Quantitative Studies of Cell Size in the Cat’s Dorsal Lateral Geniculate Nucleus Following Visual Deprivation T. L. HICKEY, PETER D. SPEAR* AND KENNETH E. KRATZS School of Optometry, The Medical Center, University of Alabama in Birmingham, Birmingham, Alabama 35294 and Department of Psychology, Kansas State University, Manhattan, Kansas 66506

ABSTRACT The effects of visual deprivation upon dorsal lateral geniculate (DLG) cell size were compared for seven kittens reared with monocular lid-suture (MD), seven with binocular lid-suture (BD), and six with one eye lid-sutured and the other eye enucleated soon after birth (MD-E). Six additional kittens were reared normally for comparison. For each kitten the cross-sectional areas of 300 cells were measured in one or both nuclei. Measurements were taken from the binocular segment of laminae A and A1 and the monocular segment of lamina A. In agreement with previous studies, cells in the binocular segment of the deprived laminae of MD cats were smaller (33-34%)than those in the non-deprived laminae. Comparisons with normal animals indicated that this difference was due to an increase (10-158) in size of cells in the non-deprived laminae as well as a decrease (23-25%)in size of cells in the deprived laminae. Cells in the monocular segment also were affected by deprivation in MD cats, and this effect increased with the age (and duration of the deprivation) of the animal. However, it was always smaller than the decrease in cell size in the binocular portion of the DLG. In BD kittens, DLG cells were smaller (7-12%)than normal in all portions of the nucleus, including both the binocular and monocular segments. Direct comparisons between the deprived laminae of MD and BD kittens indicated that the decrease in cell size was greater for MD kittens in the binocular segment, but tended to be greater for BD kittens in the monocular segment. In MD-E kittens, DLG cells in the deprived laminae were smaller (11-17%)than normal in all portions of the nucleus, including both the binocular and monocular segments. Thus, the effects of deprivation were similar to those in BD kittens, even though inputs from the deprived eye had been placed at a competitive advantage in MD-E kittens. These results indicate that two factors may affect cell size in the DLG of visually deprived cats: deprivation per se and abnormal binocular competition. Finally, separate analyses for the ten largest and the ten smallest cells in each lamina of each cat were carried out in an attempt to determine if the changes in cell size were limited to the largest cells. In every case, differences observed for the total sample of cells were paralleled by differences from normal of both the largest cells present and the smallest cells present in the deprived laminae. Since at least two alternative interpretations can account for this finding, the question of whether the large cells are selectively affected by visual deprivation remains unanswered in the cat. Supported by N. I. H. Grant lROl EY01338 and N.S.F. Grant BMS 74-23658 to T.L.H. and USPHS Grant 5 R01 EY01170 to P. D. S. * Present address: Department of Psychology, University of Wisconsin, Madison, Wisconsin 53706. Present address: Department of Physiology, University of Virginia, School of Medicine, Charlottesville, Virginia 22901.

J. COMP. NEUR , 172: 265-282.

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Following development with monocular deprivation (MD), cells in the layers of the dorsal lateral geniculate nucleus (DLG) which receive input from the deprived eye are much smaller than cells in layers which receive input from the non-de rived eye. This has been shown in the cat Wiesel and Hubel, '63; Kupfer and Palmer, '64; Guillery and Stelzner, '70; Chow and Stewart, '72; Guillery, '72; Garey et al., '73; Wan and Cragg, '76), dog (Sherman and Wilson, '75), tree shrew (Casagrande et al., '74), squirrel (Guillery and Kaas, '74a), and monkey (von Noorden, '73; von Noorden and Middleditch, '75; Headon and Powell, '73). In cats, cells in the deprived layers typically are 30-40%smaller than those in the non-deprived layers following monocular lid-suture. The effects of binocular lidsuture (BD) on DLG cell size appear to be much less severe (Chow and Stewart, '72; Guillery, '73; however, see Wiesel and Hubel, '65). For example, when comparisons were made with a large sample of normally reared kittens, Guillery ('73) found only a moderate decrease in cell size (about 5%) in BD kittens. In binocularly deprived animals, evaluation of the effects of deprivation must be made in comparison with normal kittens since all layers of the DLG have been deprived. In contrast, studies of the effects of monocular deprivation typically have compared deprived and non-deprived DLG layers within the same animal. Such comparisons assume that non-deprived cells are, in fact, normal in size. However, Sherman and Wilson ('75) have recently shown in MD dogs that cells in the non-deprived layers are actually larger than normal. Furthermore, Guillery ('73) has noted that in MD cats the differences between cell size in deprived and non-deprived layers could as easily be due to hypertrophy of nondeprived cells as to a decrease in size of deprived cells (see also discussion b . Wiesel and Hubel, '65; Garey et al., '73y. These observations raise the possibility that the decrease in cell size following MD is actually very small and that the apparent difference in severity of effects of MD and

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BD is due to hypertrophy of cells in the non-deprived layers of MD cats. This can be tested only by directly comparing cell size in the deprived layers of MD and BD animals. In order to answer this question, we undertook measurements of a large number of cells in seven MD, seven BD, and six normally reared kittens. Quantitative comparisons between DLG laminae of kittens in each group confirmed the presence of a small but significant decrease in cell size in the DLG of BD cats. In MD cats, a significant hypertrophy of cells in the nondeprived laminae were observed. Nevertheless, direct comparisons between the deprived laminae in MD and BD cats indicated that the effects of deprivation following MD were greater than those following BD. Thus, placing the two deprived eyes in relative competitive balance (BD) produces a smaller decrease in size than when a single deprived eye is placed at a competitive disadvantage (MD). We therefore asked whether placing the deprived eye at a competitive advantage (by lid-suturing one eye and enucleating the other, MD-E) would result in an even smaller effect on DLG cell size. Evidence from experiments in which a small retinal lesion was placed in the non-deprived eye of MD cats suggested that this would be the case. Morpholo ical (Guillery, '72; Sherman et al., '74 and physiological (Sherman et al., '75) studies of the DLG found little or no effect of deprivation in the portion of the deprived lamina (the "critical segment") adjacent to the denervated portion of the non-deprived lamina. In addition, behavioral studies have shown that these animals orient normally to stimuli presented in the portion of the visual field corresponding to the critical segment, but appear blind to stimuli presented elsewhere in the visual field of the deprived eye (Sherman et al., '74). However, recent experiments suggest that a different result may be observed if a larger lesion is made so that the entire eye is removed (i.e., one eye enucleated and the other lid-sutured). Behavioral studies indicate that these cats

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CHANGES IN DLGN CELL SIZE FOLLOWING DEPRIVATION

do not now orient to stimuli throughout the visual field of the deprived eye. Rather, their orientation behavior appeared similar to that of BD cats (Sherman and Guillery, '76). Furthermore, single-unit recording in striate cortex indicates that the responses of cells in cats reared with one eye lidsutured and the other eye removed are still abnormal (Kratz and Spear, '76). From these behavioral and physiological results, it might be expected that DLG cell size in cats reared with one eye deprived and the other enucleated also would still be abnormal. Our findings confirmed this, showing that the effects of deprivation in MD-E cats were similar to those in BD kittens. Finally, the present experiment addressed the question of whether visual deprivation has a selective effect upon certain cell groups in the DLG. Previous studies have suggested that the decrease in DLG cell size following deprivation is due to a selective decrease in the sue of large cells, rather than a proportional decrease in the size of all DLG cells (Sherman et al., '72). This suggestion is based upon evidence that DLG cells with Y-type receptive fields are morphologically the largest cells (Hoffmann et al., '72), and that there is a decreased probability of recording Ycells in the DLG following visual deprivation (Sherman et al,, '72; Sherman et al., '75; Hoffman and Cynader, '77). However, direct morphological evidence that the large cells are selectively affected is lacking. Sherman and Wilson ('75) demonstrated that the largest DLG cells were decreased in size following MD in the dog; hawever, they did not determine whether or not the smaller cells were also affected. We attempted to provide information on this question by making separate comparisons between rearing conditions based upon the largest cells present and the smallest cells present in each lamina. By making measurements on a large number of cells in each laminae of each cat, we were able to obtain adequate samples of the largest and smallest cells to allow direct statistical comparisons between groups. It was reasoned that if significant differences

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were present between normally reared and visually deprived kittens in the size of the largest cells present but not in the size of the smallest cells, then conclusive evidence would be provided that deprivation had a selective effect upon the large cells. However, it should be emphasized that if both the largest and the smallest cells showed a decrease in size following deprivation, alternative interpretations of the results would preclude a conclusive answer to this question. METHODS

Subjects and rearing conditions Four groups of animals were studied. The first group consisted of six normally reared kittens. The were studied at the age of four months kittens), five months (1kitten), and eight months (1kitten). The remaining two normal kittens were between four to eight months old, but the exact age was not known. The second group consisted of seven kittens reared with monocular lid-suture (MD). These kittens were studied at 4 months (1 kitten), 4.5 months (1kitten), 5 months (2 kittens), 7 months (2 kittens), and 7.5 months (1kitten) of age. The third group consisted of seven kittens reared with binocular lidsuture (BD). They were studied at 4 months (1 kitten), 4.5 months (1 kitten), 5 months (2 kittens), 7 months (2 kittens), and 7.5 months (1kitten) of age. Finally, six kittens were reared with one eye lidsutured and the other eye removed (MDE). They were studied at 3 months (2 kittens), 4 months (2 kittens), 5 months (1kitten), and 5.5 months (1 kitten) of age. Enucleations were done when the MD-E kittens were four (1kitten), five (2 kittens), six (1 kitten), or seven (2 kittens) days old. Lid-suturing was done prior to the time of natural eye opening for all the deprived kittens. The kittens were born in the laboratory breeding colony and littermates were divided into each of the four groups. Kittens were housed with their mothers in individual cages until they were weaned. They were then moved to a large group

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Histology

colony room with shelves and ramps for climbing. Throughout rearing, the animals were kept on a 12 hours light-dark cycle. The only exceptions to these rearing conditions were two normal kittens which were obtained from an animal breeder just prior to being used (their data did not differ from the other normal kittens). Many of the kittens used in the present study were included in studies of striate cortex neurophysiology (Kratz et al., '76; Kratz and Spear, '76).

Following a lethal dose of pentobarbital sodium all animals were perfused through the heart with 0.9%saline followed by 10% formol saline. For each animal a single block of tissue containing both lateral geniculate nuclei was processed routinely for celloidin embedding and serial sections cut at 52 pm. From this series every fifth section was mounted and stained with cresylviolet.

Lid suture and enucleation Lid-suturing and enucleations were done using halothane (Fluothane) anesthesia in 50% Nz0/50% 0,. For suturing, the lid margins were trimmed and sutured together after a plication of a local anesthetic (XylocaineT. Ophthalmic ointment (Neosporine) was applied to the cornea and sutured lids. For enucleation, the conjunctiva and extraocular muscles were dissected free of the eye, the optic nerve and associated blood vessels were clamped and cut, and the eyeball was completely removed. After haemostasis was achieved, a long-acting local anesthetic (Anucaine) was applied liberally to the remaining orbital tissue. The lid-margins were then trimmed and the eyelids sutured closed. Following both procedures, an intramuscular injection of 60,000 units of a broad band antibiotic (Bicillin) was administered. The kittens were checked once or twice a day throughout rearing for openings along the sutured lid-margins. Animals with large openings were discarded and are not included in the present experiment. In some animals, pin-hole openings occurred which were covered with mucous and apparently did not allow pattern vision. These openings always were resutured immediately. Openings of this sort occurred in five MD cats, three BD cats, and three MD-E cats. The remaining kittens in each group had perfect closures with no detectable openings of any kind. There were no obvious differences in the results for kittens which had pin-hole openings and those which did not.

Cell measurements The cross-sectional areas of 600 lateral geniculate nucleus cells were determined in each of the normal, MD and BD animals studied. Since only the deprived laminae were studied in the MD-E cats, 300 cells were measured in each of these animals. Measurements were made from cells near the middle of the mediolateral extent of the binocular segment of lamina A (100 cells) and A1 (100 cells) as well as from the monocular segment of lamina A (100 cells); i.e., that segment of lamina A that extends beyond the lateral edge of lamina A l . Measurements were made from both hemispheres. All measurements were made at a rostral-caudal level corresponding to a region lying just anterior to coronal 5 (anterior 6.5) described by Sanderson ('71). Outlines of cells showing a well defined nucleolus were drawn at a magnification of x 1,000 using a Zeiss camera lucida. Cells were sampled throughout the dorsal-ventral extent of each lamina studied, although we were careful not to include the large cells that lie in the interlaminar zones. Cell drawings always began at the top of each lamina studied. Once all the cells showing a well defined nucleolus were drawn the slide was moved until a new field of cells was in view just ventral to the previous field. Care was taken to move the slide along a path perpendicular to the long axis of the lamina being studied. After the ventral-most field of cells in a given lamina had been drawn the slide was moved laterally and the procedure repeated with slide movements toward the dorsal border of

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the lamina. Thus, cells were measured in successive dorsal-to-ventral and ventralto-dorsal sweeps across the lamina from border to border. This procedure was continued until 100 cell outlines had been drawn. This usually required two border to border sweeps. Once the cell outlines were drawn their cross-sectional areas were determined with the use of a Numonics Graphics Calculator (Numonics Corporation, North Wales, Pennsylvania). Since it is possible that cell drawings might be influenced by knowledge of the animal's history, i.e., extent of visual deprivation, age, etc., it is important to describe the conditions under which the cells were originally drawn. All animals in this study were sacrificed and the brains processed at Kansas State University. After slides were processed and coded they were mailed to the University of Alabama in Birmingham, where all cell outlines were drawn by T.L.H. Although certain rearing conditions were immediately obvious upon examination of the slides; e.g., monocular suture vs. normal rearing, no detailed information concerning age or length of derivation was available until after all cells gad been drawn. Even at that point this information was kept from the technicians making the area measurements.

Statistical analysis In order to consider animal-to-animal variations in cell size, statistical comparisons were made in the following way. For each cat, a mean of the cells measured in each lamina was obtained. Comparisons between cats in different conditions were then made using the mean from the appropriate lamina in each cat as a single observation. For example, comparison of cell size in lamina A1 of normal and BD kittens was made by comparing mean values from each of the six normal kittens and seven BD kittens, using a t-test for independent groups. Comparisons between laminae for kittens in the same rearing conditions were made in a similar manner, using a t-test for dependent measures. These statistical procedures provide the

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REGION IN NUCLEUS Fig. 1 Cell size in DLG laminae of normally reared kittens.The mean values were first determined in each animal for all 100 cells in each region of the nucleus, and then for the ten largest and ten smallest cells in each region. The mean values for each animal represent an average for the two sides of the brain. These values were then averaged across the six normal animals and the mean f standard error (S.E.) is shown by the shaded region. Similar measures for the ten largest cells (closed circles) and ten smallest cells (open circles) are also shown. For all of the small cell measurements the standard error fell within the area covered by the circle. In this figure, as well as in several of the later figures, the means and S.E. for A, A1 and monocular segment of normal cat have been joined together by the shading for clarity of illustration.

most conservative test that the differences in cell size between animals or between laminae were produced by the different rearing conditions and were not the result of random animal-to-animal variations. Some investigators have made betweengroup comparisons after combining all the cells across animals in each group. Since the cell sizes within a single animal are not independent of each other, combining cells across animals violates the statistical as-

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cleus in normal kittens appears to be primarily the result of differences in size of the largest cells. A systematic difference was observed in the distribution of small and large cells within each of the laminae studied. To illustrate this, cells were measured in an orderly sequence in a single border-to-borRESULTS der sweep across each of the laminae in three normal kittens. These ordered disNormally reared animals tributions of cell areas are shown for one of The six normally reared cats were used the normal kittens in figure 2b. The for making comparisons with animals in the sequential scatter plots reveal that in both other three groups. In addition, the nor- the binocular and monocular segments of mally reared animals afforded new infor- lamina A there was a tendency for the size mation concerning the size and distribution of the cells to increase as one moves away of cells in the different DLG laminae. from the dorsal border of the lamina. For Figure 1 shows the cross-sectional areas example, the ten dorsal-most cells in the of cells in different DLG laminae of these binocular segment of lamina A had a mean animals. There was a consistent within-ani- area of 241.4 p2 while the ten ventral-most ma1 difference in the average size of cells cells had a mean area of 395.9 p2.In lamina in the different portions of the nucleus. A l , on the other hand, the large cells were Lamina A1 cells averaged 9%larger (range distributed fairly evenly across the lamina. 6-14%in different animals) than cells in the Monocular1y deprived animals binocular segment of lamina A (t = 6.00, df In agreement with previous studies, cells = 5, p < 0.01; all p values two-tailed). Cells in the binocular segment of lamina A in the deprived binocular segment of were, in turn, 16%larger (range, 12-25%) lamina A were 33% smaller (range of 20than those in the monocular segment of 42%in different animals) than those in the corresponding portion of lamina A in the lamina A (t = 4.06, df = 5, p C 0.01). Separate analyses were conducted for non-deprived hemisphere. Similarly, derived lamina A1 cells were 34% smaler the largest and smallest cells in each animal. The average cross-sectional areas for 719-4670 ) than non-deprived lamina A1 the ten smallest and the ten largest cells cells in the same animals. Comparisons were computed for each portion of the with the normally reared kittens revealed DLG in each kitten. For the ten smallest that these differences were due to a hypercells, the average area varied little be- trophy of cells in the non-deprived laminae tween lamina A l , the binocular segment of as well as to a decrease in size of cells in the lamina A, and the monocular segment of deprived laminae. This is evident from lamina A in each kitten (fig. 1, open cir- figure 3A which compares the cell size in cles). In contrast, the average cross-sec- the deprived and non-deprived laminae of tional area of the ten largest cells varied MD kittens with measurements made in considerably between different portions of normally reared kittens (shaded area). The the nucleus (fig. 1, closed circles). The mean percent change from normal for the largest cells in lamina A1 were larger than MD kittens is shown in figure 7A (open and those in the binocular segment of lamina A filled dots). Statistical comparisons be(t = 3.83, df = 5, p < 0.02) and these, in tween the seven MD and six normal kittens turn, were larger than the largest cells in indicated that the increase (hypertrophy) the monocular segment ( t = 4.72, df = 5, p in cell size in the non-deprived laminae was < 0.01). Thus, the overall difference in statistically significant in both lamina A1 ( t size of cells in different portions of the nu- = 2.39, df = 11, p < 0.05) and the binocu-

sumption that observations within a sampling distribution are independent. For this reason, it is necessary to make statistical comparisons of this sort using each animal as a single observation. A similar procedure was used for the graphical presentation of the data (figs. 1,3-7).

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Fig. 2 Cell sizes and their distribution in the DLG of an 8-month-old normal cat. a, shows frequency histograms of cross-sectional areas for 100 cells measured in each region of the nucleus in one hemisphere. Each division along the abscissa represent 50 p2 increments. b, shows how the sizes of the cells varied as a function of their position in a given lamina. Each scatter plot represents one dorsal-to-ventral sweep across the indicated lamina. For each cell measured in sequence (cell number, ordinate), a point was plotted indicating the cell's cross-sectional area (abscissa).The plots show that for the lamina A binocular segment and the monocular segment, the sizes of the cells tended to increase toward the ventral border of the lamina.

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lar segment of lamina A (t = 2.54, df 11,p < 0.05).Conversely, cells in the deprived lamina A1 were significantly smaller than normal (t 4.10, df = 11, p < 0.01), as were those in the binocular segment of lamina A (t 4.74, df = 11, p < 0.01).

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The monocular segments of normal kittens were not significantly different from either the deprived or the non-deprived monocular segments of MD kittens. However, within-animal comparisons in MD kittens indicated a significantreduction in the

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Fig. 3 Cell size in DLG laminae of MD cats. The means (2S.E.)for normal kittens are represented by the shaded regions (from fig. 1).A, shows the average (+S.E.) measurements for all cells measured in both the deprived (closed circles) and non-deprived (open circles) laminae of MD kittens. B,shows similar measures for the ten largest (closed symbols) and ten smallest (open symbols) cells. For this graph the values for the deprived and non-deprived laminae are plotted using circles and squares, respectively. For many of the points the S.E. fell within the area covered by the symbol.

size of cells in the deprived monocular segment relative to the non-deprived monocular segment, with an average difference across animals of 10%( t= 2.69,df = 6, p < 0.05). The difference between the deprived and non-deprived monocular segments was related to the age of the animals, as shown in figure 4 (middle curves). Statistical comparisons were made between cells in the deprived monocular segment and those in the non-deprived monocular segment for individual kittens, using all 100 cells measured on each side. Neither of two 4-month-old MD kittens showed a significant difference in cell size between the two sides. Of two 5-monthold kittens, one showed no significant difference between the two sides, and the

other had significantly smaller cells in the deprived monocular segment than in the non-deprived monocular segment ( t = 2.29, df = 198, p < 0.05). All three kittens from six to eight months old showed a highly significant decrease in cell size in the deprived monocular segment compared to the non-deprived side ( t = 3.43 to 4.04, df 198, p < 0.001 in each case). Finally, cell sizes were measured in the deprived and non-deprived monocular segments of a 12month-old MD kitten (not used in any other comparisons), and cells in the deprived monocular segment were again significantly smaller than those in the nondeprived monocular segment (t = 4.99, df = 198, p < 0.001). Thus, monocular deprivation consistently resulted in smaller

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cells in the deprived monocular segment than in the non-deprived monocular segment for kittens six months of age or older, but not for kittens five months of age or younger.

Large vs. small cells Figure 3B shows comparisons between MD and normal kittens in terms of the ten largest and the ten smallest cells in each rtion of the DLG in each animal, and #&re 7 (B,C)shows the percent difference from normal for both large and small cells. Com arisons by age of the animal between the eprived and non-deprived monocular segments for large and small cells are shown in figure 4. In general, the results for both the largest and the smallest cells paralleled those for the total sample. For example, the decreased cell size in the deprived binocular segment of lamina A in MD cats was statistically different from normal for both large ( t 3.98, df = 11, p < 0.01) and small ( t = 3.54, df = 11, p < 0.01) cells. The same was true for lamina A1 (large 11, p < 0.01; small cells: t == 3.93, df cells: t = 2.23, df = 11, p < 0.05). Nevertheless, some differences were resent between the effects seen for the arge and small cells in MD cats. In certain portions of the nucleus, there was a tendency for the percent change in size to be greater for the large cells than the small cells. This was the case only for the decrease in size observed in the binocular segment of deprived laminae (figs. 7B,C). For example, the large cells decreased an average of 30%in the deprived binocular segment of lamina A, while the small cells decreased an average of 19%.Similar results were observed in the deprived laminae Al. In contrast, the percent hypertrophy was the same for both large and small cells in the non-deprived laminae. Further, there was little difference in the ercent change seen for large and small ce 1s in the monocular segments. Of course, since the percent change was the same for large and small cells in these portions of the nucleus, the absolute magnitude of the change in size

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Fig. 4 Increases with age in the differences between deprived and non-deprivedmonocular segment cell size in MD cats. The middle shaded region shows the average cell areas for deprived (solid triangles) and non-deprived (open triangles) lamina cells in MD animals when all cells were used. Similar plots for the ten largest (top shaded region) and ten smallest (bottom shaded region) cells are also shown. In the top and bottom graphs the means for the deprived and non-deprived laminae are plotted using circles and squares, respectively. The number of animals at each age is: two at 4 months, two at 5 months and one at each of the 6 , 7 , 8 and 12 month ages. The 12-monthold animal used here was not included in any other statistical analyses since no other deprived or normal animal was more than 8-months-old.

was consistently greater for the large cells than for the small cells (figs. 3B, 4).

Binocularly deprived animals Figure 5A shows the cross-sectional areas of cells in different portions of the DLG of BD kittens, and compares them with measures made in normal kittens. The percent change in cell size in BD kittens relative to normals is shown in figure 7A (open squares). There was a statistically significant decrease in cell size in BD kittens in both the binocular segment (12%;t = 2.72, df = 11, p < 0.02) and monocular segment (9%; t = 2.33, df = 11, p < 0.05) of lamina A. The decrease in lamina A1

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(7%) was somewhat smaller, and did not lamina A1 or the binocular segment of quite reach statistical significance ( t = 1.56, lamina A (figs. 3B, 5B, 7C). df = 11, 0.05 < p < 0.1). Based on the Animals with one eye deprived and the findings in the monocular segment of MD other enucleated cats it might be expected that the decrease Figure 6A shows the cross-sectional in cell size in BD cats would also vary with the age of the animal. However, our areas of cells in the deprived DLG laminae findings did not show this to be the case; of MD-E kittens, and compares them with i.e., the decrease in cell size in BD animals measures made in normal kittens. The perwas as great for the 4-month-old animals as cent change in cell size relative to normals is shown in figure 7A (open triangles). it was for the older animals. There was a statistically significant deLarge vs. small cells crease in cell size in MD-E kittens in both Similar comparisons were made using the binocular segment ( t = 3.33, df = 10, p the ten smallest and ten largest cells (figs. < 0.01) and monocular segment ( t = 2.23, 5B, 7B,C). The results for both large and df = 10, p < 0.05) of lamina A. There was small cells paralleled those obtained when a smaller (non-significant)decrease in cell all 100 cells sampled in each portion of the size in lamina Al. nucleus were used, and the percent change in size relative to normals was about the Large us. small cells same for both large and small cells (figs. As with the other rearing conditions, the 7B,C). Consequently, the absolute mag- results for both large and small cells paralnitude of the decrease in size was greater leled those obtained when all 100 cells for the large cells than the small cells were used (figs. 6B, 7B,C). Also as in the (fig. 5B). other conditions, the absolute decrease in size was greater for the large cells than the Comparison with MD kittens small cells (fig. 6B). Furthermore, there In the binocular segment of the DLG, was a tendency for the percent decrease to the decrease in cell size following lid- be greater for the large cells than the small suture was greater for MD kittens than for cells, but only in the binocular portion of BD kittens (figs. 3A, 5A, 7A). This was the the nucleus (figs. 7B,C). For example,in the case both in lamina A1 ( t = 3.19, df = 12, p binocular segment of lamina A, the largest < 0.01) and in the binocular segment of cells were 20% smaller than the largest lamina A ( t = 3.10, df = 12, p < 0.01). In cells in normal kittens, while the smallest contrast, the decrease in cell size in the cells were 10% smaller than those in normonocular segment was greater for BD kit- mals. Similar results were observed in tens than for MD kittens. However, the lamina Al. However, in the monocular segdifference between animals in the two ment, both the large and the small cells groups was not statistically significant. showed the same percent decrease (20%) When only the ten largest cells are con- from normal. sidered, very similar results were obtained (figs. 3B, 5B, 7B). The decrease in size of Comparison with BD kittens the largest cells was greater for MD kittens It is clear from the data presented in than BD kittens in both lamina A1 (t = figures 5 and 6 that the effects of visual de3.20, df = 12, p < 0.01) and the binocular privation upon DLG cell size were similar segment of lamina A ( t = 2.44, df = 12, p for BD and MD-E kittens in all portions of < 0.05). When only the ten smallest cells the nucleus. This is also shown in figure 7 in are considered, the effects of visual depri- terms of percent decrease from normal. vation were only slightly greater for MD kittens, and there was no significant difference in mean size of the ten smallest cells Deaflerented laminae in MD-E animals Although no cell measurements were between MD and BD kittens for either

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Fig. 5 Cell size in DLG laminae of BD cats. The means (23.E.) for normal kittens are represented by the shaded regions (from fig. 1). A, shows the average (*S.E.) measurements for all cells measured in the deprived laminae. The mean values for each animal represent an average for the two sides of the brain. B, shows similar measures for the ten largest (closed circles) and ten smallest (open circles) cells. For many of the points the S.E. fell within the area covered by the symbol.

made in the deafferented layers of the DLG in MD-E kittens, one observation is worth noting. While most cells in the deafferented laminae were shrunken due to transneuronal atrophy, a few large cells remained. As in previous studies of neo-natal monocular eye removal (Guillery, '72; Kalil, '72, '73; Hickey, '75) the surviving large cells were clustered near the interlaminar regions. Since there is a very close relationship between translaminar growth of afferentsfrom the remaining eye and the survival of large cells in the deafferented laminae (Hickey, '75; Hickey and Guillery, in preparation), it seems likely that such growth also occurs when the remaining eye is deprived, as in the MD-E kittens. Similar findi s have been reported previously by Kalig73).

DISCUSSION

We investigated the effects of visual deprivation upon DLG cell size in three conditions representing different degrees of competitive balance between the inputs from the two eyes. Comparisons of the relative effects of lid-suture on cells in the monocular and binocular segments of the DLG for kittens in each of these rearing conditions indicate that at least two different mechanisms contribute to the changes in cell size which were observed: deprivation per se and binocular competition. In the discussion that follows, we will consider the results in light of these developmental mechanisms. We will then consider the evidence that large and small cells are differentially affected by the deprivation,

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Fig. 6 Cell sue in the deprived DLG laminae of MD-E cats. The means (+S.E.) for normal kittens are represented by the shaded regions (from fig. 1). A, shows the average (2cS.E.)measurements for all cells measured in the deprived laminae. B, shows similar measures for the ten largest (closed circles) and ten smallest (open circles) cells. For many of the points the S.E.fell within the area covered by the symbol. No measurements were made in the deafferented laminae of MD-E cats.

and finally, the functional significance of the change in cell size will be discussed.

Relative eflects of lid-suture in MD, BD, and MD-E cats Binocular segment Direct comparison between MD and BD kittens indicated that, in the binocular segment of the DLG, lid-suture resulted in a much greater decrease in cell size in the deprived laminae of MD cats than in BD cats. Since the visual inputs to the deprived eyes are relatively equal in MD and BD animals, this difference may be attributed to effects of abnormal binocular competition in the MD animals resulting from placing the deprived eye at a competitive disadvantage. In addition to the decrease in

cell size in the deprived laminae of MD cats, an increase in cell size was observed in the non-deprived binocular segment laminae. This confirms earlier findings by Sherman and Wilson (‘75) in monocularly deprived dogs. Thus, the difference in cell size between deprived and non-deprived laminae in MD animals which has been reported in previous studies undoubtedly reflects both a hypertrophy of cells in the non-deprived laminae and a decrease in size of cells in the deprived laminae. Apparently, hypertrophy occurs only if the eye which is at a competitive advantage also receives normal visual stimulation, since no hypertrophy was seen in the binocular segment of MD-E cats (relative to BD cats) when the deprived eye was at a competitive advantage.

CHANGES IN DLGN CELL SIZE FOLLOWING DEPRIVATION 40

, A: All Cells

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Fig. 7 Percent change in size for cells in MD (circles), BD (squares)and MD-E (triangles)animals.The percent change in average cell size is plotted relative to the mean values for the six normal animals (N).Increases in cell size (hypertrophy) are plotted above the baseline while decreases in cell size are plotted below it. All percent changes represent an average of the individual kittens in each deprivation condition (METHODS).The values determined using all cells, the ten largest cells and the ten smallest cells for each portion of the nucleus are shown in A, B, and C, respectively.

Although the deprived binocular segments of BD animals were affected to a lesser degree than in MD animals, our results indicate that deprivation did result in abnormally small cells following binocular lid-suture, particularly in lamina A. In an earlier study, Guillery (‘73) compared cell sizes from a large sample of normal and BD kittens and found about a 5% reduction in cell size in the BD animals; however, the difference between normal and BD kittens was not statistically significant. In the present study, the decrease in cell size was about lo%,and this was statistically sig-

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nificant in the binocular and monocular segments of lamina A and marginally significant in lamina A l . The difference between the two studies can probably be attributed to differences in sampling techniques (e.g., number of cells per lamina and laminar area sampled) and the ages of the animals studied. Whereas Guillery (‘73) used animals that were three months old, all of our animals were four months of age or older. However, the point to be emphasized is that both Guillery’s (‘73) study and our own demonstrate a small but consistent decrease in cell size in the deprived DLG laminae of BD kittens. Since this decrease was the same in the binocular and monocular segments (see below), it seems likely that the effects observed in the binocular segment of BD kittens represent a pure deprivation effect, independent of abnormal binocular interactions. It has been suggested that in striate cortex of BD cats, abnormal binocular interactions (resulting from asynchronous darklight inputs through the closed lids) may contribute to the effects observed on single neuron response properties (Kratz and Spear, ’76). These asynchronous inputs apparently produce no added effects on DLG morphology in BD kittens, since there were no differences between binocular and monocular segments of these animals or between binocular segments of BD and MD-E animals. This is consistent with the apparent lack of effects on DLG morphology resulting from asynchronous inputs for the two eyes produced by strabismus or alternating monocular occlusion in the cat (Hubel and Wiesel, ’65b). This indicates that the mechanisms of the effects of deprivation may be somewhat different in the DLG and striate cortex (see also Guillery and Kaas, ’74a,b),or that striate cortex neuronal responses and DLG cell size do not measure the same functions. The effects of lid-suture upon DLG cell size were nearly identical in MD-E kittens to those observed in the BD kittens. Thus, placing the deprived eye at a competitive advantage in the MD-E kittens did not ameliorate the effects of the lid-suture.

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This result agrees with a recent behavioral study which found that the ability to orient to and approach stimuli presented in different portions of the visual field was nearly identical for BD and MD-E cats (Sherman and Guillery, '76). Similarly, single unit recording in striate cortex indicates that responses to the deprived eye are abnormal in MD-E kittens (Kratz and Spear, '76). However, the effects of deprivation in striate cortex of MD-E kittens were not as severe as those in BD kittens (Kratz and Spear, '76), and this is different from the close similarity in the effects on DLG cell size seen in the present study. This again suggests that either the effects of deprivation are somewhat different in the two structures or that striate cortex response properties are not measuring the same processes as DLG cell size. Guillery ('72) studied MD cats with a small retinal lesion in the non-deprived eye and observed that the effects of lid-suture on DLG cell size were less severe in the critical segment of the deprived lamina (adjacent to the denervated segment of lamina) than in segments adjacent to normally innervated lamina. Our results are consistent with these findings in that cells in the deprived laminae of MD-E cats were less severely affected than those in deprived laminae of MD cats. Nevertheless, a significant decrease in cell size (11-17%) was present in the deprived laminae of MD-E cats. It would be of interest to know whether this represents an effect which is different from that seen in a critical segment. Guillery ('72) reported that cells in the critical segment were about 10%smaller than cells in the non-deprived laminae of the same cats, a difference which appears to be statistically reliable. However, this difference could be due to hypertrophy in the non-deprived laminae rather than to a decrease in the critical segment, a possibility pointed out by Guillery ('72). This possibility seems very likely in view of the present results which demonstrated a 10-15%hypertrophy of cells in the non-deprived laminae of MD cats. However, conclusive evidence on whether

cells in the critical segment show no morphological effects of deprivation requires direct comparisons with normally reared animals.

Monocular segment The decreased cell size seen in the monocular segment of lid-sutured cats reflects a deprivation effect which is independent of abnormal binocular competition, since there are no binocular projections to this portion of the nucleus. In MD cats, the decrease was approximately 5% relative to normal kittens, and 10%when one side was compared with the other in the same kitten. This change is even greater when only the older kittens are considered. The presence of an effect of lid-suture upon cells in the DLG monocular segment of MD kittens is consistent with von Noorden's ('75) results in the DLG monocular segment of monocularly lid-sutured monkeys. It also is consistent with morphological abnormalities found in striate cortex monocular segment of MD squirrels (Guillery and Kaas, '74b) and physiological abnormalities in striate cortex monocular segment of MD cats (Wilson and Sherman, '75). However, our results appear to be at variance with previous studies in cats which found no effect of monocular lidsuture on monocular segment cell size in the DLG (Guillery and Stelzner, '70; Guillery, '72; Garey et al., '73). There are two possible explanations for these differences. The first concerns the manner in which cells were sampled for measurement. In previous studies, cells were sampled in the mid-portion of the lamina, away from the laminar borders. We have shown that the largest cells in the monocular segment tend to cluster near the ventral laminar border (fig. 2b), and that the absolute magnitude of the decrease in cell size following lidsuture is greatest for the large cells. In the present study, the border-to-border method of sampling cells would include these large cells, whereas they may have been omitted in previous studies. A second possible difference between the present study and previous reports concerns the

CHANGES IN DLGN CELL SIZE FOLLOWING DEPRIVATION

age of the animals. Previous studies of cell size in the monocular segment mf MD cats have used animals three to four months of age or younger (Guillery and Stelzner, '70; Guillery, '72; Garey et al., '73). Our own measurements in the monocular segments of four-month old MD kittens also failed to find significant differences between the deprived and non-deprived sides. These differences appeared consistently only in kittens six months of age or older. In BD and MD-E cats, cell size in the monocular segment was significantly smaller than in normal cats. This decrease was very similar to that seen in the binocular segment of lamina A, a finding also reported by Guillery ('73) for BD cats. The reductions in cell size in the monocular segments of BD and MD-E cats tended to be greater than those observed in the monocular segment of MD animals (fig. 7A); however, this difference was not statistically significant. Nevertheless, it is clear that in all three rearing conditions, some decrease in cell size results in the DLG monocular segment independent of abnormal binocular competition.

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i.e., that all cells (both large and small) are decreased by about the same proportion in each case, with the exception that the large cells are decreased in size by a slightly greater amount in the binocular segment of h4D and MD-E cats. If this interpretation is correct, it has several important implications. The first is that the effects of deprivation per se were about the same for both large and small cells in the DLG, since the percent change from normal was about the same for large and small cells in the binocular segment of BD kittens. A second implication is that the added decrease in cell size which occurs when abnormal binocular competition is present appears to be the result of a selective added effect upon the large cells. This is shown by the greater percent decrease in cell size for the large cells in the binocular segment of MD cats, a difference not seen in the binocular segment of BD cats or the monocular segment of any group of cats. A second ssible interpretation of these findings is t at only the large cells in the de rived laminae decrease in size and that folpowing deprivation they become smaller than the smallest cells which are normally Large vs. mull1 cells present. As a result, both the smallest cells Separate analyses for the ten largest and measured and the largest cells measured in the ten smallest cells in each lamina of the deprived laminae would be smaller each cat indicated that differences ob- than normal, even though it was only the served for the total sample of cells were large cells that actually changed. At the paralleled in every case by differences in present time, it is not possible to conclusize of both the largest cells present and sively decide between these two possibilthe smallest cells present. In most cases, ities or to rule out other possibilities which the percent change in size was the same for might combine features of the two (e.g., the large and the small cells. Consequently, both cell groups decrease in size, but in the absolute change in cell size (in terms of different proportions). Until it becomes p2 of cross-sectional area) was greater for possible to selectively label the large and the large cells. In two cases there also was small cells prior to deprivation and follow a greater percent change for the large cells their course of development, this question than for the small cells. This was observed must remain unanswered, at least for the for the decrease in size which occurred in cat. the binocular segment of MD and MD-E Functional considerations kittens. The present morphological results are in There are several possible interpretations of these findings. One possibility is good general agreement with the results of that the obtained measurements of the single unit recording in the DLG of both largest and smallest cells may accurately MD and BD cats. Physiological studies by reflect the actual processes taking place; Sherman et al. ('72) indicate that in BD

r

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cats there is a reduction in the percent of cells with Y-type receptive fields which occurs throughout the nucleus, including both the binocular and monocular segments. In addition, they found that the decrease in the proportion of Y-cells was greater in the binocular segment of MD cats than in BD cats, whereas the decrease was greater in the monocular segment of BD cats than in MD cats. Further, there is a suggestion that a greater than normal proportion of Y-cells was recorded in the nondeprived laminae of MD cats than in normal cats. These results all coincide with the mean cell-size changes described in the present study. The only qualitative point of disagreement with the resent results was that Sherman et al. ('72 did not observe a decrease in the proportion of Y-cells in the deprived monocular segment of MD cats relative to the non-deprived monocular segments, whereas our results indicate that cells in the deprived monocular segment of MD cats are consistently smaller than cells in the non-deprived monocular segment. Although our overall morphological results are in good agreement with the electrophysiology, it was not possible for us to determine whether the decrease in Y-cells following visual deprivation could be attributed to a selective loss of the large DLG cells (Sherman et al., '72).

P

Conclusions The principal findings of the present investigation are that, (a) in addition to the decrease in size of cells in the deprived binocular segment laminae of MD cats, cells in the non-deprived binocular segment laminae show a significant hypertrophy in size; (b) there is a significant decrease in size of cells in the deprived monocular segments of MD cats six months of age or older; (c) cells in both the binocular and monocular segments of BD cats are decreased in size compared to normally reared cats, but this decrease is not as great as that seen in the deprived binocular segments of MD cats; and (d) cats reared with one eye deprived and the other eye removed show effects of deprivation on DLG

cell size which are very similar to those seen in BD cats. These results indicate that both abnormal binocular competition and deprivation per se affect cell size in the DLG of visually deprived cats. ACKNOWLEDGMENTS

We thank Donna Whitfield for her excellent assistance with the histological preparation and Gloria Avery, Priscilla Robinson and Patrice Smith for making the thousands of area measurements.We also thank Jim Andrews and Patrice Smith for assistance with the illustrations and photography and Hazel Davis for secretarial help. Help with the statistical analyses and computer programming was kindly supplied by Mr. L. R. Smith and Doctor David C. Hurst from the Department of Biostatistics. We also thank R. W. Guillery and S. Murray Sherman for critical reading of the manuscript. LITERATURE CITED Casagrande, V. A., R. W. Guillery, J. K. Harting and I. T. Diamond 1974 Effects of visual deprivation on the LGN of TupaM glis. Proc. A. R.V. 0. Chow, K. L., and D. L. Stewart 1972 Reversal of structure and functional effects of long term visual deprivation in cats. Exptl. Neurol., 34: 409-433. Dursteller, M. R.,L. J. Garey and L. A. Movshon 1976 Reversal of the morphological effects of monocular deprivation in the kitten lateral geniculate. J. Physiol., 261: 189-210. Fukuda, Y., and J. Stone 1974 Retinal distribution and central projections of y, x and w cells of the cat's retina. J. Neurophysiol., 37: 749-772. Garey, L. J., R. A. Fisken and T. P. S. Powell 1973 Effects of experimentai deafferentation on cells in the lateral geniculate nucleus of the cat. Brain Res., 52: 363-369. Guillery, R. W. 1972 Binocular competition in the control of geniculate cell growth. J. Comp. Neur., 144: 117-127. -1972 Experiments to determine whether retinogeniculate axons can form translaminar collateral sprouts in the dorsal lateral geniculate nucleus of the cat. J. Comp. Neur., 146: 407-420. 1973 The effect of lid suture upon the growth of cells in the dorsal lateral geniculate nucleus of kittens. J. Comp. Neur., 148: 417-422. Guillery, R. W., and J. H.Kaas 1974a The effects of monocular lid suture upon the development of the lateral geniculate nucleus in squirrels (Sciureus Carolinensis). J. Comp. Neur., 154: 433-441. - 1974b The effects of monocular lid suture upon the development of the visual cortex in squir-

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rels (Sciureus Carolinensis). J. Comp. Neur., 154: 443-452. Guillery, R. W., and D. J. Stelzner 1970 The differential effects of unilateral lid closure upon the monocular and binocular segments of the dorsal lateral geniculate nucleus in the cat. J. Comp. Neur., 139: 413-422. Headon, M. P., and T. P. S. Powell 1973 Cellular changes in the lateral geniculate nucleus of infant monkeys after suture of the eyelids. J. Anat. (London), 116: 135-145. Hickey, T. L. 1975 Translaminar growth of axons in the kitten dorsal lateral geniculate nucleus following removal of one eye. J. Comp. Neur., 161: 359382. Hoffmann, K. P., and M. Cynader 1977 Functional aspects of plasticity in the visual system of adult cats after earl monocular deprivation. Proc. Roy. Soc.,London &), in press. Hoffmann, K. P., J. Stone and S. M. Sherman 1972 The relay of receptive field properties in the dorsal lateral geniculate nucleus in the cat. J. Neurophysiol., 35: 518-531. Kalil, R. 1972 Formation of new retinogeniculate connections in kittens after removal of one eye. Anat. Rec., 172: 339-340. 1973 Formation of new retinogeniculate connections in kittens: Effects of age and visual experience. Anat. Rec., 175: 353. Kratz, K. E., and P. D. Spear 1976 Effects of visual deprivation and alterations in binocular competition on responses of striate cortex neurons in the cat. J. Comp. Neur., 170: 141-151. Kratz, K. E., P. D. Spear and D. C. Smith 1976 Postcritical-period reversal of effects of monocular deprivation on striate cortex cells in the cat. J. Neurophysiol., 39: 501-511. Kupfer, C., and P. Palmer 1964 Lateral geniculate nucleus: Histological and cytochemical changes following afferent denervation and visual deprivation. Exp. Neurol., 9: 400-409. Sanderson, K. J. 1971 The projection of the visual field to the lateral geniculate and medial interlaminar nuclei in the cat. J. Comp. Neur., 143: 101117.

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Sherman, S. M. 1973 Visual field defects in monocularly and binocularly deprived cats. Brain Res., 49: 25-45. Sherman, S. M., and R. W. Guillery 1976 Behavioral studies of binocular competition in cats. Vis. Res., 16: 1479-1481. Sherman, S. M., R. W. Guillery, J, H. Kaas and K. J. Sanderson 1974 Behavioral, Electrophysiological and morphological studies of binocular competition in the development of the geniculo-cortical pathways of cats. J. Comp. Neur., 158: 1-18. Sherman, S. M., K. P. Hoffmann and J. Stone 1972 Loss of a specific cell type from the dorsal lateral geniculate nucleus in visually deprived cats. J. Neurophysiol., 35: 532-541. Sherman, S. M., and J. R Wilson 1975 Behavioral and morphological evidence for binocular competition in the postnatal development of the dog’s visual system. J. Comp. Neur., 161: 183-196. Sherman, S. M., J. R. Wilson and R. W. Guillery 1975 Evidence that binocular competition affects the postnatal development of Y-cells in the cat’s lateral geniculate nucleus. Brain Res., 100: 441-444. Stone, J. 1973 Sampling Properties of microelectrodes assessed in the cat’s retina. J. Neurophysiol., 36; 1071-1079. von Noorden, G. K. 1973 Histological studies of the visual system in monkeys with experimental amblyopia. Invest. Ophthalmol., 12: 727. von Noorden, G . K., and P. R. Middleditch 1975 Histology of the monkey’s lateral geniculate nucleus after unilateral lid closure and experimental strabismus: further observations. Invest. Ophthalmol., 14: 674-683. Wiesel, T., and D. Hubel 1963 Single-cell responses in striate cortex of kittens deprived of vision in one eye. J. Neurophysiol., 26: 1003-1017. 1965a Comparisons of the effects of unilateral and bilateral eye closure on cortical unit responses in kittens. J. Neurophysiol., 28: 1029-1040. Wan, Y. K., and B. C r a g 1976 Cell growth in the lateral geniculate nucleus of kittens following the opening or closing of one eye. J. Comp. Neur., 166: 365-372.