Improved use of tapetal reflection for eye-position monitoring

Transcription

1 Improved use of tapetal reflection for eye-position monitoring John D. Pettigrew, Michael L. Cooper,* and Gary G. Blasdel** A new technique is described for eye-position monitoring in species with strong tapetal reflections. A fiber optic is used to introduce light into the eye, whose optics then produce an image of the fundus on a tangent in front of the animal. The technique simplifies heretofore tedious measurement of cylotorsional changes, as well as providing a very wide view of the fundus. It has been used successfully in conjunction with single-unit recording from the visual system. Key words: eye-position monitoring, retinal fundus, tapetal reflection I n neurophysiological approaches to visual phenomena involving fine resolution, residual eye movements present a significant technical barrier in the paralyzed preparation. These small drifts resist even the most stringent precautions taken to eliminate them 1 and assume a crucial role in some investigations. For example, single neuron approaches to stereoscopic vision require highly accurate (minutes of arc) information about the relative position of both eyes. One therefore has need of a sensitive eye monitoring system which is inexpensive, reliable, and simple to operate. One that can be used con- From the Beckman Laboratories of Behavioral Biology, California Institute of Technology, Pasadena. This work was supported by the Spencer Foundation, National Institutes of Health grants EY01909 and MH25852 to J. D. P., and a Weizmann Fellowship to G. G. B. M. L. C. held a National Science Foundation Predoctoral Fellowship. Submitted for publication Nov. 8, Reprint requests: J. D. Pettigrew, Beckman Laboratories, Division of Biology, California Institute of Technology. Pasadena, Calif * Present address: Section of Neuroanatomy, Yale University School of Medicine, New Haven, Conn. **Present address: Department of Ophthalmology, University of Washington Medical School, Seattle, Wash. currently with receptive field studies of single neurons has added advantages. The method which we present fills these criteria and has been used successfully in receptive field studies of both the domestic cat 9 and the bushbaby, Galago senegalensis (Allman, J. personal communication). Both species have strong tapetal reflections. Our method, which is based upon the one first described by Fernald and Chase, 2 simultaneously projects the entire tapetal retina rather than a small portion of it and, in addition, facilitates repeated determinations. The larger field of view conveys a number of advantages such as the ready estimation of cyclotorsional movements, whose measurement is normally a cumbersome but essential task in studies of binocular disparity. 3 ' 4 ' 9 Technique The experimental animals were prepared for electrophysiological recording according to methods which have been described in detail elsewhere. 5, 6 After inserting venous and tracheal cannulas under halothane anesthesia, we transferred the cats to a stereotaxic apparatus and paralyzed them with 40 mg of Flaxedil. The animals were anesthetized by force-hyperventilating them with a mixture of N 2 0: 0 2 : C0 2 (70% : 27.5% : 2.5%) and paralyzed with a continuous infusion of Flaxedil /79/ $00.60/ Assoc, for Res. in Vis. and Ophthal., Inc.

2 Volume 18 Number 5 Eye position by tapetal reflection 491 (5 mg/kg/hr) and d-tubocurarine (0.5 mg/kg/hr) in 0.3% saline-8% dextrose (4.3 ml/hr). 1 We used cyclopentolate HCl (Cyclogyl, Alcon) to dilate the pupils and phenylephrine HCl (Neosynephrine, Winthrop) to retract the nictitating membranes. Contact lenses of zero power protected the corneas, and a thermistor-controlled heating blanket maintained the animals' temperature at 37 C. We projected images of the ocular fundi onto a tangent screen which was placed 57 cm in front of the posterior nodal points of the animal's eyes. We did this by using a 3 mm fiber-optic conduit to conduct light from a SCR-controlled light source (Edmund Scientific) to a small section of rigid fiber optic (image conduit, Edmund Scientific) which has been bent at the end (Fig. 1). The 90 bend, which was made 4 mm from the tip, allowed the fiber optic to be brought in from the side and positioned very close to the cornea. Since the diameter of the fiber optic was smaller than that of the dilated pupil and since it was projected behind the near point of the eye, this arrangement did not disrupt the cat's visual field and in fact did not even cast a noticeable shadow on the tangent screen (see photograph). By moving the end of the fiber optic to various points in front of the cornea, we were able to project images from any part of the fundus. Light brought to the eye by the fiber optic was reflected off the ocular fundus and focused onto the tangent screen by the eye's own optics. We were able to refract each eye by fitting contact lenses of various curvatures until we obtained the sharpest image of chosen retinal landmarks on the tangent screen. Usually these landmarks were retinal blood vessels, but it should be noted that it is possible with this method to bring into optimal focus other retinal planes such as the plane of the tapetal fissures containing the vessels passing forward from the choroid. Since the fiber optic blocks so little of the visual field, we were able to position a fiber optic in front of each eye for the duration of our electrophysiological experiments. We usually did this by attaching the light pipe to the stereotaxic frame in such a way as to allow visualization of the optic disk and area centralis. In this way we were able to avoid errors which might derive from repositioning the fiber optic (see below). As one can see from the photograph in Fig. 2, our method projects a very wide area of fundus. It is usually possible to visualize a region covering 40 on the tangent screen. This is considerably more than one can achieve with the method of Fernald and Chase. 2 Through an appropriate se- Fig. 1. Fiber-optic projection system in use. The black rod is a rigid fiber-optic bundle with a 90 bend 4 mm from the polished end from which the retina is illuminated. The longer end of this rigid fiber optic was fitted to a flexible fiber optic which conducted light from a variable source. In practice, fiber optics were left in place in front of each eye to facilitate studies of binocular alignment. Only one fiber optic assembly is shown in the diagram. The diameter of the fiber optic is small in relation to the diameter of the dilated pupil. There is consequently no significant visual field obstruction (see Fig. 2). lection of contact lenses with proper curvature it thus becomes possible to obtain a detailed image of a wide extent of fundus on the tangent screen and to produce a detailed drawing of the fundus (Fig. 3). This image, of course, will have the same tangent distortions as the positions of the receptive fields for visual neurons, thereby making it possible to relate the receptive field positions to various retinal landmarks (on whole-mounts, for example). The sharp image provided by our method enables accurate estimation of the position of the area centralis which, in the cat, usually appears as a darker green region against the lighter yellowgreen of the tapetum. The convergence of small retinal blood vessels in this region aids in the plotting of the area centralis on the screen. The accuracy of such estimates of the position of the area centralis is to within 1, which has been confirmed by comparisons of tangent screen drawings with retinal whole-mounts. Errors If care is taken to maintain good optics, the light-pipe method can be used to monitor the residual eye movements occurring under pa-

3 492 Pettigrew, Cooper, and Blasdel Invest. Ophthalmol. Visual Sci. May 1979 Fig. 2. A, Image of the cat's left fundus projected with fiber-optic technique onto the screen where the experimenter is plotting the position of blood vessels. Note the wide area of fundus which is surveyed simultaneously. Part of the optic disk is visible in the upper corner near the experimenter's head; the area centralis is near the center of the picture above the experimenter's hand; the dark shadow at the bottom is caused by a flexible snake-arm attached to the stereotaxic apparatus. Kodachrome II, f4, 40 sec. Canon F - l with 35mm focal length lens. B, Retinal whole mount of a cat in which ganglion cells have b e e n stained for horseradish peroxidase retrogradely transported from the contralateral L G N. The area centralis, nasto temporal division, and blood vessel pattern are all clearly defined and can therefore be related to the situation which obtains in life by correlation with the blood vessel pattern observed by the use of the projection technique (A). More details reported by Cooper and Pettigrew. 9

4 Volume 18 Number 5 Eye position by tapetal reflection 493 ralysis. By drawing blood vessel intersections symmetrically placed around the area centralis, it is possible to obtain a baseline against which translation and torsion of the eye can be measured. 3 ' 4 ' 9 The accuracy with which such eye movements can be determined is limited by two factors. The first source of error here is the slight shifts in the position of the projected image due to errors in repositioning the fiber optic for successive measurements. This error is small, being a displacement of about 0.4 in the image for a movement of the light pipe from one edge of the cornea to the other. However, as mentioned above, this source of error can be eliminated by fixing the light pipe in front of the eyes for the duration of the recording session. These errors might assume greater significance in other situations (for example, if one were looking at eye movements in a restrained, unparalyzed preparation). The second source of inaccuracy has to do with the limit of resolution in the projected image itself. We believe that retinal blood vessel intersections can be plotted to within 0.1 by our method; this accuracy allows eye translation to be determined to within 0.2 and eyeball torsion to within about 0.3. If sufficient care is taken, the quality of the projected image of the fundus is usually good, as evidenced by the fact that we have been able to observe on the tangent screen the pulsations of retinal blood vessels in time with the heartbeat. Disadvantages One concern which should be mentioned with regard to our method is that the light levels which we use to project the fundus might cause an abnormal degree of retinal bleaching or even retinal damage. This strong adapting effect of the beam may be the chief disadvantage of the method. We have not measured these light levels in the eye directly; however, the amount of light used in the fiber-optic technique can be adjusted to a such level that the reflected image has the same brightness as that observed on the screen with the method of Fernald and Chase. 2 We have often recorded from striate Fig. 3. Drawing of the retinal blood vessels made during the experiment with the fiber-optic technique (upper) compared with a fresh retinal whole mount of the same retina (lower). The whole mount had been dissected free of the posterior globe just before the photograph was taken. During dissection the experimenter produced more tears than usual since the previous exposure to the fiber-optic beam tends to lead to a more adherent retina. However, note that corresponding blood vessels can be traced in both pictures and can therefore be used to relate anatomical landmarks to those plotted on the tangent screen. The retinal whole mount was photographed with combined light and dark optics and the print has been reversed to facilitate comparison with the tangent screen drawing. cortical neurons while illuminating the fundus with the fiber optic; although we have noticed a brief loss of responsiveness attributable to retinal bleaching, this responsiveness usually returns within 1 or 2 min of extinguishing the light. This did not prove to

5 494 Pettigrew, Cooper, and Blasdel Invest. Ophthalmol. Visual Sci. May 1979 be a problem in any of our cortical experiments, where up to 30 estimations of eye position (one after plotting each binocular unit) were made over the course of as many hours, without any noticeable lasting effect on responsiveness besides the 1 to 2 min of reduced sensitivity just after a bleach. In addition, we have noticed no permanent decrease in cortical responsiveness due to retinal damage over the course of 2-day experiments in which the fiber optic was used repeatedly. In one cat, prolonged use of the highest light levels did lead to a darkening in the horizontal streak area of the fundus image by the second day of the experiment. However, even in this cat, cortical responsiveness did not seem to be affected. However, we do have the impression that the retinas of cats which have undergone prolonged exposure to the beam are more difficult to dissect from the posterior globe at postmortem examination. Advantages We believe that the method we have described has wide usefulness in visual studies of species with retinal tapeta, such as the cat and the bushbaby. The equipment required is cheap, readily obtainable, easy to put together, and reliable. The output can be directly related to receptive field estimations and visual stimuli, since all use the same screen. In addition to the greater ease of defining the vessel-free area centralis during an experiment, the method allows a detailed drawing of the retinal blood vessels to be made which can later be used to estimate the area centralis more precisely from a retinal wholemount stained for ganglion cells (see above in Fig. 3). The fine detail resolved also simplifies refraction, since an appropriate contact lens curvature or correcting lens can be chosen to bring into sharp focus the desired feature and retinal area in the projected image. In other words, some of the uncertainties of retinoscopy which are related to the site of the reflection can be avoided in the present case because the investigator can choose for himself the retinal plane which shall be brought into sharpest focus (e.g., just in front of the tapetal fissures or just behind the retinal blood vessels). It is also reassuring to be able, so directly, to verify the optical state of the eye at such a wide range of eccentricities during an experiment without uncertainties about the effects of the intervening optics of another instrument. The most notable feature of the method is the ease and accuracy with which it facilitates heretofore tedious monitoring of changes in eye position, particularly the tiny drifts and cyclotorsional movements of such crucial significance in studies of binocular disparity. A flick of a switch provides an immediate picture of any translational or cyclotorsional changes, which can therefore be virtually simultaneous with a receptive field plot. Comparison with other techniques It may help to provide a brief comparison of our technique with others used to monitor eye position in neurophysiological experiments. These include the fundus camera technique developed by Bishop's group, 3 4 ' the light-lever technique with a corneal mirror, 1 the reference cell technique, 7 and the methods preceding ours which also utilize the fundal reflection and the animal's own optics to produce an image on the same screen used for receptive field plotting. lj 8 Of all these techniques, the one most comparable with ours is the fundus camera technique, which shares most advantages, including a wide view and the ability to handle cyclotorsion. The chief disadvantages of the fundus camera approach are its cost and the complications introduced by intervening optics (such as the reverse projection beam) for which careful alignment or calibration is necessary. All the other methods share the disadvantage that they do not lend themselves readily to estimations of cyclotorsion. Although a reference cell which was very sharply tuned for orientation might theoretically be used to estimate cyclotorsion, this method 7 would still have the weakness that it hinges on the assumption that the receptive field properties of the reference cell are not altered by the experimental variables under investigation. In addition, this technique is experimen-

6 Volume 18 Number 5 Eije position hy tapetal reflection 495 tally quite demanding, since movement of the second electrode to search for units frequently results in a loss of stability at the first electrode where the reference cell is being recorded. Of all techniques we believe that the current technique and the light-lever method have the greatest accuracy because with either method, each estimation requires but a single judgment by the experimenter, with no manipulations once the beam has been put in place. REFERENCES 1. Rodieck, R.W., Pettigrew, J.D., Nikara, T., and Bishop, P.O.: Residual eye-movements in receptive field studies of paralysed cats, Vision Res. 7:107, Fernald, R., and Chase, R.: An improved method for plotting retinal landmarks and focusing the eyes. Vision Res. 11:95, Nelson, J.I., Kato, H., and Bishop, P.O.: Discrimination of orientation and position disparities hy binocularly activated neurons in cat striate cortex, J. Neurophysiol. 40:260, van der Heydt, R., Adorjani, Cs., Hanny, P., and Baumgarten, G.: Disparity sensitivity and receptive field incongruity of units in the cat striate cortex, Exp. Brain Res. 31:523, Nikara, T., Bishop, P.O., and Pettigrew, J.D.: Analysis of retinal correspondence hy studying receptive fields of binocular single units in cat striate cortex, Exp. Brain Res. 6:353, Pettigrew, J.D.: The effects of visual experience on the development of stimulus specificity by kitten cortical neurones, J. Physiol. (Loud.) 237: 49, Hubel, D.H., and Wiesel, T.N.: Cortical and callosal connections concerned with the vertical meridian of visual fields in the cat. J. Neurophysiol. 30:1561, Barlow, H.B., Blakemore, C, and Van Sluyters, R.C.: Measurements of residual eye movements during the analysis of disparity of receptive fields of visual neurons, J. Physiol. (Loud.) 242:38P, Cooper, M.L., and Pettigrew, J.D.: A neurophysiological determination of the vertical horopter in the cat and owl. J Com p. Neurol. 184:1, 1979.