Visually evoked eye movements in the mouse (Mus musculus)

Vision Rrs.

Vol.

16.

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VISUALLY

PergamonPress1976. PnntedI"GreatBt~tsn~

EVOKED EYE MOVEMENTS (MUS MUSCULUS) J. C.

MITCHINER,

IN THE MOUSE

L. H. Pnun, and J. W. VANABLE,JR.

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907. U.S.A. (Received 26 September 1975; in revised form 26 January 1976)

AImtract-Visually evoked eye movements were observed and measured in one inbred strain of the mouse (C57BL/6J) and in one inter-strain hybrid. This was done by observing pupillary movements while a mouse was restrained inside of a teat apparatus that presented the mouse with slowly moving stripes and allowed the experimenter to view the mouse’s pupil using ir. iIlumination. Slow eye movements (smooth pursuits) occurred only in the direction of drum rotation. Rapid eye movements (saccades) occurred only in the opposite direction. The eye position often appeared to follow a fixed location on the drum while the direction of drum rotation was being reversed. Eye movements did not occur when the stripes were rotated in the dark. Mutants having retina1 degeneration (C57BL/6J rd le/rd le) bad oscillatory eye movements independent of visual stimulus luminance or motion. Hybrid animals performed more frequent following movements than inbred animals. It is concluded that M. muscuhcs has visually-evoked eye movements, including the optokinetic nystagmus, and that the test described is suited to screen mice for subtle hereditary defects in vision. Key B’ords-optokinetic;

eye motions; mutants; mouse (Mus musctdus).

of vertical stripes that rotated in the horizontal plane while providing the experimenter with a “transparent” enclosure through which the mouse’s eye could be viewed. The side consisted of a continuous sheet of inexpensive polarizing material (HN32, Polaroid Co., Cambridge, Mass.) that was oriented in one direction, overlain at regular intervals by thin stripes of identical material oriented in the perpendicular direction. These stripes subtended 10” and appeared dark in visible light, but since the material from which they were made is a poor polarizer of i.r. wavelengths, the stripes were transparent to i.r. illumination. To illuminate the stripes evenly with visible light, a pair of fluorescent lamps was placed behind an opal glass window outside of the drum (subtense 150”). The drum was turned with a reversible motor. Air currents from the drum rotation were blocked with a stationary clear plastic barrier between the mouse and the wall. The mouse’s pupil was observed through an i.r. microscope, fabricated from an existing stereomicroscope that had eyepieces with parallel optical axes (American Optical Cycloptic). Surplus grade image converters (type 6914, Varo, Inc., Waco, Texas) were positioned with their pbotocathodes 3.0cm from the end of the eyepiece tubes. Standard 10x eyepieces, spaced 1 cm above their usual fully inserted position, provided nearly parfocal operation. Working distance was increased by using a 0.5 x objective lens. The images formed on the phosphor screens of the image converters were viewed with 6 x comparator lenses (No. 30325, Edmund Scientific, Barrington, N.J.). To measure distances, a reticle was placed on top of the phosphor screen of one image converter. Image converters were enclosed in a plastic housing that was painted black, and powered by a surplus grade power supply (15 kV, 100 PA). Infrared illumination of the mouse eye was provided by a 60W microscope illuminator equipped with a filter that passed wavelengths longer than 850 nm (Wratten 87C). The illuminator was placed 15 cm from the mouse’s eye. METHODS We were able to observe the mouse’s pupil during the The apparatus is a modified optokinetic drum. A mouse test with only i.r. light. The mice always closed their eyes was restrained (see Fig. 1) and placed in a drum with an when ordinary light was made bright enough to allow the immobile bottom (see Fig. 2). The rotatable side of the pupil to be seen with a stereomicroscope. The use of i.r. drum was designed to pr&ide the mouse with a pattern gave an additional benefit. Pupillary diameter could be 1169

Mammalian vision requires eye movements to ensure that the image of the object being viewed falls upon the retina. Visual targets that move continuously across the visual field in a given direction elicit an optokinetic nystagmus, which consists of two component eye movements: slow smooth pursuit movements occur during target tracking; rapid saccades in the opposite direction interleave the smooth pursuits (Alpern, 1972). The pathways for controlling each component movement are not completely known, but are probably somewhat different (Ashoff, 1974). One approach to studying these pathways employs single gene mutations that affect the visual system. The mouse (Mus musculus) is the only mammal with eye movements for which there exists extensive genetic information and a wide range of neurological mutants (see Sidman, Green and Appel, 1965). Optokinetic nystagmus has not yet been reported for “normal” M. musculus, probably because of the small size of the eye, dark pigmentation of the iris and spherical cornea. The closest genus upon which optokinetic nystagmus has been measured is Peromyscus (Vestal and King, 1968; King and Vestal, 1974). These measurements took advantage of the non-spherical curvature of the cornea of Peromyxuq which causes the reflection of a light from the surface of the cornea to appear to move as the eye moves. Unfortunately, reflections from the cornea of M. musculus remain stationary as the eye moves, which necessitated viewing the pupil in order to measure the optokinetic nystagrnus.

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MITCHI~ER.

L H. PINTOand J

measured m the dark. with low luminance (0.7gcdjml. with the starting filaments of the fluorescent lamps burnmg). and with high luminance (73cd’m’. with the fluorescent lamps running). Luminance measurements were made with a Macbeth Illuminometer (Leeds and Northrup Co.. Philadelphla. Pa.). The experImenta procedure was modified after that of Hayes and Ireland (1969). The restrained mouse was placed m the bottom of the drum and kept in darkness for a few set while the experimenter brought the eye mto focus. The fluorescent lamps were then started by applying starting current and then runnmg current. Pupillary diameter was measured m darkness and for both lamp currents. Thirty set after starting current was applied. drum rotation was begun and observations of eye movements began. After 30sec the direction of rotation was reversed. Again after 3Osec of rotation in the new direction the drum rotation was reversed and the drum was rotated in the initial direction for a final 30 set interval. The initial direction of rotation was set according to a random number table. The velocity of rotation for best responses was 5-45 deg’sec. The reversals of rotation took about 1 set each. The number of degrees of rotation of the globe that yielded I mm of pupillary motion was estimated by taking advantage of the unity angular magnification of the eye and assuming that the angular velocity of the eye equalled the angular velocity of the drum. The linear velocity of the pupil movement was measured for each of four angular velocities of drum rotation between 3.7 and I8 deg/sec for one mouse. The ratio of deg/sec to mmjsec was used to compute the desired estimated factor. the mean value of which was 56.5 degjmm (range 34.1-68.4 de&mm). The temperature of the test room varied between 21 and 22’C. but the animals were kept warmer by the i.r. illuminator inside of the drum. Testing was done between 2000 and 0200 hr. Test animals had to weigh about 9.Og in order to have eyes large enough to be seen reliably, but most animals were greater than 6 weeks old and weighed over 15 g. Altogether 36 animals were tested: 15 from the inbred strain C57BLj6J. fwo from the inbred strain degeneration 10 mutants with retinal C3H;HeJ. (C57BL/6J rd kit-d le) and 9 hybrids, B6C3HN3F2. No differences were observed between responses of male and female animals or between young (6 weeks) and old (I yr) mice.

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IAVABLL.

JK

reversing the direction of drum rotation every 30 sec. Doing this enabled the experimenter to check two additional features of the eye movements. If the anlma1 actually tracked the moving stripes. then smooth pursuit movements should always occur In the direction of drum rotation. This was observed. If the anlmal happened to be performing a smooth pursuit movement during drum reversal. then the movement

of the eyes should have had the same time course as that of the drum: gradual deceleration. stop. and gradual acceleration in the opposite direction. We observed eye movements following the drum reversal about ZSP, of the time for C57BL,6J animals. T.he maximum size of this movement was sometimes 50 The mean number of OKNs in each 30 set interlal of drum rotation is plotted in Fig. 3. It can be seen that C57BL/6J animals performed OKNs throughout the three intervals shown (Fig. 3a). However. animals with retinal degeneration (C57BL!6J rd kvrd Ir) performed no OKNs whatsoever. About half of these animals gave only occasional small oscillations In pupil position (< 7’, about ZHz) and slower oscillations in diameter. In addition, their pupillary reflexes were abnormal. Low luminance (0.78cd/m’) never caused constriction to less than 7.5”,,of maximal pupillary diameter. High luminance (73 cd/m’) sometimes caused pupillary constriction to half maximal diameter, but the constriction was sluggish and took between 10 and 60 set to occur. These general observations were repeated with two animals of the inbred

INITIAL 4

3 OMN

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NUMSER AND DIRECTION ; o

RESULTS

3

The qualitative

behavior of mice from the inbred strain C57BL/6J will be described first. In the dark, the pupil was dilated maximally (mean for eight animals, 2.05 mm dia). Low luminance (0.78 cd/m’) caused the pupil to constrict to about 65% of maximal diameter. High luminance (73 cd/m’) caused constriction to about 35% of maximal diameter. These constrictions occurred in about 1 sec. Movements of the small pupil were easy to see. The eye moved slowly in the direction of the drum’s rotation, presumably tracking the stripes (smooth pursuit motion). This slow movement proceeded for about 0.4mm (about 25”) before the rapid saccadic movement in the opposite dinction. Each pair of smooth pursuit and saccadic movements was called an optokinetic nystagmus (OKM). Animals from the C57BL/dJ inbred strain did not produce OKNs without interrup tion. The animals produced an average of at most three OKNs in a given 3Osec interval. If the drum was kept rotating continuously in one direction, the animals produced OKNs at a reduced rate after a few min. This lack of response was prevented by

RORII:

Fig. 3. Direction and mean numba of optokinetic nystagmus movements (OiCNs) for em& of three 3Qsec lest intervals. (a) Plot for 15 CS7W4.I animals. Standard errors are shown by small bars. (b) Plot for 10 animals with hereditary retinrrf -ation (C57BL/6J rd kjrd le).

Horizontal bar indicates that these mice were tested for the full 90 set and did not perform any OK%. only small oscillations in pupil dkirfWer and poaifion were noted. (c) Plot for nine hybrid animals (B6C3HN3FZ). (d) Plot of direction of drum rotation. Initial direction set randomly.

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Visually evoked eye movemenls in the mouse strain C3HiHeJ. which are also homozygous

for the rtl allele. Strong visually-evoked eye movements were elicited from hybrid animals. The particular hybrid used was derived from a stock used to maintain a recessive neurological mutation. joltirlg. for other purposes. These animals were derived from crosses between C57BLi6J and C3HiHeJ strains (designated B6C3HN3F3). and the individuals we used had no neurological mutant phenotypes. Over 50”” of these animals performed tracking of the drum rotation without interruption during the entire test. an observation that was rare for a C57BL/6J animal. However. the mean number of OKNs for these two groups was not significantly different (P > 0.05 in MannWhitney one tailed U-test). Although the OKNs of each group subtended the same number of degrees, those of the larger hybrids were easier to see. Hybrid animals also performed more following movements during drum reversal (over 70”“), and the size of these movements was larger, often exceeding 75’. Our test could not be used with animals with small eyes (for example the nlicropl&abnia mutant, C57BL/6J/jI/Ii;+) or with any albino animals. The eyes of the former were too small to see even with great magnification. and the pupils of the latter had too low contrast to be seen. DISCUSSION

The house mouse. Mus n~usculus, has visuallyevoked eye movements as indicated by the following evidence. (1) The position of the pupil was observed to move in a way that was consistent with tracking of the visual target. Slow (smooth pursuit) movements were observed only in the direction of drum rotation, and saccades were observed only in the opposite direction. (2) Systematic tracking movements did not occur when the drum was rotated in the dark. (3) Animals with retinal degeneration performed no tracking movements. Thus, our results expand those of Briickner (1951) who found small (15”-20”) spontaneous eye movements in the mouse. That the hybrid animals performed better than animals of either “parent” strain may indicate hybrid

vigor, which is also exhibited by their larger size, greater strength, and greater ability to escape handling.

The test takes about 5 min to perform. is non-destructive. is repeatable and is reliable. The test enables us to distinguish animals that have hereditary retinal degeneration from those that do not. This test is suitable for screening mutant mice that have functional deficits of the visual system without gross anatomical abnormalities. A similar approach has proved to be of great utility in the genetic dissection or the visual system of Drosopllila (Hotta and Benzer. 1969: Pak. Grossfield and White. 1969: Pak. 1974). and perhaps it can now be estended to the analysis of the vertebrate visual system. Acktlo,~/r~lycmc,tlrs-We thank Drs. John A. King and Martin Heisenbeq for their help and encouragement. and Drs. U. C. Drleer. J. A. King and A. L. Pearlman for reading the manuscript. Special thanks is also extended to Mr. Evan Hinds for construction or the apparatus. Supported by NIH grant EY01211-01. REFERENCES

Alpern M. (1972) Movements of the eyes. In TII~ Eye. Vol. III, Muscular A4echanisms (Edited by Davson H.). Academic Press, New York. Ashoff J. (1974) Reconsideration of the oculomotor pathway. In ?-he Neurosciences Third Srudy Progranr (Edited by Schmidt F. 0. and Worden F. G.). pp. 305-310. M.I.T. Press. Briickner R. (1951) Spaltlampenmikroscopie und Ophthalmoskopie am Auge von Ratte und Maus. Documenru ophth. 5-6, 452-554. Hayes W. N. and Ireland L. C. (1969) Optokinetic response of the guinea pig. J. camp. physiol. Psycho/. 68, 199-202. Hotta Y. and Benzer S. (1969) Abnormal electroretinograms in visual mutants of Drosophila. Noture, Land. 222, 354-356.

King J. A. and Vestal B. M. (1974) Visual acuity of Perontyscus. J. Marmnl. 55. 238-243. Pak W. L. (1974) Mutations affecting the vision of Drosophila nwlanogasrer. In Horldhook qf Generics (Edited b! King R. C.). Vol. 3. pp. 703-733. Plenum Press. New York. Pak W. L., Grossfield J. and White N. V. (1969) Nonphototactic mutants in a study of vision of Drosophila. Nature, Lortd. 222, 351-354. Sidman R. L., Green M. C. and Appel S. H. (1965) Cat&g oftlIe Neurological Muranrs ofthe Mouse. Harvard Univ. Press. Cambridge. Vestal B. M. and King J. A. (1968) Relationship of age at eye opening to first optokinetic response in deermice (Perom~scus). Deoel. Ps.vcltobio/. 1, 30-34.

Fig. 1. Photograph of restraint. Note the “noose” made of solid. insulated 18AWG copper wire. \vhich limits head motions, For smaller mice. additional top and side pads made of cardhoard were employed. Scratching the ventral surface of the tail often caused the mouse to open its eyes.

Fig. 2. Schematic diagram of test apparatus. The mouse was placed in the restraint (R) facing an opal glass screen behind which was a pair of fluorescent lamps. This glass. the white screen and another attached to the microscope (not shown) provided a relatively uniform background illumination that allowed light to be seen between the strips of crossed Polaroid (dark). The source (Sl) and i.r. filter (Fl) illuminated the mouse with wavelengths longer than 850nm. The half power objective (Ll) and slightly unseated eyepieces (L2) cast an inverted i.r. image of the mouse’s eye upon the photocathode of the image converter (PC). The erect image was viewed on the phosphor screen (P) with a 6x eyepiece (L3). The drum was 23 cm dia and the mouse eye was placed in its middle, Both dark and light stripes were 11.5 cm from the mouse eye and subtended IO”. uacirlg pap

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