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loss of cell bodies in the gangfion layer induced by chronic section ... Ninety and 270 days following optic nerve section the ganglion cell layer of the side.
Documenta Oph thalmologica 61, 4 1 - 4 7 (1985). 9 Dr W. Junk Publishers, Dordreeht. Printed in the Netherlands.

T h e p i g e o n p a t t e r n e l e c t r o r e t i n o g r a m is n o t a f f e c t e d b y massive loss o f cell b o d i e s in the gangfion l a y e r i n d u c e d b y c h r o n i c s e c t i o n of the optic nerve

V. PORCIATTP, W. FRANCESCONI2 and P. BAGNOLI~ 1Divisione Oculistica USL 13, Livorno, Istituto di Fisiologia deU'Universitfie (Correspondence address) Istituto di Neurofisiologia del CNR, Via S. Zeno 31, 56100 Pisa, Italy

Key words: alternating gratings, electroretinogram, ganglion cells, pigeon Abstract. In pigeons, electroretinographic responses to contrast reversal of sinusoidal gratings (pattern ERGs) were recorded before and after section of the left optic nerve. Ninety and 270 days following optic nerve section the ganglion cell layer of the side that underwent the surgical procedure showed an 80% reduction in the number of cell bodies as compared with the intact side. At these times the pattern ERGs showed comparable iamplitudes in both eyes. There is a possibility that the inne} nuclear layer of the pigeon retina plays a major role in the generation of the pattern ERG.

Introduction

In cats, monkeys and humans the electroretinogram in response to contrastreversal stimuli (pattern ERG) is thought by many authors to be related to ganglion cell activity. Indeed the pattern ERG is absent in cats and monkeys, after degeneration of ganglion cells, induced by a chronic section of the optic nerve whereas the flash or flicker ERG remains unaltered (Holl~inder et al., 1984; Maffei and Fiorentini, 1981, 1982; Maffei et al., 1985). Moreover, in human patients the pattern ERG, in contrast to the flash ERG, is affected by dysfunction of retinal ganglion cells (Fiorentini et al., 1981; Porciatti and Von Berger, 1985). The pattern ERG is also recordable from the pigeon eye. This response depends on temporal frequency of reversal, contrast, spatial frequency, and area of the stimulus (Bagnoli et al., 1984; Holden and Vaegan, 1982, 1983). Holden and Vaegan (1982, 1983) demonstrated that the intraretinally recorded pattern ERG and flash evoked b-wave showed a similar depth profile of amplitude and polarity, thus suggesting common generators for both types of responses. In the present study the role of ganglion cells in the generation of the pattern ERG in pigeon was investigated. Pattern ERG responses were recorded before and after section of the optic nerve, which causes retrograde degeneration of avian ganglion ceils (Muchnik and Hibbard, 1980; Ehrlich, 1981). Histological preparations of whole mounted retinas were performed in order to assess the degree of retrograde alterations in the ganglion cell layer.

42 Methods Experiments were performed on four adult pigeons (Columba livia) which had a chronic section of the left optic nerve. The animal was mounted in a stereotaxic apparatus and anesthetized with Equithesine. The upper eyelid was cut near the orbit, the ocular bulb was gently displaced, and with microscopic control and sterile technique the optic nerve was cut with a razor blade. The section was consistently made approximately 2 mm behind the eyeball and thus did not affect the pecteneal artery (Wood, 1917; Wingstrand and Munk, 1965). The animals were allowed to recover from anesthesia and were put back into their cages. The recording sessions were performed at different times during the first 270 days after the optic nerve section.

Electrophysiologic techniques The pigeons were anesthetized with Equithesine and mounted in a stereotaxic apparatus. The pupils were dilated with local application of D-tubocurarine chloride to a diameter of approximately 6 ram. Refraction was measured by retinoscopy and corrected when necessary with additional lenses which focused the eye at a distance of 57 cm. The ERG was recorded monocularly by using stainless steel needles inserted into the upper eyelid. The eyelid contralateral to the stimulated eye was used as reference in order to minimize the noise coming from brain and muscle activity. The central forehead was grounded.

Visual stimulation The pecten was projected by inverted ophthalmoscopy on a tangent screen 57 cm from the eye. The screen was then replaced by a circular display positioned in such a way as to center the fovea centralis of the yellow field. Visual stimuli were produced on a 27-inch TV monitor (50 Hz) peripherally masked by white cardboard to obtain a 32-cm diameter field stimulation. The room light was adjusted in such a way that the luminance of the TV screen and the surrounding cardboard were approximately the same (24 cd/m 2). Sinusoidal gratings of various spatial frequencies (0.2-4 c/deg) and fixed contrast (37%) were generated on the display and shifted in spatial phase by 180 ~ (pattern reversal) at 8.33 Hz. Light flashes were generated by a strobe lamp. The ERG responses were filtered by a band-pass between 1.5 and 65 Hz (6 dB/oct) and amplified 50 000-fold. The responses were averaged by a SAN-EI T08A signal processor and recorded by an X-Y plotter. Usually 256 repetitions for each average were sufficient to obtain clear.responses. Under these experimental conditions the ERG had an approximately sinusoidal waveform, with a temporal period of 120msec, corresponding to the second harmonic of the stimulus temporal frequency. The ERG amplitude was a function of the spatial frequency of the grating, with maximal values at 0.25-0.5 c/deg and a high cut-off at about 4 c/deg (Bagnoli et al., 1984).

43 We excluded possible artifact potentials induced by the stimulating set; no responses above noise were recorded with the pattern defocused by a + 25diopter lens placed in front of the stimulated eye.

Histologie techniques The eyes of the four pigeons with the left optic nerve sectioned were used for retinal mounts, 90 days (two cases) and 270 days (two cases) after the operation. The animals were killed by decapitation and the ocular bulbs were quickly removed; a circular cut was placed along the ora serrata to remove the cornea, iris, and lens. The vitreous humor was carefully sucked from the posterior portion of the eye ball, which was repeatedly washed in physiological solution. The retina was dissected from the pigment layer by a fine brush. The pecten was cut at the base and its superior angle was taken as reference to identify the laterally located fovea centralis. The histologic procedure was performed according to previous reports (Hughes, 1975; Hebel, 1976). Briefly, the retina was mounted on a gelatinized slide, covered with a filter paper flattened by a slight weight, and fixed overnight by immersion in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.3) at room temperature. Then the whole mounts were defatted and processed for cresyl violet staining. Results

Soon after section of the optic nerves (4 h), flash and pattern ERGs recorded from the operated eyes showed amplitudes and shapes comparable with those recorded from the unoperated eyes. This result was taken as evidence that the operations did not cause circulatory disturbances and ruled out a possible control of the centrifugal system on the ERG responses (Miles, 1975; Nye, 1968). In addition this result excluded that the ERG responses were overlaid by far-field potentials of central origin. In the four pigeons, pattern ERG responses were recorded 3 and 9 months following the optic nerve section, after which the birds were sacrificed for histologic retinal mounts. Figure 1 shows the electrophysiologic and histologic results obtained from one pigeon tested 270 days after the optic nerve section. Comparable results were obtained from the other three pigeons tested. Figure 1A shows the amplitudes of the pattern ERGs at different spatial frequencies evoked from the central yellow field of the operated (filled diamonds) and unoperated (open diamonds) eyes. It can be noticed that the pattern ERGs showed quite similar amplitudes in both eyes over the whole range of spatial frequency. The amplitude values were in the range of those reported for normal pigeons (Bagnoli et al., 1984). Figure 1B and C shows the ganglion cell layer of the central yellow field from whole mounted retinas of the unoperated (B) and operated (C) eyes. The high density of cells with small diameter shown in Figure 1B is typical of the central yellow field (Bingelli and Paule, 1959; Hayes and Holden, 1980).

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Figure 1. (A) Peak-to-trough amplitudes of the pattern ERG responses at different spatial frequencies, evoked by the stimulation of a retinal region including the fovea centratis (see insert). Open diamonds refer to unoperated eye. Filled diamonds refer to operated eye (270 days after a chronic section of the optic nerve); (B and C) wholemounted retinas showing ganglion cell layer of the central yellow field in unoperated (B) and operated (C) eye; RF: red field; (F) fovea centralis; P: pecten oculi; calibration bar: 50 ~m. The ganglion cell layer o f the operated side (Figure 1C) showed a drastic reduction (about 80%) in the number of cell bodies as compared with the unoperated side.

Discussion In agreement with previous results which showed massive degeneration of cell bodies in the ganglion cell layer of the quail (Muchnick and Hibbard, 1980) and chick retina (Ehrlich, 1981) following optic nerve section, our results demonstrate that 80% of cell bodies in the ganglion cell layer of the pigeon retina degenerate after axotomy. The cellular loss is o f the same order as that

45 reported for the quail and chick. The cells affected by axotomy were presumably ganglion cells. By light and electron microscopic observations cells that underwent degenerative changes after axotomy had previously been identified as ganglion cells (Muchnick and Hibbard, 1980) or presumed ganglion cells (Ehrlich, 1981). In addition, by retrograde transport of horseradish peroxidase from the optic tectum of the pigeon, it has been shown that 85% of the cells in the ganglion cell layer were true orthotopic ganglion cells (Hayes and Holden, 1980). The cells not affected by axotomy comprised about 20% of the total number in the central yellow field. No relationship has been reported between the survival of cells in the ganglion cell layer and fiber myelination in the optic nerve or optic fiber layer (Madison et al., 1984; Muchnick and Hibbard, 1980). The remaining cells might be identified as glial cells, displaced amacrine cells, intraretinal association neurons, ganglion cells which send collaterals within the retina, or surviving ganglion ceils which exhibited new intraretinal growth (Bingelli and Paule, 1969; Dawson and Liebermann, 1979; Ehrllch, 1981; Hayes and Holden, 1980; Madison et al., 1984; Muchnick and Hibbard, 1980). Our estimate of 20% differs from that of Bingelli and Paule (1969). These authors used the discrepancy between optic fiber counts and whole retinal counts to conclude that 43% of neuronal cells in the pigeon ganglion cell layer were displaced amacrine or intraretinal association neurons. The cellular loss in the pigeon ganglion cell layer was not equal across the retina. Indeed about 66% of cell bodies of the peripheral retina degenerated. In particular, large cells were resistant to axotomy (not shown in the results). This result agrees the findings of Muchnick and Hibbard (1980) who reported 30% remaining cells in the quail peripheral retina 1 month postaxotomy. Our study demonstrates that the pigeon pattern ERG is unaffected by massive cell degeneration in the ganglion cell layer induced by chronic section of the optic nerve. By contrast, transsection of the optic nerve in cats and monkeys led to disappearance of the pattern ERG (Holliinder et al., 1984; Maffei and Fiorentini, 1981, 1982; Maffei et al., 1985). In the same preparation, recent results reported a 30% ganglion cell loss in the cat temporal retina and a 20% mean diameter reduction in the remaining ganglion cells (Holl~inder et al., 1984). The discrepancy between our electrolchysiologic results and those obtained in the cat might be due to the different anatomic and functional organization of the avian and mammalian retina. The inner nuclear layer of the pigeon has a thickness of more than 10 cell bodies, whereas only three to four cell bodies can be found in the cat retina. It seems possible, therefore, that the pigeon inner nuclear layer plays a major role in the generation of the patternERG. This possibility is supported by recent results which demonstrated in pigeons that intraretinally recorded pattern ERG had maximal amplitude and reversed polarity in the inner nuclear layer (Holden and Vaegan, 1983). In

46 this layer, the pattern ERG might reflect, at least in part, the center-surround receptive field organization of the bipolar cells (Richter and Simon, 1975; Saito et al., 1979, 1981, 1982). Alternatively the pattern ERG might simply represent the sum of local luminance responses without regard to the spatial content of the stimulus (Spekreijse et al., 1973). On the other hand the possibility remains that degeneration-resistant cells in the ganglion layer may contribute to the generation of the pattern ERG.

Acknowledgements The authors wish to thank A. Bertini, G. Marengo, B. Margheritti, and A.M. Niccolai for their excellent technical assistance. Supported by the Consiglio Nazionale delle Ricerche (Grant No. 83. 00354.004).

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