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Department of Neurology, Case Western Reserve University, Cleveland, Ohio 44106
Submitted 3 November 2003; accepted in final form 8 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Stahl et al. 2000).
P/Q calcium channel mutants offer an opportunity to apply this approach to exploring ocular motor signal processing. The P/Q calcium channel is heavily expressed in multiple ocular motor structures, including inferior olive, cerebellar granule cells, Purkinje cells, and extraocular motor neurons (Hillman et al. 1991; Stea et al. 1994; Tanaka et al. 1995; Westenbroek et al. 1995). In all these areas the channel acts as a conduit for calcium entry during synaptic transmission (Catterall 1998), but in Purkinje cells it also shapes the electrophysiology of the dendritic tree (Genet and Delord 2002; Llinas et al. 1992; Schmolesky et al. 2002). Geneticists have identified 7 mouse strains harboring mutations of CACNA1A, the gene encoding the ion pore protein of the channel complex. Listed in rough ascending order of ataxia severity, the strains are: rocker, tottering, tottering-4J, tottering-5J, rolling-Nagoya, tottering-3J, and leaner (Zwingman et al. 2000, 2001). In vitro electrophysiological studies of cerebellar neurons from rocker, tottering, rolling-Nagoya, and leaner have demonstrated that these mutations are all associated with reduced calcium currents through P/Q channels (Dove et al. 1998; Itsukaichi et al. 2002; Lorenzon et al. 1998; Mori et al. 2000; Wakamori et al. 1998). Additionally, the tottering mutation has been shown to result in a shift from a dependency on presynaptic P/Q to N channels at the synapse between parallel fibers and Purkinje cells, and this shift secondarily renders the synapse more susceptible to inhibition by GABAergic interneurons (Zhou et al. 2000).
Although some of these strains exhibit prominent cerebellar degeneration, histology in others (i.e., rocker, tottering, tottering-4J, and tottering-5J) is relatively normal (Zwingman et al. 2000, 2001). In this latter group, the phenotypic abnormalities may be attributable to alterations in intrinsic properties, given that the normal histology implies that neuronal architecture and connectivity could be relatively unaffected. Of the 4 strains exhibiting preserved histology, rocker is particularly suitable for use in an eye movement study. Induced by ethylnitrosourea mutagenesis, the rocker mutation consists of a single amino acid exchange within one of the extracellular loops forming the mouth of the calcium channel (Zwingman et al. 2001). Homozygous animals exhibit a rocking gait and limb tremor. Like the allelic mutation tottering, rocker exhibits absence seizures, but lacks tottering's frequent dystonic episodes. Rocker cerebella exhibit normal gross morphology, molecular layer width, and Purkinje cell numbers, but in the second year of life the Purkinje cells atrophy, assuming a "weeping willow" appearance in Golgi–Cox preparations.
As noted above, the P/Q channel is concentrated at several loci within the ocular motor circuitry. Neuronal recordings can be used to assess the degree to which any site contributes to the abnormal phenotype. For instance, if recordings of Purkinje cell complex spikes reveal normal spontaneous rates, regularity, and response to motion of the visual world (Graf et al. 1988), then mutation-engendered dysfunction in the inferior olive can be excluded. Likewise, if the quantitative relationships (e.g., transfer functions) between extraocular motor neuron firing and eye movements are normal, then one can discount the effects of abnormal P/Q channels at the neuromuscular junction. However, 3 issues must be resolved by behavioral investigations before neuronal recordings can be contemplated. First, the chosen mutant must be demonstrated to have an abnormal ocular motor phenotype consistent with cerebellar dysfunction. To date, ocular motility has not been studied in any murine P/Q calcium channel mutant, and the vestibuloocular reflex (VOR) has been reported to be normal in episodic ataxia type 2 (EA-2), one of the 3 human diseases attributed to CACNA1A mutations (Baloh et al. 1997). Second, the chosen mutant must be demonstrated to exhibit ocular motor abnormalities that are congenital. Any abnormalities that appear only as the animal ages are likely attributable to secondary degenerative changes, as opposed to being a direct consequence of the congenital alterations in electrophysiology. Even the rocker mutant, which does not exhibit age-related loss of cerebellar neurons, does exhibit progressive alterations in Purkinje cell morphology (Zwingman et al. 2001), which could potentially have deleterious effects on eye movements. Third, strongly abnormal behaviors must be identified that can be elicited using stimuli that are compatible with single-neuron recording. The current experiments were conducted to address these 3 issues in the rocker mutant.
Portions of data contributing to this report were previously presented in preliminary form (Stahl 2002a,b).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Experimental use of mice was approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and conformed to the National Institutes of Health guidelines for the use and care of vertebrate animals. Rocker mutants of either sex were obtained from the breeding colony of Dr. Karl Herrup at Case Western Reserve University. Control animals were drawn from the C57BL/6J-derived colony from which the rocker colony had been developed (Zwingman et al. 2001). Additional controls of the C57BL/6J strain were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were aged 50–700 days at the time of eye movement recording. Overall, we studied 38 control animals and 14 rocker mutants. During analysis of the data, animals were divided arbitrarily into 3 age groups: 2–8 mo, 8–14 mo, and >14 mo. These groupings correspond roughly to breeding adult, postbreeding adult, and elderly periods for the laboratory mouse. Some animals were recorded repeatedly at widely separated times and consequently contributed data to more than one (usually 2) age groups, but no animal contributed more than once to any one age group. All animals did not undergo all types of testing. Numbers of animals tested in each condition and age group, as well as the average age of the animals tested in a particular condition, are summarized in Table 1.
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). Surgery was conducted under halothane general anesthesia with additional local application of bupivacaine to all wound margins. The pedestal was contrived so that during recordings the head would be maintained in a natural position in which a line drawn from the inner to the outer canthus of the eye would be parallel to, or pitched slightly down from, earth-horizontal. Before each recording session, the eye to be recorded was treated with an ophthalmic solution of 0.5% physostigmine salicylate to limit pupil dilation in darkness (Stahl 2002a). Eye movement recording and calibration
Eye movement recordings were obtained using a video-tracking method as previously described (Stahl 2002a; Stahl et al. 2000). During recordings, the animal's body was loosely restrained in an acrylic tube with its head pedestal bolted securely to an extension of the tube. The animal could be mounted so that either the left or right eye was imaged by a small video camera fixed to the axis of the turntable (see Stimulus apparatus and conditions, below). Video images of one eye were processed by a commercial pupil tracker (ETL-200, ISCAN, Burlington MA), which extracted signals proportional to the horizontal and vertical (linear) positions of the pupil center. Additionally, it reported pupil diameter and the horizontal and vertical positions of the corneal reflection of an infrared emitter mounted directly over the center of the camera's optical axis. Sampling rate was 120 Hz during measurements of nystagmus fast phases, and 60 Hz for all other purposes. Analog outputs of the eye tracker were passed through 4-pole Bessel low-pass filters (corner frequency 100 Hz), resampled at 200 Hz, and recorded on a PC-type computer. Other analog signals, including turntable position, optokinetic drum velocity, and planetarium velocity were similarly filtered, sampled, and stored.
The positions of pupil and reference corneal reflection (CR) were converted off-line to angular position as previously described (Stahl 2002a; Stahl et al. 2000). Briefly, we recorded the pupil and CR positions as the video camera was rotated over a 20° arc about an earth-vertical axis passing approximately through the recorded eye. The positions obtained at the extremes of the camera rotation were used to calculate RP, the linear distance between the plane of the pupil and the center of corneal curvature. Data were collected over a range of pupil diameters (produced by varying the ambient illumination) and linear regression performed to generate a formula relating RP to pupil size. Subsequent records could then be processed to yield horizontal eye-in-head angle (EH) by first using pupil diameter and the regression equation to determine instantaneous RP, and then applying the formula
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X is the horizontal distance in millimeters between the pupil and CR. Note that given this equation and the setup geometry, EH = 0 when the animal's pupil is positioned such that the line from the center of corneal curvature through the pupil centroid parallels the camera's optical axis.
For assessing absolute eye elevation, the reference infrared (IR) emitter was repositioned to lie at the side of the camera lens, aligned vertically with the camera optical axis. [Because the cornea closely models a spherical reflector (Remtulla and Hallett 1985), the virtual image of the reference emitter appears along a radius of the cornea that is parallel to the camera's optical axis, i.e., in the plane of the equator of the eye.] The vertical linear distance between the pupil and CR centers (Y) was converted to elevation above the ocular equator by the equation
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).
The above procedures are based on the assumption that vertical eye position remains, for the duration of the recording session, close to the elevation occupied during the original calibration procedure. This assumption is reasonable when vestibular and optokinetic stimuli are restricted to the horizontal plane because the evoked movements would also be horizontal. In addition, the head-fixed mouse makes few spontaneous eye movements (van Alphen et al. 2001) and thus is unlikely to assume and maintain an eccentric vertical position in the absence of a vertically directed stimulus. However, if eye elevation varies (as it does in the cross-axis stimulus described below), RP changes, as illustrated in Fig. 1. Consequently, we introduced a modified method to treat responses to combined horizontal and vertical stimuli. The method is based on RP0 (which differs from RP by being a "great circle" radius), and yields horizontal and vertical eye angle in Fick coordinates.
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Y(raw) between the pupil and the CR by
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), and 
is the angular elevation of the reference emitter above the optical axis of the video camera (9.3° in our setup).
This correction compensates for the fact that the reference emitter is not coaxial with the video camera in the vertical direction (i.e., the emitter rests on the top of the lens). The correction renders Y a measure of the linear elevation of the pupil above the eye's equator. Finally, we calculated RP0 from the corrected vertical elevation and RP by
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To use the vertical-sensitive calibration to determine horizontal angular position, we first used pupil diameter and the calculated regression equation to determine instantaneous RP0. Next we calculated the instantaneous, vertical-sensitive RP by
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To determine vertical angular position, we first corrected the linear vertical position Y(raw) for reference emitter elevation using Eq.3. Then vertical angle was calculated as
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It should be noted that the vertical-sensitive strategy introduces additional sources of noise (from the vertical pupil and CR positions) to the calculated horizontal eye angle. Thus we reserved the vertical-sensitive method for the single condition that evoked vertical eye movements (i.e., the cross-axis adaptation).
Stimulus apparatus and conditions
During recordings, the animal was mounted on a servocontrolled turntable, which produced rotations about the earth-vertical axis under manual or computer control. Optokinetic stimuli about the vertical axis were provided by rotation of a servocontrolled drum painted with a high-contrast white-on-black pattern, measuring 0.5 m in diameter, and enclosing the animal from the zenith to 10° below the horizon. During cross-axis adaptation (see following text), optokinetic stimuli about a rostrocaudal axis were generated by a planetarium (Leonard et al. 1988) mounted over the animal's head and projecting on a featureless gray fabric background. Multiple aspects of eye movements were characterized, including the absolute vertical position of the eye at rest, gain, and phase of the vestibuloocular reflex in dark (VOR) and light (visual VOR, or VVOR), gain of the optokinetic response (OKR), adaptability of VOR gain and direction, and velocity characteristics of nystagmus fast phases. A complete assessment required at least 4 one-hour recording sessions to complete.
The determination of average absolute vertical position was based on about 60 s of recording with the animal held motionless in the light. Before, and then once or twice during, acquisition of the data we briefly oscillated the animal and then rotated it to bring the eye (under the influence of the VVOR) back to the approximate horizontal center of the orbit. This procedure ensured that the mouse, which may not spontaneously recenter its eye, was not occupying an unusual vertical position at the moment the data were acquired. Horizontal nystagmus fast phases for analysis of peak velocity–amplitude relationships were generated by slowly rotating the animal under manual control in the light. The experimenter monitored the image of the eye and adjusted the rotation speed and direction to generate a range of fast phase amplitudes, as well as to ensure that eye velocity was low at the moment of fast phase initiation. VOR and VVOR were tested by rotating the animal in darkness and light, respectively, at 0.1 Hz (±10° amplitude), 0.2 Hz (10°), 0.4 Hz (10°), 0.8 Hz (
4.6°), and 1.6 Hz (3.9°). Corresponding velocity amplitudes were ±6, 13, 25, 23, and 39° /s. OKR was assessed during constant velocity rotations at ±2.5, 5, 10, 20, and 40° /s. Constant velocity periods alternated direction, lasted 4 s each, and were separated by 3.5-s periods of darkness during which the drum reversed direction and reaccelerated to the next test speed. The ability of the animal to increase the VOR amplitude toward a target gain of 1.5 (1.5x adaptation) was tested by oscillating the animal in the light at 0.4 Hz, 10° amplitude while simultaneously rotating the optokinetic drum in counterphase at half the amplitude of the table. VOR gain was tested before initiation of the adaptation stimulus, and then every 10 min during the 50-min adaptation period. The interim testing required that the adaptation stimulus be interrupted for about 60 s. We also recorded the response to the adaptation stimulus (i.e., table rotation in the light with the drum in motion) at similar intervals. The ability of the animal to alter the direction of its compensatory eye movements (cross-axis adaptation) was tested by oscillating the animal in the light at 0.4 Hz (10° amplitude) while simultaneously rotating the planetarium about a rostrocaudal axis at the same frequency and amplitude. The phase of the planetarium rotation was adjusted with respect to the turntable so as to promote downward eye movements in association with nasally directed horizontal compensatory eye movements. As in the 1.5x adaptation, VOR direction was assessed before adaptation, and then every 10 min during a 50-min adaptation period.
Data analysis
Fast phases of vestibular nystagmus (henceforth loosely termed "fast phases") were quantified in terms of the slopes of the relationships between peak eye velocity and fast phase amplitude. Horizontal eye velocity was calculated off-line by numerically differentiating the eye position signal after smoothing it by convolution with a Blackman window whose cutoff frequency was 40 Hz. Candidate fast phases were identified using a supervised automated program. Onsets and offsets were defined as the moments at which eye speed exceeded or fell below 5° /s. Linear regression forced to pass through the origin was used to extract the slope of the relationship between peak velocity and fast phase amplitude. Abducting and adducting fast phases were treated separately.
VOR and VVOR data were processed to extract gain and phase values with respect to head velocity as previously described (Stahl et al. 2000). Gain versus frequency and phase versus frequency (Bode) plots were compiled for each recording session, and then the curves from at least 2 sessions were averaged to generate a single Bode plot for each animal/age. These single-animal curves were in turn averaged to generate curves for each genotype and age group. OKR data were processed to extract gain values. For each period of constant velocity drum rotation in the light, fast phases were removed from the eye velocity record (by replacement with a slow movement interpolated from the flanking slow phase movements) and the average velocity for the period was calculated. Gain was then calculated as the ratio of the average eye and drum velocities. Three to 4 records were collected per session, and the gains at each stimulus velocity were averaged to generate a gain versus stimulus velocity (speed tuning) curve for the session. As with the VOR/VVOR data, curves from at least 2 sessions were averaged to yield single-animal curves, and multiple single-animal curves were averaged to generate a curve for each genotype/age group. In reporting OKR results, positive drum rotations signify temporal-to-nasal rotations with respect to the recorded eye.
The 1.5x adaptation data were processed to generate for each animal a curve relating horizontal VOR gain to duration of the adapting stimulus. Curves for multiple animals were averaged to generate a curve for each genotype/age. Cross-axis adaptation data were processed to determine horizontal VOR gain, as well as vertical VOR gain defined as the amplitude of the vertical velocity (extracted, as usual, by Fourier analysis) divided by the amplitude of the horizontal stimulus. The progression of the horizontal and vertical gain changes through the adaptation period were compiled as in the 1.5x experiment. Additional measures tracking the magnitude of vertical movements will be described in RESULTS.
Significance of interage group and intergenotype comparisons were tested as described in RESULTS. In general, curves were compared using repeated-measures ANOVA, whereas point measurements were compared using 2-tailed t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Static pupil elevation varied widely in controls animals, ranging 11–30° in the middle age group, for which most data are available. Figure 2 plots static pupil elevation in the light as a function of animal age. There was no tendency for elevation to change with age in control animals (elevation vs. age correlation r2 = 0.004). In contrast, eye elevation tended to increase with age in mutants, and the elevation–age slope was significantly different from zero (r2 = 0.35, P = 0.0015, 2-tailed t-test for correlation coefficients). Average elevations in mutants were higher than controls in all age groups (17.1 vs. 15.6°, 22.7 vs. 19.5°, and 25.3 vs. 17.5° in the young, middle, and old age groups, respectively) and the difference became statistically significant in the oldest age group (P values, respectively, 0.42, 0.10, 0.008). Human P/Q calcium channelopathy and other cerebellar disorders are frequently associated with downbeat nystagmus. In contrast, the rocker animals exhibited stable (although elevated) vertical positions, both in light and darkness.
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Figure 3 depicts a typical peak velocity versus amplitude plot (a so-called main sequence relationship) for the fast phases in one control animal. For each animal, we calculated a regression curve, forcing it to pass through the origin. There was a strong linear relationship between velocity and amplitude for both mutant and control animals. Abducting fast phases tended to be slower than adducting fast phases. For control animals of all ages, the abduction and adduction slopes were 23.9 ± 1.5 and 27.1 ± 1.3 s–1, respectively, and the difference was statistically significant (paired t-test, n = 52, P = 0.0011). Main sequence slopes were lower in rocker than in controls. For instance, the abduction and adduction slopes for rocker animals of all ages were 19.3 ± 1.8 and 23.2 ± 1.6 s–1. The differences between the average slope values for mutants and controls in all age groups were statistically significant (P < 0.0001) for both abducting and adducting fast phases. Main sequence slopes did not change appreciably with age. Figure 4 plots the slopes for mutants and controls versus age for the abducting (top panel) and adducting (bottom panel) directions. Correlation coefficients of the slope versus age regressions were low, and only one of the 4 correlation coefficients differed significantly from zero (abducting fast phases of control animals, slope =+0.0025 s–1/d, r2 = 0.076, P = 0.048, t-test for r2).
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Figure 5 shows Bode plots for VOR and VVOR of controls and mutants in each age group. In control animals of all ages, VVOR gain increased gradually, peaking near unity gain at 0.8 Hz. Eye velocity was nearly in phase with head velocity at 0.1 Hz, and a small phase lag developed with increasing stimulus frequency. In darkness, gain declined and phase lead increased with decreasing stimulus frequency. Of note, the low-frequency gain attenuation differs from results obtained using magnetic search coil recordings (van Alphen et al. 2001). Some of the tendency for the video-derived VVOR gain to decline at the lowest stimulus frequencies may relate to apparatus peculiar to the video system (i.e., the camera and IR illuminators) that travels with the animal and conflicts with the earth-fixed visual surround.
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The effect of ageing on VOR dynamics was assessed by regressing gain and phase versus animal age. Overall, there was a tendency for VOR gain to decline with age. Regression analysis on the plots yielded negative slopes for all stimulus frequencies and strains, with the exception of the control animals at 0.4 Hz, and the mutants at 0.8 Hz. However, the correlation coefficients were uniformly low, and statistical significance bettered P = 0.05 in only 2 cases (control animals at 0.1 Hz and mutants at 0.2 Hz). The low degree of significance reflects the shallow slopes and wide variability in the data, indicating that the effects of ageing are less prominent than is the interanimal variability. Analysis of phase versus age plots yielded very similar results. There was a tendency for phase lead to increase with age in both groups (negative slopes at 4 of 5 tested frequencies in both mutants and controls), but most (7/10) of the correlation coefficients failed to reach statistical significance, again indicating that the effects of ageing on VOR phase are modest at best.
A striking, qualitative difference in the Bode plots of rocker animals and controls was the manner in which VVOR gain "rolled off" at the higher stimulus frequencies. We quantified this abnormality by calculating the ratio of gains at 1.6 and 0.1 Hz versus age, thereby compensating for some of the interanimal variability in absolute gain. This ratio is plotted versus animal age in Fig. 6. Results for rocker and control were largely nonoverlapping, emphasizing the robustness of the abnormality. There was no effect of ageing on the gain ratio in rocker (r2 = 0.004, P = 0.76), indicating that the physiological abnormality underlying the finding is either congenital, or fully developed by the earliest age included in our sample. Control animals exhibited a weak tendency for the ratio to decline with age (r2 = 0.080, P = 0.09).
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Mice exhibited robust eye movements in response to drum rotation. In accordance with previous conclusions that velocity storage is minimal in this species (van Alphen et al. 2001), we observed little evidence of an increase in eye velocity over the course of prolonged rotations, or of persistent nystagmus if the lights were extinguished after a period of prolonged rotation. Figure 7 shows plots of OKR gain versus drum velocity (speed tuning curves) for normal and mutant mice in each age group. Both genotypes exhibited maximal gains of about 0.8, rolling off rapidly above drum velocities of 5° /s. Although control and mutant gains overlapped extensively, the mutant averages consistently fell below the controls at all drum speeds and in all age groups, suggesting a mild optokinetic deficit. The curves differed significantly only in the oldest age group (repeated-measures ANOVA, P values of 0.12, 0.39, 0.004 for 2–8, 8–14, and >14 mo ages, respectively). At earlier ages the significance levels were certainly reduced by the wide variability in OKR gains, which was particularly marked in the mutants. Regression analysis of rocker OKR gain at each drum speed versus animal age (analogous to the VOR/VVOR analyses in Fig. 6) yielded essentially flat slopes that were not significantly different from zero (P values 0.19–0.90). Thus the increased separation of the tuning curves in the oldest age group does not reflect a steady decline in rocker OKR gain through life.
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Figure 8 displays the progress of gain changes for rotation in the light (response to the adapting stimulus) and dark (VOR) during the course of the 50-min 1.5x adaptation period. Gains were normalized to the initial (preadaptation) gain. Qualitative inspection of averaged gain curves for the individual age groups revealed no consistent age-related changes, so animals of all ages were pooled for this plot. Control animals exhibited, on average, a gradual and mild increase in gain in response to the adaptation stimulus. VOR gain, however, tended to rise only minimally during the first 20 min of the adaptation, after which point it fell in most animals. The results could be explained by postulating that the process(es) responsible for augmenting VOR gain is(are) antagonized by a gain-depressing, habituation process, and habituation "wins out" as the experiment proceeds. The fact that gain did increase in the light indicates that the habituation applies more strongly to rotation in darkness, or that the optokinetic component of the response to rotation in the light also undergoes an increase in gain during the course of the adaptation period, offsetting the decline in VOR gain. In contrast to control animals, rockers generally exhibited declining gains for rotation in both the light and dark, as well as greater animal-to-animal variability. Both the lack of an increase in gain in light, as well as the more pronounced decline in gain in darkness, could be explained if the gain augmentation process is weaker in rocker, resulting in a more obvious expression of the concomitant habituation process. Alternatively, the habituation process could be stronger in rocker than in controls, masking any gain augmentation to a greater degree. The possibility of a stronger habituation process is supported by the cross-axis data, below.
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Figure 9 displays the progress of gain changes for rotation in the light (response to adaptation stimulus) and dark during the course of the 50-min cross-axis adaptation period. Qualitative inspection of averaged curves for the individual age groups revealed no striking age-related changes, so animals of all ages were pooled for this plot. On average, control animals exhibited a mild decrement in the horizontal response to rotation in the dark, and a mild increase in the response to rotation in the light, over the course of the adaptation period. The vertical eye movement in the light (i.e., in the presence of the optokinetic stimulus) increased continuously. Finally, the vertical eye movement in the dark (i.e., the cross-coupled response) increased gradually, plateauing within the first 30–40 min. The behavior of rocker animals, as a group, differed considerably. Horizontal gain in light and dark fell off continuously throughout the adaptation period. The baseline vertical response in the light, while present, was slightly smaller than for controls (0.169 ± 0.067 vs. 0.217 ± 0.052, respectively, difference significant, P = 0.007) and increased only minimally with time. Finally, the mutant and control vertical gains in darkness were identical at baseline, but the mutants, as a group, developed little or no cross-coupling during the course of the experiment. The averaged 30- and 40-min vertical gains in darkness for mutants and controls were 0.089 ± 0.042 and 0.194 ± 0.052, respectively, and differed significantly (P < 0.0001).
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Figure 10 plots the corrected gain values in light and dark. Although the mutant corrected vertical gains do increase slightly throughout the experiment, they remain considerably below the control values, and the averages of the 30- and 40-min corrected gains differ significantly for mutants and controls (P < 0.0001, P = 0.0002 for darkness and light, respectively).
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As a group, the mutants exhibited abnormalities in a variety of ocular motor characteristics. If the calcium channel mutation is directly responsible for each of the abnormalities, then the severity of these abnormalities might be expected to correlate. We addressed this question by pairwise linear regressions of 9 of the measures discussed earlier, including: static elevation, main sequence slope, VOR phase at 0.2 Hz, VOR gain at 1.6 Hz, the ratio of the VVOR gains at 1.6 and 0.1 Hz, OKR gain at ±10° /s (i.e., averaging the gains for the 2 directions), and the averaged corrected vertical gains in darkness after 30 and 40 min of cross-axis stimulation. The correlation coefficients (r2) for which the t-test for r2 yielded P values of 0.2 or better are presented in Table 2, with control animals occupying the upper-right half of the matrix and mutants occupying the lower left. Correlation coefficients are signed according to the signs of the associated regression slope. Overall, correlation coefficients were low, and none would be significant if corrections for multiple comparisons had been applied. Nevertheless, inspection of the patterns in Table 2 is instructive. For instance, in mutants, 6 of the 8 variables exhibited negative correlation with static elevation, suggesting that greater static elevation was associated with greater aberrancy of other ocular motor measures. In contrast, in control animals, no ocular motor measure exhibited a correlation with static elevation with P < 0.2 or better. Likewise, mutants exhibited a significant linkage between increased VOR phase lead at 0.2 Hz and decreased VOR gain at 1.6 Hz, whereas control animals did not. Several of the measures are logically related and were accordingly correlated in both controls and mutants, including fast phase abduction versus adduction, VOR gain at 1.6 Hz versus the 1.6:0.1 Hz VVOR gain ratio, and the vertical gains in light versus dark after cross-axis adaptation. If one discounts these linked measures, then the normal animals exhibited P < 0.2 correlations in only 4 of 33 comparisons, and in 2 cases the sign of the correlations suggests a random relationship (e.g., it is difficult to explain why faster abducting fast phases should be associated with lesser cross-axis adaptation, or why more rapid adducting fast phases should associate with a decline in VVOR gain with increasing stimulus frequency). In contrast, mutants exhibited P < 0.2 correlations in 14 of 33 informative comparisons and, in every case, the sign of the correlation was appropriate for more abnormal values in one measure to associate with more abnormal values in the second measure. Overall, the results in Table 2 support a linkage between some of the abnormalities found in the mutant group, but emphasize the degree of variability in the findings.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), we needed to assess a variety of different behaviors. Below, we discuss the results of each type of testing with regard to the study goals. Static elevation
When applied to mammals lacking fixation-specialized retinas, most oculographic techniques are limited to reporting positions relative to some arbitrary zero position. In contrast, our technique allowed us to determine absolute vertical position. Our study is the first to report static elevation in mice, but it has been investigated using other techniques in a variety of other species, including rabbits and rats, in which the elevation measures 13 and 30°, respectively (Hughes 1977, 1979). These values are comparable to the average value of 18.0° obtained in our study.
Older rocker animals exhibited more elevated optical axes. We have speculated (Stahl 2002a) that this elevation could represent the mouse homolog of the downbeat nystagmus seen in humans with various disorders of the cerebellum (Leigh and Zee 1999), including the P/Q channelopathies episodic ataxia type 2 (EA-2) (Baloh et al. 1997) and spinocerebellar ataxia type 6 (SCA6) (Gomez et al. 1997). Downbeat nystagmus refers to a pattern of abnormal eye movement in which upward drifts of the eyes alternate with downward fast phases ("beats"). Several of the explanations that have been proposed for downbeat nystagmus involve the loss of a signal from the cerebellar flocculus that ordinarily cancels an upward bias of the angular VOR (Baloh and Spooner 1981; Bohmer and Straumann 1998). The mouse could potentially manifest the same VOR imbalance as a static elevation because, lacking an obvious retinal region specialized for fixation (Jeon et al. 1998), it may not maintain a stringent orientation of the eye with respect to the horizon. As such, vertical drifts may not generate recentering fast phases until the eye is carried to an extreme position. Because the mouse neural integrator is also weak (van Alphen et al. 2001), the postulated upward drift may come into equilibrium with the centripetal, integrator-related drift before such an extreme position is reached. This hypothesis predicts that other murine cerebellar disorders should be associated with increased elevation, a prediction supported by limited data from lurcher (Stahl 2002a), a mutant that suffers postnatal degeneration of all Purkinje cells. Because static elevation increased with age, this ocular motor abnormality is less likely to be a direct consequence of the lifelong alteration in channel biophysics. Rather, it may be related to this mutant's age-related atrophy of Purkinje cell dendrites (Zwingman et al. 2001).
Fast phase dynamics
This study is the first to describe the velocity–amplitude relationships of vestibular-induced nystagmus in mice. These relationships were linear, and in this respect mice resemble guinea pigs (Escudero et al. 1993) and rats (Fuller 1985) more than humans (Bahill et al. 1981), cats (Evinger and Fuchs 1978), or rabbits (Collewijn 1977), in which the velocity–amplitude relationships exhibit exponential profiles. Mouse fast phases were consistently faster in the adducting direction. Humans have also been reported to exhibit more rapid adducting saccades, although the validity of this result has been questioned (Becker 1989). Reports of fast phase dynamics in other nonfoveate mammals did not separate abducting and adducting directions (Collewijn 1977; Escudero et al. 1993; Fuller 1985).
The slowing of fast phases in rocker may or may not reflect dysfunction at the level of the cerebellar cortex. Slowing of saccades has been reported after anatomical lesions of oculomotor vermis in monkeys (Takagi et al. 1998), as well as in humans with inherited spinocerebellar ataxia (Buttner et al. 1998). It is unclear, however, whether these effects on primate voluntary saccades are relevant to fast phases of vestibular nystagmus made by nonfoveate mammals. Saccade slowing in the spinocerebellar ataxias has actually been attributed to pontine, rather than cerebellar, pathology (Buttner et al. 1998). No pontine atrophy was reported in rocker (Zwingman et al. 2001), although the coarse histological examination would probably not have detected subtle degeneration restricted to ocular motor structures. Studies that assessed P/Q channel distribution suggest the channel is only lightly expressed in the paramedian pontine reticular formation, wherein lie the neurons responsible for generating the burst of neuronal activity that accelerates the eyes during fast phases (Craig et al. 1998; Hillman et al. 1991; Stea et al. 1994). However, as in the case of the histological evaluations in rocker, none of the expression studies was of sufficient resolution to exclude the possibility of a heavier concentration of P/Q channels on the burst neurons themselves. Fast phase slowing could also be a consequence of extraocular muscle weakness, given that the P/Q channel is heavily expressed at neuromuscular junctions, and defects of neuromuscular transmission have been demonstrated in tottering mice (Plomp et al. 2000) and EA-2 patients (Jen et al. 2001). Single-neuron recordings from extraocular motor neurons could be used to generate transfer functions from which one could determine whether junctional defects are of sufficient magnitude to explain the observed slowing. Whatever the locus of the dysfunction responsible for fast phase slowing, the slowing was independent of animal age, and thus is potentially a direct consequence of the altered calcium biophysics.
It should be noted that the current study of fast phase dynamics was limited by the relatively low sampling rate (120 Hz) of the video oculography and the 40-Hz low-pass numerical filtering applied during calculation of eye velocities. The net effect is to underestimate the peak velocities, and thus the contrasts between the velocity–amplitude relationships of rocker and controls, or between abducting and adducting fast phases, may also be underestimated.
Compensatory eye movements
Rocker mutants exhibited an increase in phase lead at low stimulus frequencies. This abnormality is consistent with impairment of the neural integrator (Skavenski and Robinson 1973), which can be induced by lesions of the flocculus (Zee et al. 1981) and is a fairly common finding in human cerebellar disorders (Leigh and Zee 1999) including EA-2 (Baloh et al. 1997; Jen et al. 1999) and SCA6 (Gomez et al. 1997). The mild OKR gain deficit is likewise consistent with cerebellar dysfunction in mammals whose retinas lack areas specialized for fixation, based on results of bilateral flocculus lesions in rabbits (Nagao 1983, 1989; van Neerven et al. 1991), and recordings from the mouse mutant lurcher (van Alphen et al. 2002) [although a flocculus lesion experiment in mice yielded a contradictory result (Katoh et al. 1998)]. The optokinetic deficit in rocker is sufficiently mild that it seems unlikely the VOR deficit is a consequence of abnormal visual experience. OKR gain has been reported to be abnormal in EA-2 (Baloh et al. 1997), but this finding is probably not equivalent to ours because human OKR reflects major contributions from the pursuit system, which is lacking in mice and other nonfoveate species. In mice, the OKR gain reduction was clearest in aged animals, and thus may be partly attributable to age-related Purkinje cell dendritic atrophy. There may also be a congenital component, given that at all ages the mutants, as a group, tended to have slightly lower gains. However, the disparity between mutants and controls in the younger animals was not sufficiently large to be attractive for investigation using neuronal recordings.
From a qualitative standpoint, the most striking abnormality of compensatory eye movements was the manner in which VVOR gains rolled off with increasing stimulus frequency, a finding we attribute to a deficiency in the gain of the underlying VOR. The deficit was present at all ages and thus may be a direct consequence of the inborn alteration in P/Q calcium channel function. VOR gain has been reported to be normal in EA-2 (Baloh et al. 1997), but the study was restricted to stimulus frequencies of 0.4 Hz and below. Recordings in rabbits indicate that, although Purkinje cells are inhibitory, the phase of their modulation is appropriate to enhance VOR gain (De Zeeuw et al. 1995; Stahl and Simpson 1995). Thus the mouse gain deficit could arise in a simple fashion by virtue of impaired synaptic transmission from parallel fibers to Purkinje cells, or from Purkinje cells to vestibular nucleus neurons. The finding of a worsened deficit at higher stimulus frequencies could arise if the mouse flocculus normally provides a signal in which velocity components (i.e., high-frequency signals) are emphasized, as has been suggested for the rabbit (De Zeeuw et al. 1995; Stahl and Simpson 1995). Alternatively, channelopathy-related defects in synaptic transmission or dendritic integration could vary with stimulus frequency. The origin of the high-frequency deficit may be clarified by in vivo recordings from floccular Purkinje cells during vestibular stimulation, or by in vitro experiments that assess synaptic transmission of electrically evoked spike trains comparable to those generated during natural vestibular stimulation.
VOR adaptation
The ability of the VOR circuits to adjust their gain is essential to the production of compensatory eye movements of proper amplitude and direction. The cerebellar flocculus is well known to participate in the gain adjusting process (duLac et al. 1995; Ito 1982; Miles and Lisberger 1981), and thus deficits in VOR gain plasticity are consistent with floccular dysfunction. We attempted to modulate VOR in 2 different ways, either increasing its gain (1.5x adaptation) or changing its direction (cross-axis adaptation). Because our ultimate goal is to record single neurons during the adaptation process, we employed single-session adaptation procedures. In this respect our approach differed from that of investigators (Boyden and Raymond 2003; van Alphen and De Zeeuw 2002) who altered gain using multiple training sessions spaced across several days.
The 1.5x adaptation experiments were confounded by the tendency of the mice to habituate to repetitive sinusoidal rotation. This tendency appeared more prominent in the mutants, and the divergence of the gain curves for mutants and controls could reflect either differences in the ability to increase gain, differences in the prominence of habituation, or both. The cross-axis experiments circumvented the problem of habituation; vertical gain in darkness generally plateaued (as opposed to declined) toward the end of the adaptation period, and we could use changes in the horizontal gain to compensate provisionally for habituation in the vertical response. The experiments demonstrated robust cross-axis adaptation, and constitute the first time that this capability has been demonstrated in a mammal lacking retinal specialization for fixation. As a group, rocker animals exhibited weaker cross-axis plasticity, and this difference remained even after compensation for rocker's greater degree of habituation. This difference is probably not attributable to inability to respond to the vertical optokinetic stimulus, given that rockers exhibited only slightly lower gains than controls at the baseline assessment (see Fig. 9, top). Moreover, younger mutants did exhibit the ability to increase their optokinetically driven vertical eye movements (i.e., the movements made in the presence of the adapting optokinetic stimulus) over the course of the training period. The deficiency in cross-axis gain measured in darkness was present throughout life, and thus may be a direct consequence of the alterations in P/Q channel biophysics. In contrast, the ability to augment the vertical optokinetic response declined with age. This decline may relate to the age-related deterioration of horizontal optokinetic gain, and also may indicate that distinct processes underlie the augmentation of vertical movements demonstrated in light and dark during the cross-axis experiment.
Effects of ageing
In the course of obtaining control data, these experiments generated the first assessment of the effect of ageing on compensatory eye movements in normal, C57BL/6 mice. In humans, multiple aspects of ocular motor performance decline with age (Baloh et al. 1993; Demer 1994; Furman and Redfern 2001; Paige 1994; Tian et al. 2001). Given that the life span of the laboratory mouse is measured in months, ageing effects have the potential to confound experiments in this species. This concern is particularly great in the case of the C57BL/6 strain, which undergoes severe age-related cochlear degeneration (Henry and Chole 1980; Hultcrantz and Li 1993) and lesser (but still detectable) alterations in peripheral vestibular structures (Cohen et al. 1990; Park et al. 1987). In fact, we found that ageing had little effect in normal mice, with the exception of a weak tendency for VOR gain to decrease and phase lead to increase through life. Similar changes have been described for the horizontal angular VOR in humans (Baloh et al. 2001; Paige 1994).
Apart from the tendency for vertical elevation to increase with age, the abnormalities we detected in mutants were present lifelong, and thus may be direct consequences of the inborn abnormalities of calcium channel biophysics. The current experiments do not exclude the possibility that age-related correlations might be detected in a more highly powered sample, or the possibility that the ocular motor abnormalities develop sometime between birth and the earliest age we studied. The experiments do, however, prove that most of the phenotypic abnormalities are present well before, and do not accelerate with, the appearance of the histological abnormalities.
In conclusion, rocker mutants exhibit abnormalities of ocular motor behavior that are consistent with dysfunction of vestibulocerebellar circuitry. Several of these abnormalities are present lifelong and thus are potentially a direct consequence of alterations of calcium channel biophysics. Alternatively, they may reflect secondary, but still congenital, alterations in circuit properties engendered by the channel mutation. Some abnormalities (e.g., abnormal eye elevation, deterioration of OKR gain) appear later and may stem from age-related changes, such as the late atrophy of the Purkinje cell dendritic arbor that has been reported in this mutant. Such later alterations are interesting in their own right because they offer the opportunity to investigate the effects of changes in neuron structure on signal processing. Studying additional calcium channelopathy strains is desirable, given that having data from 3 or more strains would allow one to use the covariation of biophysical and behavioral measures to gain insight into the linkages between the biophysical and behavioral abnormalities.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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GRANTS
This work was supported by National Eye Institute Grants EY-13370 and EY-11373.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Dr. John Stahl, Dept. of Neurology, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-5040 (E-mail: jss6{at}po.cwru.edu).
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REFERENCES |
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