Central neurophysiological correlates of constant light-induced retinal degeneration

EXPERIMENTAL

NEUROLOGY

63, 5-O-75 (1979)

Central Neurophysiological Correlates of Constant Light-Induced Retinal Degeneration VANCE LEMMON

AND KENNETH V. ANDERSON’

Department of Anatomy, Emory University, Atlanta, Georgia 30322 Received June 30, 1978 Single-unit electrophysiological experiments were conducted on albino rats housed in either cyclic light or constant light for as long as 24 weeks. Histological examination of the retinas of the constant light-exposed rats confirmed that there was a massive degeneration of the photoreceptor cells. Despite this degeneration, visually responsive cells were found in the lateral geniculate nucleus (LGN) and the visual cortex (VC) at all exposure times. With increasing exposure times, we found that cells in the LGN and VC had longer response latencies and less vigorous responses and that it was necessary to illuminate larger areas of the visual field in order to produce responses. Studies of LGN cells in control and experimental rats showed that the spectral sensitivities of those cells were similar in shape and peak sensitivity to the spectral absorption properties of rhodopsin. In view of this fact, it seems most likely that rhodopsin is the primary light-transducing molecule in the retina of rats exposed to long-term constant light. The spectral sensitivity curves of some cells in control animals and light-exposed animals had minor peaks at 600 nm, which may have been due to some additional visual pigment molecule.

INTRODUCTION Photoreceptors in albino rats undergo an irreversible degeneration as a result of exposure to continuous illumination. Despite this degeneration, behavioral studies showed (2,3,35) that rats exposed to constant light can perform visually guided tasks remarkably well. Because of those studies Abbreviations: LGN-lateral geniculate nucleus; VC-visual cortex; OT-optic tract; PSTH-poststimulus time histogram; P cells-principal cells; I cells-intemeurons; ONL, INL-outer, inner nuclear layer. 1 The present address of Dr. Lemmon is Department of Physiology and Biophysics, Washington University, St. Louis, MO 63110. The excellent technical assistance of D.A. Prince is gratefully acknowledged. This research was supported, in part, by a grant from the Thompson Fund and National Institutes of Health grant EY 01%7.

50 0014-4886/79/010050-26$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

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there has been a continuing controversy as to the type of cell serving to transduce light in the retinas of constant light-exposed rats. Recently it was suggested that residual cones may be serving this role (6, 14). Whereas there have been numerous studies of the electroretinograms of constant light-exposed rats, there have been only two investigations of visually evoked responses recorded from the central nervous system of lightexposed rats (23, 26). In both those studies, visually evoked potentials were found in the visual cortex after exposure to constant light had produced a massive degeneration of photoreceptors. In a companion paper on the behavioral capabilities of rats exposed to long-term constant light (15), we reported that rats exposed to 24 weeks of constant light performed a pattern discrimination poorly, although they performed intensity discriminations and color discriminations quite well. The object of the present work was to examine the response properties of cells in the lateral geniculate nucleus (LGN) and the visual cortex (VC) of light-exposed rats. This was done to compare in light-exposed rats the response properties of visual cells with behaviorally determined visual capabilities. In addition, the spectral sensitivities of LGN cells were examined to determine if cones were likely to be the principal lighttransducing element in rats with constant light-induced degeneration. METHODS Neurophysiologic Techniques. The lighting conditions used were described in detail elsewhere (15). The constant light-exposed rats were exposed to 440 lux of continuous illumination. Neurophysiologic subjects were anesthetized with urethane (1.5 g/kg, i.p.) or Nembutal (60 mg/kg, i.p.) and injected with atropine (0.15 mg/ kg, i.p.) prior to surgery. Maintenance doses of anesthetic agent were given as needed. The animals were placed in a specially designed head holder that provided a minimum of obstruction of the visual fields. The electrocardiogram was monitored on a Tektronix Model 561A oscilloscope and an audio system. Body temperature was maintained in the range of 33 to 36°C by placing the animal on a Gorman-Rupp constant-temperature heating pad, and the rectal temperature was monitored by a Yellow Springs Instrument Co. telethermometer. The cranial skin was reflected, and the calvaria and dura overlying the VC and LGN was removed unilaterally. Warm mineral oil was placed on the cortex to prevent drying. A stainlesssteel screw was placed in the bone overlying the olfactory bulb for attachment of an indifferent electrode. The lids of the contralateral eye were removed. Microelectrodes used in the experiments were glass capillary tubes

52

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pulled to a tip diameter of 1.5 to 3 .O pm and filled with saturated fast green dye in 2 M NaCl. All neuronal responses were recorded through a Grass high impedance probe, Model HIP5 1 lC, and fed through a Grass Model P5 11 preamplifier to a Tektronix Model 549 storage oscilloscope for direct visualization. Neuronal responses were also monitored on an audio system. Visual stimuli were projected onto a tangent screen that was oriented to cover most of the visual field of the eye under study, which was 30 cm from the screen. Visually responsive units were searched for by presenting flashes of light, generated by a Grass PS-2 photic stimulator, and by projecting moving stimuli on the screen. In three animals the responses of LGN cells to electrical stimulation of the optic tract were studied. All units were tested for responsiveness to auditory stimuli in addition to visual stimuli. A Lietz Wetzlar (Model 3 l-044-000) slide projector, mounted on a tripod, was used to present circles, annuli, bars, and squares of various sizes and colors including white. The bars and squares could be presented at any angle by an image rotator built into the projector. When mounted on the tripod, the projector could be moved smoothly, by hand, in any direction to produce moving stimuli. Attached to the front of the projector was an electromechanical shutter that was driven by a Grass S-4 stimulator. Normally, a stimulus was presented for 1 s every 10 s. All stimuli were presented at low ambient illumination. A separate series of experiments was conducted in which the spectral sensitivity of cells in the LGN was examined. The animal was placed in a Kopf small animal stereotaxic apparatus, and the calvaria over the right LGN was removed, as was the left eyelid. A hemispherical light diffuser (approximately two-fifths of a white, Halex two-star Ping-Pong ball) was then placed over the left eye. Narrow band light was produced by placing 5 x 5-cm (2 x 2-in.) interference filters, having half-band widths of 10 to 15 nm, in the Lietz projector. The intensity of the lights was varied by placing Balzers neutral density filters, singly or in combination, in the path of the light beam. The intensity of the light reaching the eye was calculated by multiplying the intensity of the light source at a particular wavelength by the percentage transmission of the interference filter, the neutral density filters, and the light diffuser that were in the light path. No correction was made for the chromatic properties of the rat eye itself. The intensity of the light source was measured with an Instrumentation Specialties Co. (1X0) spectroradiometer for each of the bulbs used during the experiments. The transmission properties of the light diffuser were also determined with the ISCO spectroradiometer. The stimuli were repeated every 10 s and had durations of 300 ms.

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The threshold of a cell’s response to a given wavelength was defined as the intensity of light to which a cell responded two of three times at a particular latency. The threshold latency chosen for a cell was normally 10 to 40 ms longer than the cell’s latency to a suprathreshold stimulus of white light. We felt that the latency must be included in the threshold criterion, because response latencies near a cell’s absolute threshold were quite variable and introduced an unacceptable scatter into the data of two trial experiments. At the recording sites of single units of special interest, fast green dye was ejected from the electrode by passing 6 PA current for 10 min (30). At the conclusion of an experiment, the animal was killed with an overdose of anesthetic agent and perfused transcardially with saline followed by 10% formol-saline. The brain was then removed and immersed in 30% sucrose-lo% formol-saline. A block of the posterior two-thirds of the brain was sectioned at 40 pm and stained in cresyl Echt violet acetate for tissue examination and confirmation of electrode placement. Data Acquisition and Analysis. A signal from the microelectrode preamplifer was relayed to a DEC PDP-8L computer for on-line analysis, as was a trigger pulse from the stimulator that controlled the photic stimulator and the electromechanical shutter on the projector. Using these two inputs, poststimulus time histograms (PSTHs) were generated by the computer. The action potentials were detected with a Schmitt trigger adjusted for each cell to insure that the computer was responding to the activity of one cell and not to several cells or to random noise. The PSTHs were composed of 200 individual time bins, with each bin having a duration of 10 ms. Normally, 25 consecutive stimulus presentations were used to generate each PSTH, and control time histograms, showing the cell’s spontaneous activity in the absence of stimulation, were also made. The response latencies to a flash were analyzed using a one-sided MannWhitney U-test to compare latencies of VC and LGN cells of experimental animals with those of control animals. The hypothesis tested was that visual cells in experimental animals would have longer response latencies than those in control animals. The data from the spectral sensitivity experiments were first analyzed by calculating a spectral sensitivity curve for a cell and then normalized by assigning a value of 100 to the most sensitive point on the spectral sensitivity curve. The remaining points on the curve were assigned new values from 0 to 100, based on their value relative to the peak. The normalized data were compared with several different absorption curves for rhodopsin obtained from the Dar-mall nomogram (9), using the leastsquares method. The rhodopsin curves were chosen based on the cone mechanisms proposed by Cicerone (6).

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Histological Studies. At the conclusion of each recording session, one eye was removed and fixed in Bouin’s solution, dehydrated, cleared, and embedded in paraffin. The other eye was prepared for plastic embedding by first removing the cornea and lens and immersing it in paraformaldehydeglutaraldehyde. It was then postfixed with osmium, dehydrated through a series of alcohols and propylene oxide, and embedded in Epon. The eye embedded in paraffin was cut serially at 7 pm on the anterior-posterior axis and stained with toludine blue and eosin. Three sections that included the optic disk and were separated by at least 70 pm were selected for cell counts. Two-hundred-micron lengths of the retina 1,2, and 3 mm from the ciliary body were examined at a 400x magnification, and the number of nuclei in the outer nuclear layer (ONL) were counted. The number of cell bodies in the inner nuclear layer (INL) and the ganglion cell layer also were counted. The eyes embedded in plastic were cut at approximately 1.5 to 2.0 pm on the anterior-posterior axis on an LKB ultramicrotome. Sections that contained the ciliary body and the optic disk were stained with toludine blue and examined under oil at a 1000x magnification. The number of nuclei in the ONL were determined in 100~pm lengths of the retina 1, 2, and 3 mm from the ciliary body in three sections separated by at least 10 pm. Additionally, the nuclei were classified as being either rod or cone nuclei based on their heterochromatin patterns. A nucleus in the ONL with more than one clump of heterochromatin was classified as a cone nucleus (14), with only one clump of heterochromatin as a rod nucleus. After the plastic sections were examined it was apparent that these criteria were unreliable; this point will be considered further in the Discussion. Data Analysis. Cell counts from control and experimental retinas were compared by a one-sided Student’s t-test. The hypothesis tested was that there were fewer cells in the retinas of experimental animals than in control animals. RESULTS Neurophysiofogical Studies. The receptive fields and response characteristics of cells in the VC and LGN were studied in animals that had been exposed to constant light and in control animals. The durations of light exposure chosen for examination were 1, 4, 10, 16, and 24 weeks. The l-week exposure was chosen because visually evoked potentials can be recorded at this time, even though significant retinal degeneration has already occurred. A 4-week exposure was chosen because most visually evoked potentials have disappeared at this time, and retinal degeneration has reached an advanced stage. Because preliminary behavioral experiments had shown that pattern vision still existed at 90 days of exposure,

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but was significantly decreased after 150 days, experimental animals were examined after 70 days (10 weeks), 112 days (16 weeks), and 168 days (24 weeks). The data from preliminary experiments on the response patterns of LGN cells to flash and electrical stimuli in normal rats (1) provided a basis for interpreting much of the data collected in light-exposed animals. The responses of LGN cells in control rats to flash stimuli were quite vigorous, with any given cell responding to almost every flash. LGN cells were classified as being principal cells (P cells) or interneurons (I cells) based on the number of spikes in the initial discharge following a flash. P cells had one to three spikes following a flash, and I cells had at least five spikes following each flash (1). As reported (l), the response patterns of Pcells can be separated into three classes. In the first class (E cells) the excitatory period after a flash was followed by a return to the spontaneous level of firing. In the second class (E-S cells) the initial period of excitation was followed by suppression of firing. The third class (S cells) was characterized by the first response after a flash being a suppression of firing. I cells all had similar response patterns, consisting of an early discharge followed by a period of no activity, which was followed by one or more late discharges. Figure 1 presents typical response patterns of the three classes of P and I cells. There was a large degree of variability among the response patterns in the seven control animals in which the VC was studied. Approximately one-half of the cells responded with a short-latency, short-duration period of excitation similar to the response patterns shown in the top of Fig. 2. The remaining cells had responses that could not be categorized simple. Some exhibited a generalized excitation, and others showed a period of suppression followed by a return to the spontaneous rate of firing. The PSTHs shown in the bottom of Fig. 2 are examples of the types of response patterns of cells in this second “class.” The responses of visual cells in light-exposed animals were less vigorous than in normal animals, and the response latencies were also more variable. This was particularly obvious for geniculate cells. The response of many cortical cells are variable in normal animals, so the variability seen in experimental animals was not remarkable. Figures 3 and 4 show typical response patterns for geniculate and cortical cells of animals exposed 10 and 16 weeks to constant light. With increasing exposure durations, it became more difficult to find cells in the cortex that had visually evoked responses. This observation was in contrast to the findings noted in the LGN where it was fairly easy to find responsive cells, even after 24 weeks of exposure. The response latencies to an intense flash stimulus were determined for cells in the LGN and VC. The response latency was defined as the time be-

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1 set FIG. 1. Poststimulus time histograms of lateral geniculate neurons in control rats in response to a flash. I-C-the response properties of an E cell; there was a short latency excitation (arrowhead), followed by a return to the spontaneous rate of firing. N-8-the response pattern of an S cell; after the flash there was a period of suppression (open arrow), followed by a late period of excitation (marked by solid arrow). D-17-the response pattern of an E-S cell; an early discharge (arrowhead), followed by a period of suppression of firing. AF-l-response pattern of an I cell to a flash; the early discharge (arrowhead) was followed by a late discharge having a variable latency.

tween the stimulus and the peak of the first excitatory period after the stimulus. These values were obtained by examining the PSTHs that were generated with data from 25 consecutive stimulus presentations. The mean response latency for LGN cells in control animals was 78 + 31.27 ms (mean ? SE). If the response latencies of two S cells were excluded from the calculation, the values dropped to 32.7 + 1.8 ms. The median, minimum, and maximum and mean ? SE response latencies of the geniculate cells in control and light-exposed animals are shown in Table 1. The response latencies of cells in light-exposed animals differed (P < 0.01) from control animals. Cells in the VC had longer response latencies than cells in the LGN. The average response latencies of VC cells at the different exposure times are shown in Table 1. The response latencies of VC cells in control animals were shorter than those of VC cells in experimental animals. Statistical analysis revealed, however. that the response latencies of cortical cells in

RETINAL

75-2

DEGENERATION

AND CNS NEUROPHYSIOLOGY

57

BK-17

1 set FIG. 2. Response patterns of visual cortex neurons in control rats, showing the PSTHs after a flash stimulus. 75-2 and BK-17 responded with a short-latency, short-duration period of excitation (arrowheads). 75-2 had a period of decreased activity (open arrow) after the period of excitation. 80-l produced an extended burst of spikes (solid arrow) after a flash, the first spike (arrowhead) of the burst having a fixed latency. 67-7 responded to a flash with a short-duration reduction of ongoing activity.

1 set FIG. 3. Response patterns of visual cells in rats exposed for 10 weeks to constant light. 49-7 and 49-6 are records from geniculate cells, and 46-7 and 43-7 are from cortical cells. 49-7 was a P cell, and 49-6 was an I cell. Note the vigorous responses (arrowheads) of these two cells.

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30 15 0 lsec FIG. 4. Response patterns of visual cells in rats exposed 16 weeks to constant light. 53-7 (a P cell) and 56-10 (an I cell) are records from geniculate cells, and 53-l and 47-3 are from cortical cells.

experimental animals were significantly different from those of cortical cells in the control animals at the P < 0.1 level for all time points, except 24 weeks, when only two cells were studied. This finding is considered in U-test comthe Discussion. The P values obtained with a Mann-Whitney paring experimental groups with the control group are shown in Table 1. An attempt was made to examine the receptive field properties of the cells in the LGN and visual cortex. The normal procedure consisted of studying a cell’s response patterns before examining its receptive field properties. Because of the time involved in generating a PSTH, many cells were lost before a careful receptive field analysis could be completed. Of the geniculate cells studied, it was found that 70% of the receptive fields consisted of a single, dominant region that produced either an “off” response or an “on” response. In 30% of the geniculate cells in control animals, peripheral regions of the receptive field gave rise to an antagonistic response compared to the center of the receptive field. Of the cortical cells studied in normal animals, no receptive field could be found for 38% of the cells. Cortical cells that had identifiable receptive fields were classified as being either simple or complex. Simple cells had receptive fields that could be separated into areas that gave “on” or “off” responses to stationary, flashing stimuli (12). Complex cells responded to stationary, flashing stimuli, if at all, with an “on-off” discharge (12). In control animals, 27% of the cells had simple receptive fields, and 33% had complex ones. These geniculate and cortical cells normally could be activated

RETINAL

DEGENERATION

TABLE

1

Latency Analysis of Cells in Control and Experimental Exposure duration (weeks)

No. of animals

No. of cells

Median

59

AND CNS NEUROPHYSIOLOGY

Mean

SE

Minimum

Animals

Maximum

P

Response latencies of lateral geniculate nucleus cells (ms) Control 1 4 10 16 24

7 5 5 5 4 4

14 22 12 17 10 8

33 105 80 90 120 190

78 190 135 119 175 192

31.27 32.19 39.55 22.56 41.74 42.07

25 40 55 60

70 150

-

400 500 450 200 500 400

0.0001 0.0005 0.0003 0.0011 0.003 1

500 500 620 630 250 260

0.027 0.061 0.016 0.079 0.138

Response latencies of visual cortex cells (ms) Control 1 4 10 16 24

7 5 5 5 4 4

23 15 17 15 17 2

62.5 200 100 210 130 230

1.50 232 187 230 146 230

30.6 35.3 45.6 36.4 12.6 29.7

30 60 50

70 90 200

-

by stimuli that were only 2 to 4” in diameter and had receptive fields that were 20 to 40” in diameter. The results of receptive field analyses of cells in light-exposed animals are shown in Table 2. In the LGN, no cells having antagonistic surrounds were found after 4 or more weeks of exposure to constant light, whereas cells giving “on” or “off” responses were found at all exposure times. With increasing exposure durations it was necessary to use larger visual stimuli to activate cells. For example, after 10 weeks of constant light it was necessary to use stimuli that were 40 to 90” in diameter. In the cortex there was a decrease in the number of cells having complex receptive fields properties as well as a decrease in the number of ceils having simple receptive fields (although the decrease in simple cells appeared to have a longer time course than the decrease in complex cells). As in the LGN, extremely large stimuli were needed to activate cortical cells in animals exposed to long durations of constant light. An additional observation was that visually responsive cells were found in the cortex of only two of the seven animals exposed to 1 week of constant light. This was in spite of the numerous electrode penetrations made in what appeared to be healthy, undamaged cortices. When a geniculate cell’s response pattern to a flash had been studied and

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2

Receptive Field Types Exposure duration (weeks)

Lateral geniculate Center only Center + surround Cortex Simple Complex No receptive field

Control

1

4

10

16

24

I

7

5

10

4

4

3

3

0

0

0

0

5 6

4 2

5 0

4 2

4 0

0 0

7

2

5

3

2

2

the receptive field had been analyzed, we determined if the cell had opponent color properties. Of the seven cells studied in control animals, one cell had opponent color properties (Fig. 5). None of the 22 cells examined in the light-exposed animals had opponent color properties. The responses to different colors of a cell in an animal exposed to 16 weeks of constant light are shown in Fig. 6. Note that red stimuli were very ineffective in activating cells in animals exposed to constant light. The spectral sensitivity of cells in the geniculate were examined in a separate series of experiments. In two control animals, 10 cells were studied completely; in two rats exposed to constant light for 12 weeks, 12 cells were examined, and in three rats exposed to constant light for 24 weeks, 10 cells were studied (Fig. 7). The spectral sensitivity of each cell was compared to the absorption curves of rhodopsin having peak absorptions at 450, 480, 500, 520, and 550 nm, using a least-squares method. A rhodopsin curve having an absorption peak at 500 nm provided the best fit to the data from 19 of 29 cells. Five cells had spectral sensitivities that were best fit by a rhodopsin curve with a peak at 480 nm and six others at 520 nm. Absorption curves at 450 and 550 nm were not best-fits to the data of any of the cells. Two cells having extremely unusual spectral sensitivity curves were excluded from the least-squares analysis. These latter cells had spectral sensitivity curves that were not smooth and did not resemble any single rhodopsin curve or a combination of two or three different rhodopsin absorption curves. It was felt that the unusual data from these two cells was due to a nonstationarity of their responses. There were no obvious differences between the spectral sensitivities of cells in control and in light-exposed animals. In each of the three groups at least

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67-14

1 set FIG. 5. Opponent color responses from a P cell in the geniculate of an animal housed in cyclic light. The three PSTHs on the left were compiled from responses to a 4” spot projected on the center of the cell’s receptive field for 1 s (note the black time bar below each PSTH). This cell gave a strong “on” response to green and a less vigorous response to blue. A red stimulus in this position had no effect on the cell. The PSTHs on the right show the responses from the cell’s receptive field periphery. The response to green changed from a strong “on” response to a response that was difficult to classify as being strictly “on” or “off ‘. The response to red, however, was a strong “on” response. Thus, this cell can be said to have red-green opponent color properties.

one cell was found with spectral sensitivities that had a small peak at 600 nm associated with the larger peaks at 500 nm (Fig. 7). By comparing the location of the fast green dye markers and electrode tracts with the stereotaxic coordinates of the cells that were studied in the LGN, we confirmed that those cells were, indeed, in the LGN. Of the fast green dye markers placed in the LGN, 60% were found upon histological examination; those placed early in an experiment were less likely to be found than those placed later. It was also frequently impossible to find markers placed with electrodes having very high impedances. No fast green dye markers were found outside the LGN, and those found were not localized to any particular region of the nucleus.

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1 set FIG. 6. Responses to color from a P cell in the geniculate of an animal exposed 16 weeks to constant light. The top left PSTH shows the response to a flash of white light, and the remaining PSTHs show the response to a 30” spot of colored light on for 1 s. The responses to different colors were not qualitatively different, with the exception of a lack of a response to red. This latter observation was representative of all cells in light-exposed animals that were studied for opponent color properties.

Histological Studies. Exposure to constant light produced a decrease in the number of photoreceptor nuclei present in the retina. Also, constant light produced small but significant changes in the number of cells in the inner nuclear layer (INL) and in the ganglion cell layer. The retinas of control animals had all the traditionally described layers including well-defined inner and outer segments and outer plexiform layers. In’ the posterior retina the outer nuclear layer (ONL) was normally .about 12 nuclei thick and decreased in thickness peripherally to only five to six nuclei thick. The nuclei of cells classified as rods were round and about 5 pm in diameter, and those classified as cones were oval and normally at least 7 pm on the long axis (Fig. 8). Cones also had three or four small clumps of heterochromatin. After 1 week of exposure to constant light, the inner and outer segments of the photoreceptors were disrupted, and the distance between

FIG. 7. Spectral sensitivity curves of cells in the lateral geniculate nucleus (LGN), 0, and the absorption curve for rhodopsin having a peak at 500 nm, 0. The wavelength in nanometers is plotted on the X-axis. The normalized relative threshold for LGN cells and the absorption, as a percentage of the maximum, for rhodopsin are plotted on the Y-axis. The top two frames show data from cells in normal animals, and the middle and bottom frames are from animals exposed to 12 and 24 weeks of constant light, respectively. Note the small side peaks at 600 nm in cells 93-l 1 and 41-2. 63

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the ONL and the pigment epithelium was decreased from the normal condition. The outer plexiform layer was still present. The thickness of the ONL had decreased to only two to three nuclei in the posterior retina. The retinas from animals exposed to 4 weeks of constant light were qualitatively similar to the l-week-exposed animals. A small bacillary layer of inner segments was still present, and an outer plexiform layer still separated the remaining photoreceptor nuclei from the INL. With 10 or more weeks of exposure there was a marked difference in the condition of the retina. The INL was immediately adjacent to the pigment epithelium, and the few remaining photoreceptor nuclei were scattered along the retina intimately touching both the pigment epithelium and the INL. Rarely, when two or three photoreceptor nuclei were clumped together, there appeared to be a very small plexiform layer between them and the INL. The photoreceptor nuclei were pyknotic, about 4 pm in diameter, and were frequently elongated along a line parallel to the pigmented epithelium. Many of the photoreceptor nuclei had two clumps of heterochromatin, rather than one as observed in control rod nuclei, or three or four as observed in control cone nuclei (Fig. 8). The eyes embedded in paraffin were examined to determine the number of cells present in the ONL, INL, and the GCL. Table 3 shows the number of photoreceptor nuclei in the posterior, intermediate, and peripheral retina at each exposure period. The data suggest that photoreceptor degeneration occurred at a faster rate in the posterior retina than in the periphery during the first 4 weeks. However, from 10 to 24 weeks the degeneration appeared to proceed at the same rate throughout the retina. The number of cells present at all experimental times were significantly different from the controls at P < 0.01. After long-term exposure to constant light there was a small but significant change in the number of cell bodies in the INL and the ganglion cell layer. Table 4 shows the mean number of cells in these layers in the intermediate region of the retina at each exposure period. A one-sided r-test was used to compare the control retinas to those of light-exposed animals. After 24 weeks there were significant differences from controls (P < 0.05) in two of the three regions of the INL and the GCL. Only one or two plastic-embedded retinas at each time point provided usable data for ONL cell numbers in the posterior, intermediate, and peripheral retina. The principal reason for this was the extreme difficulty of obtaining the 2-pm sections that included the entire retina from the ciliary body to the optic disk. However, the number of nuclei in the ONL at any particular time point was within the range found in the paraffinembedded eyes when similar regions of the retina were compared. By using

RETINAL

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AND CNS NEUROPHYSIOLOGY TABLE

3

Number of Nuclei in 2OOpm Lengths of the Outer Nuclear Layer Exposure duration (weeks) Control Number of eyes examined Posterior retina Mean SE

P

1

7

6

410 32.8

106 14.04

-

Intermediate retina Mean SE

P

383 31.3

P

327 15.8

-

10

5

0.001 105 18.82

-

Peripheral retina Mean SE

4

0.001 119 24.14 0.001

16

24

5

5

4

73.6 20.08 0.001

1.34 0.50 0.001

1.92 0.73 0.001

1.09 0.88 0.003

55 20.12 0.001

2.1 0.94 0.001

2.8 0.71 0.001

1.7 0.84 0.003

83.3 23.21 0.001

3.4 1.01 0.001

2.1 0.63 0.001

1.2 0.70 0.003

the criterion of one clump of densely staining heterochromatin as characterizing a rod nucleus, it was found that after 10 weeks of constant light there were almost no rod nuclei remaining. On the other hand, the counts of cone nuclei were remarkable because in two of the three regions of the retina we found an apparent increase in the number of cone nuclei TABLE

4

Number of Cells in 2OOpm Lengths of the Inner Nuclear Layer (INL) and the Ganglion Cell Layer (GCL) in the Intermediate Retina Exposure duration (weeks) Control Number of eyes examined INL Mean SE

P GCL Mean SE

P

1

4

10

16

24

5

5

4

7

6

5

128 3.93

129 9.06 >O.l

133 6.26 >o. 1

115 2.73 0.012

109 8.32 0.022

107 6.05 0.006

17 0.44 >O.l

15 1.28 >O.l

13 1.68 0.016

11 1.10 0.002

15 0.75

-

13 1.91 0.042

FIG. 8. Sections from plastic-embedded retinas. Frames A through F show selected regions of retinas from a control rat and from rats exposed to 1, 4, 10, 16, and 24 weeks of constant light, respectively. The pictures were taken at a magnification of 250x and show sections of retina approximately 1 mm from the ciliary body. The frames were selected to show examples of photoreceptor nuclei at each exposure duration. The long, narrow, black arrows indicate the pigmented epithelium (PE). A-the bold, short arrows indicate two cone nuclei. Note the oval shape of these two nuclei and that they have more than one clump of densely staining heterochromatin. These two nuclei are also oriented perpendicular to the PE. The inner and outer segments of the photoreceptors are well defined and separate the PE from the photoreceptor nuclei. B-the disruption of the inner and outer segments and the decrease in the distance between the PE and the photoreceptor nuclei. C-an example of the more extensive disruption of the inner and outer segments in retinas from animals exposed 4 weeks to constant light. D-one photoreceptor nucleus is seen immediately adjacent to the PE. This nucleus had three small clumps of heterochromatin. E and F-nuclei with more than one clump of heterochromatin. However, these nuclei are not oval, nor are they oriented perpendicular to the PE, as are the cone nuclei in control animals.

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AND ANDERSON TABLE

5

Mean Number of Rod Nuclei and Cone Nuclei in lOO+m Lengths of the Intermediate Retina Exposure duration (weeks)

Number of eyes examined Rod nuclei Cone nuclei

Control

1

4

10

16

24

1 145 7

2 33 10

1 33 12

1 0 2

2 0 1

2 0 1

present after an exposure of 4 weeks. After longer exposures, there was a decrease in the number of presumptive cone nuclei. Table 5 lists the number of cone and rod nuclei counted in 100~pm lengths of the intermediate retina. DISCUSSION Retinal Anatomy. The results of light microscopic examination of retinas from control and experimental animals show that exposure to low levels of constant light produces a severe loss of photoreceptor cells, leaving the remaining layers of the retina relatively intact. These results are in agreement with those of other workers (3, 22). The cell counts in this study, indicating there was a more rapid cell loss in the posterior than in the peripheral retina, confirmed the results of O’Steen er al. (24), who used measurements of retinal thickness. The apparent absence of photoreceptors with intact inner and outer segments in the retinas of animals exposed 10 or more weeks to light is consistent with the results of O’Steen and Anderson (23), and of LaVail (14), who used the electron microscope to show a lack of intact photoreceptors in animals housed under similar conditions. The condition of the pigment epithelium in the retinas of light-exposed animals appeared to be good regardless of the exposure duration. This is similar to the results of O’Steen et al. (29, but is in contradistinction to those of Noel1 et al. (21), who used more intense illumination than those used by O’Steen er al. (25) or in the present study. The results of the cell counts of the INL and the GCL require special comment. After 24 weeks of exposure to constant light, there were fewer cells in the INL and GCL in two of the three regions of the retina than in control retinas (P < 0.05). The data were examined to determine if the age of the animals could account for the differences. Although there was a trend for older control rats to have slightly fewer cells in the INL and the

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GCL than younger control rats, the differences were not large enough to explain the differences between controls and animals exposed 24 weeks to constant light. It is possible that the decrease was due to a direct effect of light on the ganglion cells and the cells of the INL, or it may have been due to transneuronal degeneration. In many systems, long survival times after deafferentation are required to show any transneuronal effects (8). The fact that 20 weeks beyond the time when the majority of the photoreceptors degenerated was required to produce significant changes in the INL and the GCL is consistent with this hypothesis. The changes in the INL and the GCL reported here are paralleled by changes in the inner plexiform layer (5) and are consistent with changes in the thickness of the INL (27). The counts of the number of rod and cone nuclei present at different exposure times are particularly important in this study. Initially, the criteria we used to identify rod and cone nuclei were those originally defined by LaVail(14). According to that author, those photoreceptor nuclei containing two or more densely staining clumps of heterochromatin are cone nuclei, whereas nuclei having only one clump of heterochromatin are rod nuclei. LaVail (14) did not utilize information relating to the size and/or shape of the rod and cone nuclei in helping to discriminate between them. In retrospect, we believe that one should consider size and/ or shape factors when attempting to identify rods and cones, as the failure to do so would appear to lead to a significant overestimation of the number of cone nuclei present in the retinas of animals exposed to constant light. For example, in the present experiment, the ratio of rods to cones in the retinas of control rats was found to be about 20: 1. This observation is in contrast to the data of Walls (32) and LaVail(14) himself, who reported a ratio of about 100: 1. If size and shape were included in the criterion for cones, then the ratio of rods to cones found in control rats was about 100: 1. Apparently not all rod nuclei in the retinas of control rats had only one clump of heterochromatin. In the present study we found that 4 weeks of exposure to constant light produced an increase in the absolute number of photoreceptor nuclei having two or more clumps of heterochromatin. There is no reason to suggest this was due to the development of additional cones. Rather, it was probably due to disruption of pyknotic rod nuclei as a result of exposure to constant light which resulted in an increase in the number of rod nuclei having more than one clump of heterochromatin. Almost all photoreceptor nuclei found in the retinas of animals exposed to 10 or more weeks of constant light had more than one clump of heterochromatin. None of those nuclei, however, looked like oval cone nuclei. When an occasional cluster of several photoreceptor nuclei was observed,

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each nucleus would have two to three clumps of heterochromatin. But the fact that those nuclei were clustered together suggests that they were not cone nuclei, because oval cone nuclei in control retinas were never observed in a similar close association with one another. In control retinas, cone nuclei were found only in the outer one-third to one-half of the ONL and were normally at least 50 pm apart, so a loss of rod nuclei would not be expected to result in cone nuclei coming together. Taken together, these data suggest that criteria for identifying rods and cones based solely on heterochromatin staining patterns are unreliable. Using criteria that did not consider size and/or shape factors, LaVail (14) concluded that rods were more susceptible than cones to the effects of constant light. However, Tso et al. (31) reported that in monkeys cones were found to be more sensitive to light-induced degeneration than rods. Further studies will be needed to determine whether the differences between LaVail’s (14) results and those of Tso et al. (31) reflect interspecies factors or whether methodological factors, such as the criteria used to identify photoreceptors, can account for the difference. Neurophysiological Studies. In the animals exposed to constant light there were E, S, and E-S cells at all exposure times except at 24 weeks, when no S cells were observed. Many of the cells in animals exposed to 10 or more weeks of constant light had extremely low spontaneous activity. As a result, some cells that were suppressed after a flash may have been classified inadvertently as E cells, because it was impossible to detect suppression in the absence of significant spontaneous activity. The I cells observed in animals exposed to 1, 10, and 16 weeks of constant light had response patterns comparable to those of I cells in the control animals of the preliminary study. That response patterns similar to those of P and I cells in control rats were found in the light-exposed rats implies that no major changes occurred in the functional connections of cells in the LGN as a result of exposure to constant light. The latencies in response to a flash of both geniculate and cortical cells increased with increasing durations of constant light exposure. Evidence from cats (7, 16) suggests that the firing of one ganglion cell can result in the production of an action potential in a geniculate cell. Also, very few ganglion cells (six or less) are thought to send excitatory input to one geniculate cell (7, 16). If those results can be extrapolated to rats, then temporal or spatial summation of inputs from ganglion cells is not necessary to produce an action potential in a geniculate P cell. Therefore, the increased response latencies of geniculate cells is most likely due to an increase in the response latencies of retinal ganglion cells. However, a decrease in the functional convergence on LGN cells may also account

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for this finding. It is not surprising that the response latencies of the cells in the VC increase with exposure to constant light, because the LGN cells that provide the primary input to the cortex themselves have longer response latencies. If one compares the minimum response latencies of geniculate cells with the minimum response latencies of the cortical cells at each exposure time, one finds that the LGN latencies are shorter than the cortical latencies at all exposure times, with the exception of 4 weeks, when the minimum latency in the cortex was 50 ms and the minimum LGN latency was 55 ms. The receptive field studies of geniculate cells in control animals showed that, in 7 of the 10 cells studied, there were no antagonistic surrounds, while the remaining three cells had antagonistic peripheries. Other workers (17,28) found that about half of the P cells had receptive fields with antagonistic surrounds, and 2 to 3% of their cells were directionally selective. The remaining P cells had receptive fields with a dominant center and no antagonistic surround. No geniculate cells were found in the present study that had confirmed, directionally selective properties which is most likely due to the fact that only a very small percentage of geniculate cells have this type of receptive field organization. After 4 or more weeks of constant light, no cells having antagonistic surrounds were found, and very large stimuli were required to produce a response from geniculate cells. These changes in the receptive field properties could be due to a major reorganization of the synaptic input to the geniculate cells or to changes in the response properties of the retinal ganglion cells. Of the two possibilities, the latter is much more likely, because of the changes that have been reported previously in the INL and the IPL (5). During the receptive field analyses of cortical cells in control animals, there were a significant number of cells (39%) for which no receptive field could be found. There are several possible explanations. One obvious possibility is that the visual stimuli were grossly out of focus on the animals’ retinae. This is unlikely, becuase the tangent screen was 33 cm from each rat’s eye in the present experiment, and Hughes (13) convincingly showed that the near point for rats is about 30 cm and that they have a large depth of field. The most likely explanation is that either the proper position in the visual field was not examined for a particular cell or that stimuli having the proper temporal and spatial characteristics were not used to test the cell. In any case, the results reported here are not inconsistent with those of other investigators (29, 36), who reported finding significant numbers of cells in the rat cortex for which they were unable to identify receptive fields. The results of our behavioral experiments (15), together with those of the

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electrophysiological experiments reported here, suggest a striking relationship between the receptive field properties of visual cortex cells and the ability of animals to perform pattern discriminations. The results of the neurophysiological experiments revealed that there was a decrease in the number of cells having complex receptive fields as a result of exposure of the rat to constant light, so that after 16 or more weeks in constant light no complex cells were found. No simple cells were found in rats exposed to constant light for 24 weeks. This is similar to the time course seen for the decrease in the ability of the rats to perform pattern discriminations. In contrast to these results, light-exposed rats performed intensity discriminations and color discriminations throughout the 24 weeks in constant light. Similarly, cells responsive to light were found in the LGN of rats exposed to 24 weeks of constant light. Another important observation was that during the first week in constant light, rats showed a small but significant impairment on pattern tasks that recovered during the second week. This was paralleled by an extreme difficulty in finding any visually responsive cells in the cortex of rats exposed to 1 week of constant light. It has been known for at least 40 years that rats can discriminate between blue and red and between green and red on a basis of wavelength (18, 20, 33, 34). It was also shown that, although rats can learn to discriminate between red and yellow and blue and yellow, they do so only after an extremely large number of trials [>500 (34)]. Rats have not been trained to discriminate between blue and green or between green and yellow, despite the fact that different investigators have attempted to do so (19, 34). The work of the latter investigators showed that although the rat has color vision, it is extremely limited compared to that of primates or even squirrels. For a rat to discriminate two colors on the basis of hue, the two colors must have significantly different dominant wavelengths. In view of these observations, it seems extremely unlikely that rats have three different cone mechanisms with peak sensitivities at 450, 520, and 560 nm, as proposed by Cicerone (6). If rats had those cone mechanisms, then one would expect them to be able to discriminate between blue (about 450 nm) and green (about 520 nm) or between green (about 520 nm) and yellow (about 560 nm). If, however, rats use a rod pigment (498 nm) and a single cone pigment (about 600 nm) in its color system, then one would predict that rats would be able to discriminate between colors only if their peak wavelengths were relatively far apart, as was observed in several behavioral experiments (18-20, 33, 34). In our behavioral experiments (15), we found that rats exposed 24 weeks to constant light were still able to discriminate between blue and red. This capability would seem to require at least two different visual pigments. The results of the neurophysiological experiments on light-exposed an-

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imals indicated that, although all of the LGN cells were most sensitive at the point predicted by the rat rhodopsin absorption curve (4), some cells had spectral sensitivity curves that could not be explained simply on a basis of a rhodopsin absorption spectrum. The threshold at 600 nm for these cells was from 400 to 800% higher than the threshold expected from a rhodopsin absorption mechanism. These latter findings are interesting because Granit (11) obtained spectral sensitivity curves with minor peaks at 600 nm for rat retinal ganglion cells that were strikingly similar to those determined for geniculate cells in the present study. Granit (11) concluded that this sensitivity to red was due to a cone mechanism and not to a rod mechanism or an error in measurement becuse the two peaks of the spectral sensitivity curves had different recovery rates from light adaptation. If this is the case, then it is possible that both a cone pigment and rhodopsin are involved in the light transduction process in animals exposed to constant light and, thereby, provide a mechanism for color vision in the light-exposed rats. While this manuscript was in preparation, a study on hereditary retinal degeneration in mice was published (lo), in which the receptive field properties of cells in the visual cortex and superior colliculi were examined. Of particular interest was the fact that no electrophysiologic evidence was found for cones being the primary light-transducing element in mice with hereditary retinal degeneration. Neither a Purkinje shift nor a rod-cone break in the dark-adaptation curve was found in the tectal cells studied. These findings are important because cones have been thought to be the primary remaining photoreceptor cell type in mice with hereditary retinal degeneration (10). Because the evidence from mice and that in the present paper from rats indicates that a rhodopsin mechanism and not a cone mechanism is the primary light-transducing mechanism in rodents with retinal degeneration, it seems that accurate and reliable criteria for distinguishing between residual rods and cones are essential. Our behavioral studies showed there was a decrease in the ability of rats to perform pattern discrimination, which was followed, much later,’ by a decrease in the ability of the rats to perform intensity discriminations. The neurophysiological experiments showed that there were important changes in the response latencies and receptive field properties of geniculate and cortical cells as a result of exposure to constant light. In view of the fact that there were photoreceptor nuclei present in the retinas of the lightexposed animals that could perform visual discriminations and in the retinas of the animals with visually responsive geniculate and cortical cells, it is possible that these residual photoreceptor nuclei might be involved in the transduction of light and the transmission of information to the cells of the INL. Because the spectral sensitivity measurements suggest that the

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light-transducing molecules are the same in control animals and lightexposed animals, this seems particularly likely. Therefore, there appears to be a correlation between the presence of residual photoreceptor nuclei and the ability of rats to perform visually guided tasks. Our data do not eliminate the possibility that some novel retinal element is also involved in the transduction of light in animals exposed to long-term, constant light. However, the observations concerning color vision and the spectral sensitivity of the light-transducing element would, at present, seem to argue against it. REFERENCES 1. ANDERSON, K. V., V. LEMMON, AND H. S. ROSING. 1977. Response properties ofcells in the dorsal lateral geniculate nucleus of the albino rat. J. Neurosci. Res. 3: 143- 152. 2. ANDERSON, K. V., AND W. K. O’STEEN. 1972. Black-white and pattern discrimination in rats without photoreceptors. Exp. Neural. 34: 446-454. 3. BENNE~, M. H., R. F. DYER, AND J. D. DUNN. 1972. Light induced retinal degeneration: effect upon light-dark discrimination. Exp. Neurol. 34: 434-445. 4. BRIDGES, C. D. B. 1959. Visual pigments of some common laboratory animals. Nature (London) 184: 1727- 1728. 5. CHERNENKO, G. A., AND R. W. WEST. 1976. Are-examination of anatomical plasticity in the rat retina. J. Comp. Neurol. 167: 49-62. 6. CICERONE, C. M., 1976. Cones survive rods in the light-damaged eye of the albino rat. Science 194~ 1183- 1185. 7. CLELAND, B. G., M. W. DUBIN, AND W. R. LEVICK. 1971. Sustained and transient neurons in the cat’s retina and lateral geniculate nucleus. J. Physiol. (London) 217: 473- 4%. 8. COWAN, W. M. 1970. Anterograde and retrograde transneuronal degeneration in the central and peripheral nervous system. Pages 217-25 1 in W. J. H. NAUTA AND S. 0. E. EBBESSON, Eds. Contemporary Research Methods in Neuroanatomy. SpringerVerlag, New York. 9. DARTNALL, H. J. A. 1953. The interpretation of spectral sensitivity curves. Br. Med. Bull.

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