This reflects the greater uncertainty with which the optimal response plane can be determined compared with the determination of the null plane.
Optokinetic stimulation
The rate table was 55 cm from the center of a rear-projection tangent screen subtending 70 × 70° of visual angle. An optokinetic stimulus was rear-projected onto the screen by beaming the image of a random dot contour-rich pattern, projected by a 35-mm slide projector, off three first surface mirrors, two of which were mounted orthogonally on electroencephalograph pen motors. Appropriate voltage ramps to the pen motors generated constant velocity movement of the projected image on the tangent screen in the horizontal and vertical axes. The optokinetic sensitivity of Purkinje cells was tested for stimulation about both the horizontal and vertical axes.
Microelectrode recording
The posterior cerebellum was approached by reflecting the muscles overlying the cisterna magna and enlarging the dorsal aspect of the foramen magnum. The outer layer of the dura mater was removed carefully, leaving intact a thin inner layer of dura mater overlying folia 9a-9c. A hydraulic microdrive, attached to the head restraint bar, advanced indium-filled glass pipettes or tungsten microelectrodes through folia 9a-9d toward folium 10, the nodulus. Action potentials of single neurons were recorded extracellularly in these folia.
CFRs had the classic multiple-spike waveform lasting several milliseconds (Granit and Phillips 1956
). The signal from the microelectrode was amplified (bandwidth 0.1-10,000 Hz). Individual action potentials were discriminated with a window discriminator-Schmitt trigger and then connected to a computer. Evoked single-unit activity was displayed on-line as a peristimulus histogram. Histograms were constructed from different numbers of trials at different stimulus frequencies: n = 10, (0.005-0.040 Hz); n = 20, (0.050-0.400 Hz). During data acquisition, each stimulus cycle was divided into 360 bins (for data analysis the stimulus bins were "collapsed" into 180 bins). Into these bins were stored interspike intervals for spike occurrences. The reciprocal of these interspike intervals, spike frequency, was averaged for the number of spike occurrences in each bin. This allowed a more accurate representation of the discharge of CFRs that discharge at very low frequencies rather than accumulating CFR occurrences in each bin and ignoring information concerning interspike intervals.
Histological verification of recording sites
The location of each neuron from which recordings were obtained was marked electrolytically (
5 µA, 15 s). At the conclusion of the experiment, each rabbit was anesthetized deeply and perfused transcardially with 0.9% saline, followed by 10% paraformaldehyde. The brain was removed and cryoprotected with 10, 20, and 30% sucrose in 0.1 M phosphate buffered saline (PBS),pH 7.2. The cerebellum was blocked sagittally, mounted onto cork with OCT compound, and frozen in isopentane cooled with dry ice. Sagittal frozen sections (40 µm) were cut and collected in cold 0.1 M PBS and mounted serially. The location of each recorded neuron was reconstructed from the locations of the marking lesions. These marking lesions were recovered from sagittal sections of the uvula-nodulus and marked onto a schematic of the uvula-nodulus in the sagittal plane at 0.2 mm mediolateral intervals. These locations were then transferred onto a two-dimensional map of the surface of the uvula-nodulus.
 |
RESULTS |
Activity evoked in uvula-nodular Purkinje cells by vestibular stimulation optimal and null response planes
Sinusoidal oscillation of the rabbit about its longitudinal axis evoked modulation of both CFRs and SSs depending on the orientation of the rabbits head about the vertical axis. When the rabbit was rotated in the optimal response plane, orthogonally to the null plane, CFRs from the left uvula-nodulus increased with leftward roll-tilt and decreased with rightward roll-tilt (Fig. 1). For the Purkinje cell illustrated in Fig. 1, the optimal plane of rotation corresponded to the anatomic orientation of the LPC-RAC (Fig. 1B). The null plane for this neuron and for all Purkinje cells from which we recorded was measured as follows: initially the longitudinal axis of the rabbit was collinear with longitudinal axis of the rate table. The rabbit was oscillated at 0.1-0.2 Hz with a stimulus amplitude of 10-20° peak to peak. When a modulated response was encountered, the orientation of the rabbit was shifted 30° to the left or to the right. There were three possibilities: the response was little influenced by the change in head position, the response was decreased but still in phase with the response evoked during stimulation about the initial head orientation, or the response was decreased and phase reversed. If there was no or little change in the response, then the head of the rabbit was oriented 30° to the other side of the initial longitudinal orientation. If the response were reduced, but the phase remained the same, the head was oriented an additional 15° in the same direction as the original deviation. If the response were phase reversed, the head was moved back 15° toward the initial position. In making such systematic changes in head orientation, measuring a null plane was usually possible within 3-5 min. After determining the null plane, a measurement to static tilt in the optimal plane was done (see METHODS). After the measurement of static tilt, the rabbit was exposed to several cycles of stimulation in both the optimal plane, and the null plane and peristimulus histograms were constructed of the responses of both SSs and CFRs.

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| FIG. 1.
Microelectrode recording from a Purkinje cell during vestibular roll stimulation. Sinusoidal roll vestibular stimulation was used to evoke both climbing fiber responses (CFRs) and simple spike (SS) activity of Purkinje cells in the uvula-nodulus. A1: illustration of the uvula-nodulus from which microelectrode recordings were obtained (shaded region). A2: magnified illustration of folia 9a-9d and 10. A primary afferent and secondary afferent vestibular mossy fiber projection is illustrated in folium 10 and the ventral half of folium 9d. , location of a Purkinje cell, in folium 9c, from which CFRs and SSs were evoked, as illustrated in B and C. This neuron was found 220 µm to the left of the midline. Positive action potentials in each trace are CFRs, and the negative action potentials are SSs. Sinusoidal trace indicates head position as the rabbit was rolled about the longitudinal axis of the rate table. By shifting the position of the rabbit's head about the vertical axis during roll stimulation, it was possible to determine both a null plane and an optimal plane. B: recording illustrating both CFRs and SSs. Bottom: head position about the longitudinal axis (roll). The optimal plane was the plane of stimulation for which there was maximal modulation of CFRs. At this optimal plane, the head of the rabbit was oriented 36° clockwise with respect to the longitudinal axis of the rate table. C: null plane was the plane of stimulation for which there was minimal modulation of CFRs. At the null plane, the rabbit was oriented 54° counter clockwise with respect to the longitudinal axis of the rate table. Figurines: head orientation at which the response was obtained. In this Purkinje cell, the optimal plane corresponded approximately to the maximal stimulation of the left posterior semicircular and right anterior semicircular canals, LPC-RAC.
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The responses of a neuron recorded from folium 9c of the uvula, 220 µm from the midline, are illustrated in Figs. 1 and 2. The CFRs increased when the rabbit was rolled onto its left side and decreased when the rabbit was rolled onto its right side. Conversely, the SSs decreased when the rabbit was rolled onto its left side and increased when the rabbit was rolled onto its right side (Figs. 1B and 2A). When the rabbit was stimulated in the null plane, neither the CFR nor the SS was modulated.

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| FIG. 2.
Peristimulus histograms of climbing fiber responses and simple spike responses of a uvula-nodular Purkinje cell evoked by vestibular roll stimulation in the null and optimal planes. Roll vestibular stimulation at 0.20 Hz evoked both SSs and CFRs from a Purkinje cell in the left nodulus. Primary data from this cell are illustrated in Fig. 1. Bottom: head position about the longitudinal axis (roll). A: during stimulation in the optimal plane, CFRs increased when the rabbit was rolled onto its left side (upward deflection of the stimulus trace). Conversely, the frequency of SSs increased when the rabbit was rolled onto its right side. B: during stimulation in the null plane, only a few CFRs were evoked, and there was no modulation of SSs. Figurines: head orientation during stimulation in the optimal and null planes. The binwidth for the peristimulus histogram was 28 ms.
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Of 155 vestibularly responsive Purkinje cells from which we recorded identified on the basis of their CFRs, 98 had CFRs that lacked static sensitivity as determined by the static roll test (see METHODS). Ninety-six of these cells increased their discharge during left side down rotation (Figs. 1 and 2). Two cells responded to roll onto the right side. Of the 155 vestibularly responsive Purkinje cells, 46 had CFRs were modulated by static roll onto the left side and two responded to static roll onto the right side (Fig. 3). An example of Purkinje cell with static sensitivity can be seen in Fig. 3. During step-roll stimulation, the CFRs in this Purkinje cell had both a transient response to the step onset and offset as well as a maintained response during the left side down tilt. The transient increases in CFRs were accompanied by transient decreases in SSs. Conversely, transient decreases in CFRs were accompanied by transient increases in SSs. Nine of the 155 vestibularly responsive Purkinje cells were not isolated long enough to permit static analysis and were not included in Fig. 4.

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| FIG. 3.
CFRs and SSs evoked by vestibular stimulation of the vertical semicircular canals and otoliths. Step-roll stimulation was used to characterize the otolithic sensitivity of uvula-nodular Purkinje cells. In this Purkinje cell, there was a clear "transient response" of both the simple spikes (SSs, top) and climbing fiber responses (CFRs, middle) to step-roll stimulation about the optimal plane. CFR also had a static response to the step-roll. Bottom: step-roll of the rate table. Figurine: the roll optimal plane was close to the plane of the left posterior-right anterior semicircular canals. Histogram is composed of an average of five cycles of the step-roll stimulus. Binwidth for the peristimulus histogram was 278 ms.
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| FIG. 4.
Distribution of optimal planes of canal and otolith-related Purkinje cell climbing fiber responses. Optimal planes of climbing responses are illustrated for 2 different populations of Purkinje cells: those that had no static roll responses (A) and those with static roll responses (B). Lines on the left of the figurines show that the CFRs increased during left side down roll tilts. Lines on the right show that CFRs increased during right side down roll tilts. A: optimal planes are indicated for 98 CFRs found in uvula-nodular Purkinje neurons that had no static roll tilt response. Mean optimal planes are indicated for the grouping related to the left anterior semicircular canal and the grouping related to the left posterior semicircular canal. B: optimal planes are indicated for 48 CFRs that had an otolithic response. Figurines: angle shown of the optimal planes relative to the anatomic orientation of the vertical semicircular canals. A mean optimal plane is illustrated for the CFRs with static roll sensitivity. LAC and RPC, left anterior and right posterior semicircular canals.
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Distribution of optimal response planes of cfrs in uvula-nodular Purkinje cells
The null response planes were determined for CFRs during sinusoidal stimulation at 0.1-0.2 Hz. Optimal response planes were assumed to be orthogonal to the null planes. We plotted the optimal response planes of the 98 Purkinje cells that lacked static vestibular sensitivity. On the basis of the orientation of the optimal response planes, we divided the 96 Purkinje cells that increased their discharge during left side down roll into two groups. One group consisted of 58 Purkinje cells with optimal response planes corresponding to stimulation in the plane of the LAC-RPC. This group had a mean optimal response plane orientation of 46°. A second group consisted of 38 Purkinje cells with optimal response planes corresponding to the anatomic orientation of either the LPC-RAC. This group had a mean optimal response plane orientation of 135°. Two of the Purkinje cells the CFRs of which lacked static sensitivity responded to right side down rotation (Fig. 4A).
The CFRs of the 48 Purkinje cells with a sensitivity to static roll had response planes that did not cluster as neatly around the orientations of the vertical semicircular canals (Fig. 4B). The responses of two of these statically sensitive neurons increased during static roll-tilt onto the right side. Of the 46 Purkinje cells that responded to static roll onto the left side, 38 had optimal planes of stimulation that were within ±25° of the orientation of either LPC-RAC (135°) or the LAC-RPC (46°; Fig. 4B). Because the null plane measurements were made at frequencies for which there is both otolithic and semicircular canal sensitivity, we could not rule out the possibility that the statically sensitive CFRs also were driven by convergent vertical semicircular canal signals.
Spatial distribution of cfr optimal response planes within the uvula-nodulus
The spatial distribution of Purkinje cells from which climbing fiber optimal response planes were determined was plotted on a two-dimensional representation of the surface of the uvula-nodulus. Each Purkinje cell was marked at the completion of recording with a small microlesion. The exact position of the Purkinje cell within a particular folium and its distance from the midline was measured in histological reconstructions of sagittal sections. We marked 155 Purkinje cells with vestibular sensitivity. Of these cells, we recovered the microlesions of 142. We also recovered microlesions for five Purkinje cells the CFRs of which were driven by horizontal optokinetic stimulation. In addition, we marked the locations of 10 Purkinje cells that were unresponsive to either vertical or horizontal vestibular or optokinetic stimulation. The midline in these sections was determined as the midpoint of the area that was free of cells between the left and right fastigial nuclei.
This two-dimensional map of the uvula-nodulus confirms previous observations that CFRs of Purkinje cells in the nodulus and folium d of the uvula are organized into sagittal bands having a width of 0.5-1.0 mm. A sagittal band of CFRs lacking static roll-tilt sensitivity and having optimal response planes corresponding to stimulation of the LPC-RAC, was found nearest the midline (Fig. 5,
). A sagittal band of CFRs lacking static roll-tilt sensitivity and having optimal response planes corresponding to stimulation of the LAC-RPC, was found 0.7-1.6 mm lateral to the midline (Fig. 5,
). These sagittal bands were maintained in folia 9c as well as 9d.

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| FIG. 5.
Spatial distribution within the uvula-nodulus of CFRs related to vestibular and optokinetic stimulation. A: surface of the left uvula-nodulus is represented as an unfolded two dimensional sheet. Midline of the uvula-nodulus is at 0 mm. , CFRs having optimal planes near the orientation of the left posterior-right anterior semicircular canals, LPC. , CFRs with otolithic sensitivity that also had optimal planes within ±25° of the orientation of the LPC. , CFRs with optimal planes near the plane of the left anterior-right posterior semicircular canals, LAC. , CFRs with otolithic sensitivity that also have optimal planes within ±25° of the orientation of the LAC. , (otolith only) static CFRs that did not align with either vertical semicircular canal zone. , distribution of CFRs modulated exclusively by horizontal optokinetic stimulation (posterior anterior with respect to the left eye). ×, indicates CFRs that were nonresponsive (NR) to either vestibular or optokinetic stimulation. A sagittal view of an inverted uvula-nodulus, B, illustrates how the uvula-nodulus was mapped onto the 2-dimensional plot in A.
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Included in this two-dimensional reconstruction of the uvula-nodulus are the locations of Purkinje neurons that responded to static roll-tilt and for which it was possible to determine an optimal response plane. If the optimal response plane for a statically sensitive CFR was within the range of 20-70°, for the purposes of the two-dimensional plot, it was characterized as having an optimal response plane corresponding to the LAC-RPC zone (Fig. 5, shaded squares). If the optimal response plane for a statically sensitive CFR was within the range of 110-160°, it was characterized as in the plane of the LPC-RAC zone (Fig. 5, shaded circles). The eight static CFRs that did not correspond to either of these canal-related zones were mapped in Fig. 5 as otolith-only(Fig. 5,
).
In the ventral aspect of the nodulus, we recorded from 24 Purkinje cells having CFRs that were responsive to posterior
anterior optokinetic stimulation of the left eye (Fig. 5,
). These data confirm the presence of an optokinetically sensitive sagittal zone, restricted primarily to the ventral nodulus. This zone is lodged between the two sagittal zones defined by CFRs with optimal response planes aligned with the vertical semicircular canals (Barmack and Shojaku 1995
).
Although Purkinje cells with vestibularly sensitive CFRs were localized to folia 9a and 9b, we recorded from fewer neurons in these folia. A clear distinction between zones of Purkinje cells having CFRs with optimal response planes began to break down in folium 9b and was absent in folium 9a. The lateral distribution of Purkinje cells that were sensitive to vestibular stimulation was restricted to the central 2 mm on either side of the midline of the uvula-nodular surface. We electrolytically marked the locations of several Purkinje cells with CFRs that were unresponsive to either vestibular or optokinetic stimulation in the horizontal or vertical planes (Fig. 5, ×). We recorded from many more such unresponsive cells that were located more laterally than 1.75 mm from the midline.
Interactions of CFRs and SSs
We recorded from 29 Purkinje cells during vestibular stimulation in both the optimal and null planes, from which we could maintain good isolation of both the CFR and SS. Impulse modulation was measured by a computer program that generated separate peristimulus histograms for SSs and CFRs during 10 cycles of vestibular stimulation at 0.2 Hz. The histograms were composed of 180 bins. The computer counted the impulses in 90° segments (45 bins). It then subtracted from total impulses in one 90° segment, the impulses from a second 90° segment that was 180° out of phase with the first segment. This subtraction was repeated iteratively for all possible segment pairs for both CFRs and SSs. The phase of the segments that yielded maximal differences were measured from the midpoint of each of the 90° segments and determined respect to the sinusoidal forcing function.
For most of the 29 Purkinje cells, stimulus-modulated discharge of CFRs was associated with a decreased discharge of SSs (Fig. 6), although this SS modulation was weak when the CFR modulation was <2 impulses/cycle. The greater the depth of modulation of CFRs, the greater was the modulation of SSs (r =
0.39, P
0.001). In none of these Purkinje cells was an increase in CFRs coupled with an increasein SSs.

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| FIG. 6.
Modulation of CFRs and SS responses during vestibular stimulation in the optimal plane. CFRs and SSs were recorded simultaneously in 29 uvula-nodular Purkinje cells during vestibular roll stimulation in the optimal and null planes. Impulse modulation was determined by a computer program that examined impulses in 90° segments of a peristimulus histogram, consisting of 180 bins and compiled from 10 cycles of vestibular stimulation at 0.2 Hz (see text). In individual Purkinje cells, the vestibularly evoked increase in frequency of CFRs was related to the reduction in frequency of SSs (r = 0.39, P 0.001).
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Using the technique described above, independent estimates of the phase of the CFRs and SSs were made with respect to the sinusoidal forcing function. Decreases in SSs that were 180° out of phase with increases in CFRs were plotted as having a phase difference of 0°. For the population of 29 Purkinje cells, the decrease in SSs lagged the increase in CFRs. In 20 of the 29 Purkinje cells, the maximum decrease in the SSs lagged the maximum increase in CFRs (Fig. 7, A and B). In the other nine Purkinje cells, the maximum decrease in SSs led the maximum increase in CFRs. The depth of SS modulation decreased as the phase between the SS and CFR approached ±90° (Fig. 7B). Because the modulation of SSs of some Purkinje cells actually preceded the modulation of CFRs in the same Purkinje cell, the CFRs from these particular Purkinje cells could not be responsible for the modulation of the SSs recorded from these neurons.

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| FIG. 7.
Phase of simple spike modulation relative to climbing fiber modulation during vestibular roll stimulation in the optimal plane. Phase of 29e pairs of CFRs and SSs was measured during vestibular-roll stimulation in the optimal plane. A phase of 0° indicates that the modulation in CFRs was 180° out of phase with the modulation in SSs. A phase lag of the modulated SSs relative to the CFRs is indicated as negative. A phase lead of the SSs relative to the CFRs is indicated as positive. A: In 20 of the 29 CFR-SS pairs, the peak SSs lagged the peak CFRs. B: a polar plot of the phase and amplitude of the SS modulation relative to CFR modulation. Note that the modulation decreases as the phase approaches ±90°.
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In each of the 29 Purkinje cells from which both CFRs and SSs were well isolated, we recorded during vestibular roll stimulation in both the optimal and null planes for the CFR. The mean modulation of CFRs in the optimal plane was 3.3 impulses/cycle and the mean SS modulation was
14.4 impulses/cycle. In the null plane, the mean CFR modulation was 0.08 impulses/cycle and the mean SS modulation was
0.4 impulses/cycle (Fig. 8). The lack of SS modulation when the rabbit was oscillated in the null plane showed that the influence of CFRs on SSs must be confined to the sagittal zone in which both the CFRs and SSs are recorded. In other words, vestibular stimulation in the optimal climbing fiber plane in one zone does not influence the activity of SSs in an adjacent zone. Specifically, lateral to the zone containing Purkinje cells having CFRs with optimal responses in the plane of the LPC-RAC are Purkinje cells with CFRs having optimal responses in the plane of the LAC-RPC. We also investigated the possibility of lateral interactions between optokinetically evoked CFRs in the horizontal optokinetic zone and SSs recorded from Purkinje cells located in either the more medial LPC-RAC zone or the more lateral LAC-RPC zone. In no instance did we observe such modulation. These data rule out the possibility of significant lateral interactions between CFRs of one zone on SSs of an adjacent zone.

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| FIG. 8.
Mean modulation of CFRs and SSs during vestibular roll stimulation in the optimal and null planes. The maximal modulation of CFRs and SSs was determined for each of 29 Purkinje cells during vestibular roll stimulation in the optimal and null planes. During vestibular stimulation in the optimal plane, there was a mean increase of 3.3 impulses/cycle (CFRs) and a mean decrease of 14.4 impulses/cycle (SSs). Conversely, during stimulation in the null plane, there was a mean increase of 0.08 impulses/s (CFRs) and a mean decrease of 0.4 impulses/cycle (SSs).
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Lateral distribution of climbing fiber-evoked modulation of simple spikes within the uvula-nodulus
In the 29 Purkinje cells from which we recorded well-isolated CFRs and SSs, 18 had optimal planes that corresponded to stimulation in the plane of the LAC-RPC, and 11 had optimal planes corresponding to stimulation in the plane of the LPC-RAC. We plotted the depth of modulation of these SSs as a function of lateral distance from the midline (Fig. 9). These data show that the depth of modulation for SSs with climbing fiber optimal planes corresponding to stimulation of the LPC-RAC had a peak ~0.3 mm from the midline (Fig. 9,
). On either side of the peak, the depth of modulation decreased. Similarly, the depth of modulation of SSs with climbing fiber optimal planes corresponding to stimulation of the LAC-RPC, had a peak at ~1.3 mm from the midline, and on either side of this peak, the depth of modulation of the SSs decreased. These data demonstrate that the depth of CFR-associated modulation of SSs depends on the location of a particular Purkinje cell within a climbing fiber zone. This result could explain why the correlation of CFR modulation with SS modulation is relatively low (r =
0.39, Fig. 6) when cells distributed across all regions of the vestibular zones are included in the calculation.

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| FIG. 9.
Variation in vestibular modulation of Purkinje cell simple spikes as a function of distance from the midline. In the 29 Purkinje cells from which well-isolated SSs and CFRs were recorded, the relationship between the depth of SS modulation and distance from the midline was plotted. SSs with optimal planes near the plane of the LPC-RAC ( ) and LAC-RPC ( ) are plotted separately. In each of the 2 canal-related zones, the peak modulation occurred in the spatial center of the zone.
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DISCUSSION |
Functional topography of vestibularly evoked activity in the uvula-nodulus
The present results confirm and extend the results from a previous investigation in which it was shown that folia 9d and 10 of the uvula-nodulus received vestibular information in vertical semicircular canal-related zones (Barmack and Shojaku 1995
). In the left uvula-nodulus, the most medial zone, centered ~0.3 mm to the left of the midline, is characterized by CFRs that respond to vestibular roll-tilt in the plane of the LPC-RAC. This zone is flanked by another zone, which is characterized by CFRs that respond to vestibular stimulation in the plane of the LAC-RPC and centered ~1.3 mm from the midline. In the ventral nodulus, a third zone characterized by CFRs responsive to posterior
anterior optokinetic stimulation of the ipsilateral eye, is interposed between the vestibular zones. In the present experiment, we concentrated our efforts at recording from the canal-related zones. Consequently, we sampled only a few cells from the horizontal optokinetic zone, which has been documented previously (Barmack and Shojaku 1995
; Wylie et al. 1995
). Although Purkinje cells responding to vestibular stimulation are distributed throughout the uvula, a clear distinction between canal-specific sagittal zones begins to disappear in folium 9b (Fig. 5).
The uvula-nodulus lacks any climbing fiber representation of the horizontal semicircular canals (Barmack and Shojaku 1995
). The absence of horizontal semicircular canal mediated climbing fiber input to the uvula-nodulus is consistent with recordings from both the
-nucleus and dorsomedial cell column of the inferior olive in which only vertical semicircular canal and otolithic information is represented (Barmack 1996
; Barmack et al. 1993b). It is possible that the representation of the vertical semicircular canals and otoliths in the olivo-cerebellar system may reflect an earlier stage of evolution of the vestibular system, in which the horizontal semicircular canals were absent (Lowenstein 1971
).
Although there is no climbing fiber representation of the horizontal semicircular canals, it seems likely that the nodulus receives a horizontal canal-related mossy fiber projection. In a previous experiment, we identified recordings from mossy fiber terminals, some of which were responsive to horizontal vestibular stimulation (Barmack and Shojaku 1995
). It would be of interest to know more about the topography of vestibular mossy fiber projections. Does the mossy fiber topography conform to the canal-specific climbing fiber representation of the vestibular system? If so, where do the vestibular primary afferents that convey signals from the horizontal semicircular canals terminate as mossy fibers?
Climbing fiber-associated pauses in SS activity
In a sample of 29 Purkinje cells in which both the CFR and SS were well isolated, vestibularly evoked increases in the modulation of CFRs never were accompanied by increases in SSs (Figs. 6 and 7). This observation is supported by similar observations concerning this reciprocal behavior of SSs and CFRs in both the nodulus as well as the flocculus (Barmack 1979
; Barmack and Shojaku 1995
; De Zeeuw et al. 1995
; Graf et al. 1988
; Kano et al. 1991
). In 9 of the 29 Purkinje cell CFR-SS pairs, a decrease in SSs actually preceded the increase in CFRs, suggesting that climbing fiber-evoked pauses in SS activity are not necessarily the consequence of the climbing fiber acting on the same Purkinje cell from which the SS is recorded. Rather other climbing fibers, within the same zone and having the same optimal planes, could influence SSs through an interneuronal mechanism. As discussed in greater detail later, the Golgi cell is the only interneuron that receives a climbing fiber input. Hence, it is possible that Golgi cell activity, modulated by a climbing fiber input, would reduce the activity of granule cells within the field of the Golgi cell's axonal distribution. This in turn would modulate the SSs recorded from adjacent Purkinje cells. The phase of this SS modulation could differ from the expected antiphasic responses of CFRs and SSs recorded from the same Purkinje cell even if the climbing fiber innervating the Golgi cell and the climbing fiber innervating the Purkinje cell shared the same optimal response plane. This speculation is consistent with an earlier finding showing that electrical stimulation of the inferior olive, subthreshold for evoking a CFR in a particular Purkinje cell, nevertheless evoked a decrease in the probability of antidromic invasion of the same Purkinje cell (Bloedel and Roberts 1971
). It is also possible that other interneuronal circuits, involving basket, stellate, or unipolar brush neurons but without direct climbing fiber inputs, could influence the modulation of SSs.
Synchronicity and antiphasic behavior of CFRs and SSs
In both anesthetized and unanesthetized preparations, there is an orderly functional recruitment of inferior olivary neurons (Barmack and Hess 1980
; De Zeeuw et al. 1996
; Wylie et al. 1995
). Consequently, it is possible to observe modulation of SSs that are not strictly 180° out of phase with the CFR recorded from the same Purkinje cell. Although recent statistical evidence indicates the tendency of CFRs to fire more synchronously than would be predicted by chance, this evidence does not bear on the question of the antiphasic behavior of CFRs and SSs.
CFR and SS modulation evoked by separate mossy and climbing fiber pathways?
It is possible that the antiphasic behavior of SSs might be modulated by a separate mossy fiber pathway whose activity is antiphasic to the climbing fiber pathway. For example, partial inactivation of the dorsal cap of the inferior olive by a local lidocaine injection leaves visually modulated SSs intact in floccular Purkinje cells (Leonard and Simpson 1986
). This observation suggests that the residual SS modulation could be evoked by a coactivated mossy fiber-granule cell optokinetic pathway. However, the residual SS modulation also could have been the consequence of interneuronal mechanisms activated by optokinetic climbing fiber, unblocked by the lidocaine injection. These data are consistent with the observation that an olivary stimulus that does not evoke a CFR in a particular Purkinje cell nevertheless reduces the excitability of the Purkinje cell (Bloedel and Roberts 1971
).
The idea that the modulation of SSs can be attributed to the modulation of vestibularly related mossy fiber inputs receives no support from the known physiological characteristics of vestibularly modulated mossy fibers. Both large primary and secondary vestibular afferent inputs are conveyed as mossy fiber projections to the granule cell layers of folia 9d and 10 (Barmack et al. 1992
, 1993a
; Brodal and Hoivik 1964
; Epema et al. 1985
, 1990
; Magras and Voogd 1985
; Miles et al. 1980
; Sato et al. 1989
; Thunnissen et al. 1989
). However, the SSs recorded from Purkinje cells in these regions are modulated out of phase with this vestibular mossy fiber input (Barmack and Shojaku 1995
). If these SSs were modulated by a primary vestibular mossy fiber input, the mossy fiber input would have to undergo a reversal in sign at the level of the granule cell or at the parallel fiber synapse on the Purkinje cell dendrite to evoke the observed SSs. It is also possible that an apparent reversal in sign of SSs could be accomplished by a preponderance of a type II secondary vestibular afferent input to the uvula-nodulus. There are no relevant data that bear on this question.
Folia 9d and 10 receive an extensive vestibular primary and secondary afferent mossy fiber input (Fig. 1A2). However, the reciprocal activity of vestibularly evoked CFRs and SSs also was observed in folia 9a-9c, where there are virtually no primary vestibular mossy fiber inputs (Barmack et al. 1993a
; Korte and Mugnaini 1979
) and much reduced secondary afferent mossy fiber inputs (Akaogi et al. 1994
; Barmack et al. 1992
; Thunnissen et al. 1989
). These anatomic data argue against the speculation that the modulation of SSs is the consequence of a separate primary or secondary vestibular mossy fiber afferent pathway.
Role of anesthesia in the generation of the climbing fiber-associated pause
The climbing fiber-associated pause is of variable duration and depends on anesthesia (Bloedel and Roberts 1971
) as well as the specific method of stimulating climbing fibers. This could account for some of the variability in experimental descriptions of the prevalence and duration of the pause (Bell and Grimm 1969
; McDevitt et al. 1982
; Sato et al. 1992
). In the present experiment, our use of
-chloralose-urethan anesthesia could have reduced other synaptic inputs and thereby emphasized the occurrence of climbing fiber-associated pauses. Others have observed climbing fiber-associated pauses in unanesthetized rabbits (De Zeeuw et al. 1995
), in rabbits anesthetized with halothane (Kano et al. 1991
), and in rabbits anesthetized with a pentobarbital sodium (Nembutal)/
-chloralose mixture (Graf et al. 1988
). Although anesthesia affects the parameters of the pause, the pause is not an artifact of anesthesia.
Cerebellar circuitry accounting for climbing fiber-associated pause in SSs
Climbing fiber-associated decreases in SSs could be achieved through the action of a variety of hypothetical cerebellar circuits. Other cerebellar interneurons, such as basket and stellate cells (Eccles et al. 1967
), as well as unipolar brush cells (Mugnaini and Floris 1994
) in principle could account for the reciprocal relationship between CFRs and SSs. However, these interneurons do not receive climbing fiber inputs. Purkinje cell axon collaterals also could account for the observation that a CFR in one Purkinje cell evokes a pause in SSs in an adjacent Purkinje cell (Fox et al. 1967
; Larramendi and Lemkey-Johston 1970
). However, this form of recurrent inhibition could as easily be evoked by SSs as by CFRs. The pause is seen only after CFRs not SSs.
Golgi inhibitory interneurons are the only interneurons that receive synaptic contacts from climbing fibers (Chan-Palay and Palay 1971
; Desclin 1976
; Hamori and Szentagothai 1980
). The axons of Golgi cells innervate each glomerular synapse between mossy fibers and granule cell dendrites (Fox et al. 1967
). Physiological studies indicate that the excitatory activation of granule cells by stimulation of mossy fibers is depressed for hundreds of milliseconds after a local electrical stimulation that presumably activates Golgi cells (Eccles et al. 1966b
).
A spatial arrangement of key elements in cerebellar circuitry that could account for the CFR-induced SS pause is illustrated in Fig. 10. Climbing fiber zones originate in the inferior olive (Fig. 10B) and reach the contralateral ventral nodulus (Fig. 10A). A transverse slice taken through ventral nodulus, representing the central 3 mm of a folium taken through the ventral nodulus, is depicted in Fig. 10C. Activation of a small cluster of olivary neurons, say in the caudal right beta nucleus by rotation in the plane of the LPC-RAC, evokes increased climbing fiber inputs to both Purkinje cells and Golgi cells in the most medial zone of the left nodulus (Fig. 10, B and C). Golgi cell activation would reduce granule cell discharge in the region of axonal endings of the Golgi cells, thereby reducing parallel fiber input originating from granule cells in the LPC-RAC zone. The overlap (or nests) of different Golgi cell axonal terminals (Cajal 1911; Fox et al. 1967
) within a climbing fiber zone could account for the increased depth of SS modulation in the center of the zone (Fig. 9). Overlap of Golgi cell axon terminals across zones (De Zeeuw et al. 1994
) or at the borders of zones would have a less potent effect in modulating the frequency of SSs. For the sake of simplicity, the proposed role of climbing fiber-evoked modulation of SSs by Golgi cell inhibition of granule cells ignores the potential role of parallel fiber inputs to the dendritic trees of Golgi cells. If mossy fiber vestibular primary and secondary afferents were restricted to the vestibular climbing fiber zones with similar optimal response planes, then it would be possible for the antiphasic activity of CFRs and SSs to be reinforced by a parallel fiber input to Golgi cells in the nodulus as well as folium 9d of the uvula. The inhibitory action of Golgi cells would provide the necessary sign reversal, after activation of granule cells, to subsequently reduce the activity of a local population of granule cells and decrease SSs in adjacent Purkinje cells. This would result in antiphasic behavior of SSs relative to vestibular CFRs.

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| FIG. 10.
Hypothetical circuitry underlying vestibular-related CFRs and SSs in the nodulus. A: sagittal view of the cerebellum of the rabbit. A cylindrical section through the ventral nodulus (folium 10) is "removed". Central 3 mm of this cylinder is represented in transverse section in C. In B, 2 transverse sections through the right inferior olive show regions from which vestibular-related and optokinetic-related CFRs, recorded in the left uvula-nodulus originate. Numbers in each transverse section indicate the distance from the caudal pole of the inferior olive. C: schematic view of a transverse section through the ventral nodulus. Bar above the schematic of the folium indicates the approximate location of sagittal zones of Purkinje cells responding to vestibular and optokinetic stimulation. Only cerebellar neurons represented are: Purkinje cells, granule cells, and Golgi cells. Sagittal zones related to the climbing fiber inputs are shown originating from the rostral nucleus, (LAC-RPC), caudal dorsal cap (HOKL) and caudal nucleus (LPC-RAC). These climbing fibers synapse on Purkinje neurons (P) and Golgi cells (Go). Golgi cells are illustrated as spherical cell bodies in the upper granule cell layer (GL) with conical (open) dendrites distributed in the molecular layer (ML) and conical (shaded) inhibitory axon terminals distributed to granule cells. Purkinje cells are illustrated with spherical cell bodies comprising the Purkinje cell layer (PL) and with cylindrical dendrites extending into the ML. Four different mossy fiber afferents (MF1-4) are shown making synaptic contact with granule cells on either side of the midline in the GL. The granule cells give rise to parallel fibers that traverse every Purkinje dendritic tree, both to the left and right of the midline, a distance of 3 mm. Central bifurcation of the parallel fibers is illustrated in black, and the distal extremes are illustrated in a lighter shade, to indicate variability in length. DAO, dorsal accessory olive; dc, dorsal cap; Cu, cuneate nucleus; Gr, gracile nucleus; HOKL, HOKR, left and right horizontal optokinetic stimulation; LRN, lateral reticular nucleus; NS and TS, nucleus and tractus solitarius; Pyr, pyramidal tract; V, spinal trigeminal nucleus; X, dorsal motor nucleus of the vagus; XII, hypoglossal nucleus; vlo, ventrolateral outgrowth of the dorsal cap; 12n, hypoglossal nerve.
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Hypothetical function of CFRs in Purkinje cells
If the climbing fiber-associated decrease in SSs is caused primarily by Golgi cell inhibition, then what is the function of the climbing fiber innervation of Purkinje cells? If the output of Purkinje cells consists predominantly of SSs discharging at a frequency of 20-50 impulses/s, what would be the function of an increase in CFRs of 1-3 impulses/s? The model for long-term depression (LTD) developed by several laboratories during the last decade is relevant to this question (Crépel and Jaillard 1991
; Ito and Karachot 1989
; Ito et al. 1982
; Sakurai 1987
). LTD is characterized by reduced synaptic efficacy of parallel fibers on Purkinje cell dendrites after conjunctive stimulation of parallel fibers and climbing fibers (Narasimhan and Linden 1996
).
In the context of the present experiment, the low-frequency discharge of CFRs could act as a conjunctive filter for simultaneously active parallel fibers. Parallel fibers active within a window of time associated with a CFR would have a reduced synaptic effect on the particular Purkinje cell in which the CFR was evoked. This conjunctive effect would allow a single climbing fiber to shape the synaptic influence of parallel fibers originating from spatially remote granule cells. Because single parallel fibers attain lengths of
7 mm (Brand et al. 1976
; Mugnaini 1983
), virtually every granule cell in a beam extending across the entire width of the nodulus potentially could innervate every Purkinje cell (Fig. 10C). The dendritic tree of each Purkinje cell receives synaptic contacts from
120,000 parallel fibers (Fox et al. 1967
). By depolarizing the entire Purkinje cell dendritic tree, a single CFR could influence the synaptic efficacy of these parallel fibers, depending on their conjunctive activity with respect to the CFR.
The central 3 mm of a single folia in the ventral nodulus is represented in Fig. 10. This schematic demonstrates the hypothetical spatial relationships among CFRs, Purkinje cells, granule cells, and Golgi cells. It ignores other interneurons (basket cells, stellate cells, and unipolar brush cells) as well as contacts with processes other than Purkinje cell dendrites in the molecular layer. Each parallel fiber is represented as having a minimal length of ~0.7 mm (black line) and a maximal length of 3 mm (gray line). Thus a wide distribution of granule cell activity could result from variable parallel fiber lengths. Longer parallel fibers would cross several functional climbing fiber zones within the nodulus.
Given this basic architecture, LTD of parallel fiber synapses, evoked by conjunctive CFRs, could provide a mechanism for the selective control of the mossy fiber-granule cell pathways. The excitability of a parallel fiber on a particular Purkinje cell would vary as a function of the temporal coincidence of the parallel fiber activity with the CFR of the Purkinje cell. This effect of climbing fibers on parallel fiber synapses would extend to parallel fibers that originated from granule cells located outside of a particular climbing fiber zone.
This circuitry leads to at least two consequences of climbing fiber activation. First, by activating Golgi interneurons, climbing fibers reduce the excitation of granule cells in a spatially discrete area defined by a climbing fiber zone and the overlap of Golgi cell axon terminals. Reduced parallel fiber input to the Purkinje cell dendritic tree causes a reduction in SSs within a particular climbing fiber zone. Second, by depolarizing the entire Purkinje cell dendritic tree, a CFR reduces conjunctively evoked parallel fiber excitation. Conjunctive reduction in the efficacy of parallel fiber synapses would allow a single climbing fiber to regulate the input to a Purkinje cell from as many as 120,000 granule cells distributed across several functional zones. Both the Golgi-mediated granule cell inhibition and the conjunctive effect would result in the reciprocal relation of CFRs and SSs.