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Electronic Letters to:
- DevelopmentPlasticityRepair:
Ying-Yu Huang, Oliver Rinner, Patrik Hedinger, Shih-Chii Liu, and Stephan C. F. Neuhauss- Oculomotor Instabilities in Zebrafish Mutant belladonna: A Behavioral Model for Congenital Nystagmus Caused by Axonal Misrouting
J. Neurosci. 2006; 26: 9873-9880[Abstract] [Full text] [PDF]
eLetters: Submit a response to this article
Electronic letters published:
An oculomotor phenotype in question.
- James C Beck (21 March 2007)
- Stephan C.F. Neuhauss, Ying-Yu Huang, Patrik Hedinger (7 May 2007)
An oculomotor phenotype in question. 21 March 2007 
James C Beck,
Assistant Professor
Biology Dept Long Island Univ. Brooklyn, NY & Dept of Physiology NYU School of Medicine NY NY 10016Send letter to journal:
Re: An oculomotor phenotype in question.
james.beck{at}nyu.edu James C Beck
Huang et al. (2006) propose an intriguing link between an oculomotor phenotype of spontaneous oscillations observed in mutant belladonna (bel and bel rev) zebrafish to the pathological condition of congenital nystagmus found in humans. Unfortunately, problems in the methodological approach and interpretation leave this conclusion in question.
The experimental technique used for eye movement measurements was originally created for rapid screening of mutant animals rather than critically evaluating motor performance. The highly viscous methylcellulose solution used here allows animals to be quickly restrained for simple evaluation of eye movements; however, it markedly affects motor system performance and, in particular, appreciably retards eye velocity in wildtype (wt) zebrafish (Beck et al., 2004a). In the methylcellulose solution, the bel zebrafish performed worse than their wt counterparts during optokinetic tests (Fig 2A-C), as might be expected when placing an animal with compromises in central components of oculomotor behavior into a medium that diminishes peripheral oculomotor performance as well.
The poor oculomotor performance of bel zebrafish necessitated a direct comparison of peak saccadic velocity between wt and bel/bel rev to demonstrate that these mutant phenotypes were not physically impaired. However, a sampling rate of only once every 80 ms (12.5 Hz) did not provide sufficient temporal resolution to evaluate the dynamics of saccades that reach peak velocity in ~25 ms (Beck et al. 2004; Mensh et al., 2004). An adequate sampling rate is a key technical requirement to use this type of measurement and is essential to support their conclusion of normal motor performance in bel.
The combined effects of the methylcellulose restraint and low sampling rate may explain the unusually-low average peak saccade eye velocity of 15°/s observed for both wt and bel zebrafish in Fig 3C. Alternatively, the authors’ exclusive use of monocular visual stimulation in the absence of a ganzfeld for the non-viewing eye could also produce marked drops in optokinetic performance (Beck et al., 2004b). Even so, monocular stimulation alone would still not explain why peak slow phase eye velocity recorded in wt equaled peak saccadic velocity (cf. Figs 2A and 3C). The point here is that saccades in wt zebrafish reach a peak velocity of at least 24°/s even with small, 2° changes in eye position (as calculated from Beck et al. 2004b). Because saccade velocity directly correlates with change in eye position (Mensh et al., 2004), the large changes in zebrafish eye position measured in this study (up to 10°, see Figs 1, 7) should have yielded considerably larger saccadic velocities.
The appealing aspect of the achiasmatic bel rev zebrafish is their spontaneous oscillatory eye movements and reversed optokinetic behavior. However, the description and explanation of spontaneous oscillations were not consistent with current interpretations of the complex waveform etiology underlying congenital nystagmus. The zebrafish oscillations were sensory dependent, i.e., they required vision (Fig 4D); yet pendular eye motions of congenital nystagmus persist even in the dark (Leigh and Zee, 2006). The comparison of zebrafish oscillations with human periodic alternating nystagmus was also problematic. Although both conditions may present with “active” and “quiet” phases, bel rev zebrafish never exhibited the slow drifts in eye position and rapid resetting saccades that define periodic alternating nystagmus (Leigh and Zee, 2006). Support for the authors’ interpretation could have been found if data from both eyes of bel rev zebrafish had been included, demonstrating the presence of monocular (independent) saccades characteristic of achiasmatic mammals (Dell'Osso and Williams, 1995).
In addition, the mechanistic model generated to replicate the bel rev behaviors (Fig 6) was not based upon existing oculomotor models nor did it integrate the known anatomical connections and neuronal signaling properties of the oculomotor system (Büttner-Ennever, 2005). Although not a requirement, a biological based model could have provided a more general understanding of how defects in neural circuits yield alterations in eye movements, lending additional support to the utility of comparing this zebrafish phenotype with human pathology.
Beck JC, Gilland E, Baker R, Tank DW (2004a) Instrumentation for measuring oculomotor performance and plasticity in larval organisms. Methods Cell Biol 76:385-413.
Beck JC, Gilland E, Tank DW, Baker R (2004b) Quantifying the ontogeny of optokinetic and vestibuloocular behaviors in zebrafish, medaka, and goldfish. J Neurophysiol 92:3546-3561.
Büttner-Ennever JA (2005) Neuroanatomy of the oculomotor system. New York: Elsevier.
Dell'Osso LF, Williams RW (1995) Ocular motor abnormalities in achiasmatic mutant Belgian sheepdogs: unyoked eye movements in a mammal. Vision Res 35:109-116.
Huang Y-Y, Rinner O, Hedinger P, Liu S-C, Neuhauss SCF (2006) Oculomotor Instabilities in Zebrafish Mutant belladonna: A Behavioral Model for Congenital Nystagmus Caused by Axonal Misrouting. J Neurosci 26:9873-9880.
Leigh RJ, Zee DS (2006) The neurology of eye movements. New York: Oxford University Press.
Mensh BD, Aksay E, Lee DD, Seung HS, Tank DW (2004) Spontaneous eye movements in goldfish: oculomotor integrator performance, plasticity, and dependence on visual feedback. Vision Res 44:711-726.
Re: An oculomotor phenotype in question. 7 May 2007 
Stephan C.F. Neuhauss,
Professor
University of Zurich, CH - 8057 Zurich,
Ying-Yu Huang, Patrik HedingerSend letter to journal:
Re: Re: An oculomotor phenotype in question.
stephan.neuhauss{at}zool.uzh.ch Stephan C.F. Neuhauss, et al.
Achiasmatic zebrafish mutant belladonna as a model for congenital nystagmus in humans: Reply to Beck.
Recently, we argued that the spontaneous oscillations observed in achiasmatic zebrafish mutant belladonna (bel rev) may be a valuable animal model for congenital nystagmus (CN) in humans (Huang et al., 2006). Beck calls this conclusion into question owing to apparent shortcomings in methodology and interpretation. We want to take the opportunity to respond and clarify the raised issues.
The unusually low peak saccade velocity reported in Figure 3C can be attributed to two factors: the “smoothing-effect” of digital sampling and the application of a Gaussian filter to remove noise from the eye position data. A sampling rate of 12.5 Hz reduces the true peak saccade velocity on average by a factor of 0.7 (as estimated based on the saccade velocity profiles presented by Mensh [2004 et al., Figure 2C bottom, 8B], scaled such that the velocity-amplitude ratio was 12 1/s [Beck et al., 2004, Figure 5B]). Gaussian filtering that was originally optimized for the analysis of slow phases additionally decreased the peak saccade velocity by a factor of 0.3. Thus, the mean saccade amplitude of the data presented in Figure 3C of 6 deg corresponds to a true peak saccade velocity of 72 deg/s (6 deg * 12 1/s), but was scaled down to 15 deg/s (6 deg * 12 1/sec * 0.7 * 0.3).
Yet, this reduction is unlikely to have distorted our ANOVA-based comparison among wt, bel fwd, and bel rev due to its linearity. This was further confirmed through reanalysis of the data using the slope of raw peak saccade velocity vs. saccadic amplitude. Also here, no significant differences in saccadic performance were found. Even if such differences emerge at higher sampling rates, they will be subtle at best. Taken together, the potential impairment of motor performance under methylcellulose restraint in is small and will have a similar effect on wildtype and mutant larvae.
The fact that congenital nystagmus (CN) can persist in darkness does not weaken the link between the vision-dependent spontaneous oscillations of bel rev and human CN. Observations in patients indicate that CN persists at a lower rate in the dark or dies away (personal communication of Drs. David Zee and Dominik Straumann), suggesting that vision plays a key role also in human CN.
The type of spontaneous oscillations that are highly similar to human periodic alternating nystagmus (PAN) are described here in some detail to address the doubts raised by Beck. When presented a still grating, a significant number of bel rev show alternations between left-beating (large subsequent saccades toward left side) and right-beating oscillations (large subsequent saccades toward right side) frequently interrupted by “quiet” periods that are characterized by slow drifts with few smaller saccades, or no saccades, in either direction, or slow pendular motion without saccades. The eye position and velocity traces of these fish possess such a striking resemblance to those presented in Shallow-Hoffmann et al. (1999) that, in our view, there is no doubt about the occurrence of PAN in some bel rev.
Eye movements of bel rev are always conjugate. The lack of monocular saccades does not diminish the value of bel rev as a model of human CN in any way because monocular saccades are neither a feature of human CN in general (Abadi & Workfolk, 1989), nor of CN in achiasmatic humans in particular (Dell’Osso et al., 1998).
When building our model (Figure 6, supplemental materials), we were aware of existing highly complex and comprehensive oculomotor models that, to some extent, integrate known anatomical connections and neuronal signaling patterns (e.g., Jacobs & Dell’Osso, 2004). However, instead of creating a comprehensive and neurophysiologicaly accurate model of the OKR, it was sufficient, if not to say advantageous, for our purpose to derive a model that replicated the behavior of the normal OKR and to test how this behavior was altered if the sign of the retinal slip velocity input was reversed. This was to support our hypothesis that the oculomotor pathology of bel rev (reversed OKR, spontaneous oscillations) is caused by the ipsilateral projection of the RGC via a sign-reversed retinal slip velocity input to the optokinetic system. To put it differently, we did not attempt to explain in general how defects in neural circuits alter eye movements, but how the specific defect of the RGC misprojection does so. Our approach turned out to be quite successful as our parsimonious model was able to generate reversed OKR and the waveform characteristics of the major types of spontaneous oscillations we observed in bel rev, including left-beating, right beating, and bidirectional jerk nystagmus (see Dell’Osso & Daroff, 1975).
Ying-Yu Huang, Patrik Hedinger & Stephan C.F. Neuhauss Institute of Zoology University of Zürich CH – 8057 Zürich Switzerland
References
Abadi RV, Worfolk R (1989) Retinal slip velocities in congenital nystagmus. Vision Res 29:195–205.
Beck JC, Gilland E, Tank DW, Baker R (2004) Quantifying the ontogeny of optokinetic and vestibuloocular behaviors in zebrafish, medaka, and goldfish. J Neurophysiol 92:3546–3561.
Dell’osso LF, Daroff RB (1975) Congenital nystagmus waveforms and foveation strategy. Doc Ophthalmol 39:155–182.
Dell’Osso LF, Williams RW, Jacobs JB, Erchul DM (1998) The congenital and see-saw nystagmus in the prototypical achiasma of canines: comparison to the human achiasmatic prototype. Vision Res 38:1629–1641.
Huang YY, Rinner O, Hedinger P, Liu SC, Neuhauss SCF (2006) Oculomotor instabilities in zebrafish mutant belladonna: a behavioral model for congenital nystagmus caused by axonal misrouting. J Neurosci 26: 9873–9880.
Jacobs JB, Dell’Osso LF (2004) Congenital nystagmus: hypotheses for its genesis and complex waveforms within a behavioral ocular motor system model. J Vis 4:604–625.
Mensh BD, Aksay E, Lee DD, Seung HS, Tank DW (2004) Spontaneous eye movements in goldfish: oculomotor integrator performance, plasticity, and dependence on visual feedback. Vision Res 44:711–726.
Shallo-Hoffmann J, Faldon M, Tusa RJ (1999) The incidence and waveform characteristics of periodic alternating nystagmus in congenital nystagmus. Invest Ophthalmol Vis Sci 40:2546–2553.
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