Transneuronal retrograde degeneration of retinal ganglion cells after damage to striate cortex in macaque monkeys: Selective loss of Pβ cells

Neuroscience Vol. 29, No. I, pp. 65-80, 1989 Printed in Great Britain

0306-4522/89 $3.00 + 0.00 Pergamon Press plc 0 1989 IBRO

TRANSNEURONAL RETROGRADE DEGENERATION OF RETINAL GANGLION CELLS AFTER DAMAGE TO STRIATE CORTEX IN MACAQUE MONKEYS: SELECTIVE LOSS OF P/? CELLS* A. COWEY,~ P. STOERIG~ and V. H. PERRY? TDepartment SInstitute

of Experimental for Medical

Psychology,

Psychology,

University of Oxford, U.K. Ludwig-Maximilians-University,

South Parks

Road,

Goethestrasse

Oxford

OX1 3UD,

31, Munich,

F.R.G.

Abstract-We examined the retinae of two monkeys whose left striate cortex had been removed eight years previously and compared the transneuronally degenerated hemiretina of each eye with the normal hemiretina, and with the retinae of normal monkeys. All retinae were prepared as whole mounts. One from each pair was stained with Cresyl Violet; the other was reacted for horseradish peroxidase two days after placing pellets of the enzyme in the optic nerve. Measurements of ganglion cell density in the Nissl-stained retina of the contralateral right eye showed that approximately 80% of retinal ganglion cells were missing in the central 30” of the degenerated hemiretinae. More peripherally the percentage loss was less extensive. Measurements of cell soma size and dendritic field size of peroxidase-labelled classified surviving cells in the degenerated temporal hemiretina of the ipsilateral eye showed them to be morphologically normal. In comparison with the normal hemiretina, however, the mean soma size at three selected eccentricities was larger than normal, suggesting selective loss of smaller ganglion cells. Classification of peroxidase-labelled ganglion cells in the normal and degenerated hemiretinae revealed that the population of PB cells was reduced by as much as 85% in the degenerated region. There was no comparable change in the density of Pa or Py cells. The degeneration of the great majority of Pp cells, which are believed to be the morphological substrate of ganglion cells with small and colour-opponent receptive fields, must set limits on the visual sensitivity and discrimination that survive damage to striate cortex.

In the retina of primates the ganglion cells form three major morphological classes,27.42.43.46with well segregated sub-cortical targets. Pa cells (called M cells by Shapley and Perry, 55A cells by Leventhal et aL2’ and probably corresponding to the parasol cells of Polyak,@’ and to the diffuse stratified cells of Boycott and Dowhng’) make up not more than 10% of the total population. They project to the two magnocellular layers of the dorsal lateral geniculate nucleus (dLGN), where neurons have large receptive fields, no clear wavelength opponency, high contrast gain2’ and are heterogeneous with respect to the extent of linear (X-like) or non-linear (Y-like) spatial summation (see Shapley and Perry,” for review). Morphologically PO:cells resemble the alpha cells of a large variety of other mammals.38 Pp cells (called P cells by Shapley and Perry,55 B cells by Leventhal et aL2’ and probably corresponding to the midget ganglion cells of Polyak4’) project to the parvocellular layers of the dLGN, where the neurons have small and predominantly

*There is still no agreed terminology for retinal

ganglion cells in monkeys. Several synonymous terms are in common use. Here we refer to Pa, PB and Py cells, as in our previous papers, without wishing to imply that they are necessarily homologous with or analogous to G(,/I and y cells of other mammals, or that other terminologies are inferior or incorrect. Abbreviations: dLGN, dorsal lateral geniculate nucleus; HRP, horseradish peroxidase.

wavelength-opponent receptive fields, low contrast gain and linear spatial summation. With a proportion of at least 80% they form the largest class of retinal ganglion cells. The third class, the Py cells, constitute the remainder at not more than 1O%.43 They are morphologically heterogeneous and project to the mid-brain, where their target cells are likewise physiologically heterogeneous. Relatively little is known about their functional properties, but wavelength opponency is rare or absent.“.29.52 A lesion in the optic radiations or striate cortex that produces a homonymous visual field defect, causes extensive retrograde degeneration in the dLGN within weeks,32 and leads to a much slower transneuronal degeneration in the retinal ganglion cell layer.60,6’ Eight years after total removal of striate cortex, 80% of ganglion cells in the macular region have died.’ Are all types of ganglion cell subject to retrograde transneuronal generation? Of the three classes of ganglion cells, the Py cells are the least likely to degenerate because they project to the mid-brain, not to the dLGN. Although this has not been investigated in the monkey, y cells do not degenerate after removal of striate cortex in cats.37’58In contrast to the Py cells, the Pa cells do project to the dLGN and might consequently be expected to degenerate. However, despite the fact that both magno- and parvocellular dLGN layers degenerate after damage to striate cortex, and less than 1% of the principal cells remain, a retinal 65

66

A.

COWEY

projection to the nearly empty layers can still be revealed by anterograde transport of radioactive amino acid6* or horseradish peroxidase (HRP) (Cowey et al., unpublished observations). This residual input appears more prominent in the magnocellular layers, possibly indicating a lesser loss of Pa cells. However, van Buren60,6’ reported that the degeneration particularly affected the largest ganglion cells which are now known to be Pc1.46,47 Finally, the P/3 ganglion cells must be involved in the degeneration. As approximately 80% of ganglion cells die in the central retina, Pee and Py cells that together form about 20% of the population cannot alone account for such loss. This conclusion is in accordance with the massively diminished projection to the parvocellular layers of dLGN,68 as well as with results that show a transneuronal loss of beta cells in the cat, although the extent of this loss is only of the order of 20% when striate cortex is removed in the adult.‘.” It would be informative to know whether all three classes of ganglion cells are equally involved in the degeneration, or whether they are differentially affected. The population of retina1 ganglion cells determines what information enters the visual system, and thereby sets the limits to the extent of visual processing that is still possible in the field defects that result from post-geniculate lesions. Although the changes in visual sensitivity that occur as a result of post-geniculate damage are present immediately, and therefore do not depend on retinal degeneration per se, the latter presumably indicates which ganglion cells have been disconnected from the cortex and are therefore not contributing to residual sensitivity. The differential reduction in the retinogeniculate projection led Weller et a1.68 to suggest that these residual visual functions (that include detection, localization and discrimination of stimuli presented within the blind field”) were mediated by neurons with transient and broadband (Y-like) physiological properties. However, as there is evidence that both X- and Y-like cells (classified according to the linearity of their spatial summation) occur in the magnocellular layers,*” this proposal cannot be correct unless only the Y-like magnocellular LGN are selectively preserved. A similar proposal by Cowey was based on the supposition that Y-like cells project to both the dLGN and the midbrain, as they do in cats, but in fact only a tiny minority of Pa cells projects to the tectum in primates.43 In addition, there is some evidence for the discrimination of shape3’,“6 and wavelength2.4,57 in “blindsight”, although this is not unequivocal.‘~‘s~28~4’~~ These functions are generally assigned to the parvocellular system with its small and predominantly wavelengthopponent receptive fields, leading to the expectation that at least some Pb cells that provide its retinal input should still be present. Most often, however, the residual visual functions have been attributed to the retinocollicular projection,‘.40.49.66.70 which is assumed to be unaffected by the cortical lesion. The present

e/ al

RIGHT

LEFT STRIATE CORTEX

dLGN

RETINA

NISSL STAINED

HRP-FILLEO

Fig. I. Schematic diagram of the experimental procedure. In animals LSI and LS2 the left striate cortex was removed, the left dLGN was degenerated, and the ganglion cells of the left half of each retina showed transneuronal degeneration. In the left eye the degenerated temporal hemiretina and normal nasal hemiretina were compared by labelling ganglion cells from the left optic nerve. In the right eye the nasal hemiretina was degenerated and it was compared with the normal temporal hemiretina by staining all ganglion cells with Cresyl Violet. In the control animals both sides of the retinae were normal. N = nasal retina; T = temporal retina.

anatomical investigation may help to solve these riddles by showing which ganglion cells survive even though it cannot show whether the functional properties of the survivors are unaltered. Selective loss could also help to explain the still mysterious causes of retrograde transneuronal degeneration, which has been reported in only a handful of other pathways, notably in the ventral tegmental nucleus following cortical lesions, Betz cells of the motor cortex following amputation of a limb, and inferior olive after cerebellar damage (see Ref. 7 for review). We therefore examined the retinae of two monkeys whose entire left striate cortex had been removed eight years before the retina was labelled. In the left eye ganglion cells in the normal nasal hemiretina and in the transneuronally degenerated temporal hemiretina were labelled with pellets of HRP placed into the left optic nerve. These cells were classified according to soma size and dendritic branching pattern in order to estimate the proportion of Pa, P/3 and Py cells. The retina of the right eye was stained with Cresyl Violet and ganglion cell density was assessed in the degenerated nasal retina and normal temporal retina. These arrangements are shown in Fig. 1. The results were compared with those from monkeys whose retinae were normal. EXPERIMENTAL

PROCEDURES

Subjecrs We used four wild born male monkeys (Macaca mulatta). Monkey C2 was normal and formed part of a previous study

Transneuronal

retinal

of retinal ganglion cells in monkeysti The second animal (Cl) had had its rostral inferior temporal cortex removed several years earlier. As there is no evidence that this has any effect on the eye, this animal served as a second control. In the other two animals (LSl and LS2) the left striate cortex was removed eight years earlier in an investigation of the behavioural effects of unilateral brain lesions. The three operated animals were young adults when the cortical ablations were made. All were mature adults when the retina was labelled. In addition we examined the retinae of a fifth macaque monkey in which one optic tract had been severed for a year and in which HRP was then placed in one optic nerve as in the other four monkeys. Surgery All surgical procedures were performed under strict aseptic conditions with the aid of a stereo operating microscope. The animal was first sedated with 10 mg/kg of Ketamine hydrochloride (Ketalar, Parke-Davis) i.m., then deeply anaesthetized for the duration of the surgery with sodium thiopentone i.v. (Intraval, May and Baker). At the end of the operation each monkey received 300,000 units of penicillin G. i.m. (Penidural L-A, Wyeth). All animals recovered promptly and without complications. Removal ofstriate cortex. We cut skin and fascia over the left occipital lobe, removed the bone over the lateral surface of the left striate cortex, and cut the dura to expose the cortex caudal to lunate sulcus. A brain spatula was used to severe and remove the occipital lobe approximately 5 mm behind the lunate sulcus. The remaining striate cortex on the lateral surface caudal to the lunate sulcus, and within the anterior calcarine sulcus, was then removed by sub-pial aspiration. Optic nerve deposits. The left optic nerve was exposed just behind the sclera by a dorsolateral approach through the bony orbit. A small cut was made in the lateral and medial aspects of the nerve (corresponding to temporal and nasal retina respectively) and a gel of HRP15 was placed in each cut. The membranous sheath around the nerve was drawn together and sealed with cyanoacrylate glue and a small piece of cellophane. The cut edges of fascia and skin were smeared with sterile xylocaine ointment before being sutured. The control animals then recovered from anaesthesia. The two unilaterally destriated animals remained anaesthetized for the next 48 h while electrophysiological recordings were made from cortical and subcortical visual structures in experiments that are not reported here. Although anaesthesia conceivablv influences retrograde transport of HRP, this would affect the entire retina and therefore not influence naso-temporal comparisons. HRP was also implanted into pre-striate cortex rostra1 to the occipital lobectomy and the results of this will be presented separately. Histology After 48 h the animal was given a lethal dose of sodium pentobarbitone, i.v., and first perfused transcardially with 5OOml of 0.9% saline. After removing the eyes into 1% paraformaldehyde in 0.1 M phosphate buffer at pH 7.2 at 4%, the retinae were rapidly dissected free in the same solution. The left retina was then coverslipped on a glass slide in 2% glutaraldehyde and drawn at a magnification of x 5. After 1 h it was washed in phosphate buffer for a further hour, then reacted for HRP activity according to the method of Hanker et al.16 as modified by Perry and Linden.45 The retina was again washed in phosphate buffer, mounted on a gelatinized slide, dehydrated and cleared, and then redrawn in order to assess shrinkage. There was little or no shrinkage in the central 30”. within which the measurements in the present experiment were made. The right retina was dissected free into formal-saline, drawn, mounted on a gelatinized slide in formal alcohol for 24 h, then stained with Cresyl Fast Violet. After removing the eyes the perfusion of the brain was continued with 2 1 of a mixture of 1.25% paraformaldehyde

degeneration

61

and 2.5% glutaraldehyde over 30min. The brain was removed, photographed, blocked in the coronal plane just rostra1 to the dLGN, and infiltrated with 30% sucrose in phosphate buffer at 4°C. It was then sectioned at 50 pm on a freezing microtome and a I-in-10 series of sections throughout the block was stained with Cresyl Fast Violet. Several other series were reacted for HRP using the same method, or with tetramethyl benzidene as the chromagen and ammonium heptomolybdate as the stabilizing agent.3s Analysis Extent of loss of ganglion cells. In order to estimate the total extent of ganglion cell degeneration, without respect to cell type, we counted ganglion cells in the flat-mounted Cresyl-stained right retinae of the two animals with long standing removal of the left striate cortex. This was done as follows. At a magnification of 1000 x , two of us independently counted ganglion cells in a 90 x 90 pm grid at intervals of 0.5 mm along the horizontal meridian in nasal and temporal retina. The same procedure was used to count ganglion cells in dorsal and ventral retina along a line parallel to the vertical meridian and 2mm into the nasal (degenerated) hemiretina. Glial cells and amacrine cells were excluded from the counts. Amacrine cells were distinguished from ganglion cells by their darker nucleus, inconspicuous or absent nucleolus and sparse cytoplasm and Nissl substance. As identical methods and the same two observers were used in examining normal retina,& the present results could be compared with those obtained from four normal retinae in the previous study. However, in the present study the retina of the right eye had to be fixed with formalin by immersion after dissection rather than by formalin perfusion, which would have impaired the HRP reaction in the other eye. As a result the fixation was not sufficiently good to provide the excellent Nissl staining needed to count ganglion cells and discriminate them from amacrine cells in the central 1-2 mm of the retina, where the cells are most densely packed. Consequently this region was excluded from the present analysis. Cell body size and dendritic jield size of identtjied cells. In a degenerated retina the surviving cells might be unusual in that their dimensions may have changed, or they could form a sub-group of normal cells with dimensions that differ from the mean of their group. We therefore drew and measured identified Pa and Pp cells from the degenerated temporal retina of the two experimental animals and compared them with our previously published results for normal temporal retina.& The same criteria were used to select cells for measurement, i.e. densely-filled dendrites that could be followed and drawn without being confused with dendrites from neighbouring cells. We selected cells to cover eccentricities from 0.5 to 13 mm, i.e. approx. 2-60”.” Cell body size. At any particular eccentricity and in a similar radial direction from the centre of the fovea P/l cells have smaller cell bodies on average than Pa cells.46 We therefore compared cell body size at three eccentricities (5, IO and 20”, see Fig. 3) in the degenerated temporal and normal nasal retina of the left eye of the two unilaterally destriated monkeys, and made similar measurements in the two control monkeys. The precise positions chosen could not be identical in all four animals because the retinal distribution of the most densely labelled cells was not identical in all four. Within 2mm (about IO’) of the centre of the fovea, ganglion cell density at any particular eccentricity is similar in all radial directions.46 It was therefore not necessary to select identical radial directions in order to match cell density, which might itself correlate with cell size. However, cell density at greater eccentricities differs substantially in different radial directions, especially nasally and temporally along the horizontal meridian. The measurements at 20” were therefore made in nasal and temporal retina close to the vertical meridian, as shown in Fig. 3. At each region chosen for measurement of cell body size we drew the outline of the soma of the first 200 labelled cells, provided the labelling

68

A. COWEY et al

Fig 2. A and B are diagrams of a lateral view of the left hemisphere of the unilaterally destriated monkeys. Ablated tissue is shown by shading. Coronal sections at the levels indicated alongside each brain show the extent of the missing tissue in the calcarine fissure and on the ventral surface. Striate cortex of the normal right hemisphere is shown by the dashed line. C and D are photomicrographs of coronal sections through the degenerated and normal dLGN respectively. Cresyl Violet stain. E shows a solitary surviving neuron, centre, in layer 4 of the degenerated dLGN and F shows neurons from the matching part of the normal dLGN. Scale bars: C and D = I mm; E and F = 50pm.

was sufficiently intense to reveal the outline unambiguously. The same criterion was used in nasal and temporal retina. Under oil immersion at a magnification of 1000 x , and using a IO x 10 eyepiece graticule with a drawing tube attached to the microscope, we drew the outline of cells in a square array covering 100 x 100 pm. The graticule was then moved 100 ltrn laterally or vertically until 200 cells had been encountered. The total area sampled depended both on cell density and the extent of the labelling and varied from 200 x 300 pm to

500 x 900 pm. Cell soma size varies slightly with eccentricity even within a sampled area, of course, and this variation will be greater the larger the area. However, the mean soma size should not depend on small differences in the size of the area sampled as long as the geometric centre of the area remains in the same place. Using an X-Y plotter attached to linear potentiometers on the microscope stage the outline of the retina was drawn and each area sampled was accurately marked. Its centre is shown in Fig. 3. The angular eccentricity

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from neighbouring cells, were recorded in order to assess their proportion. Cell bodies containing only a few granules of reaction product were not recorded at all. Classification was carried out using the same sampling procedure as for cell body size. However, in order to match the area of the sampled regions the number of cells classified varied considerably, as shown in Table 1; however, at least 50 cells were positively classified in each sample.

RESULTS

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Fig. 3. Diagrams of the retinal positions at which the size of HRP-labelled cell bodies was determined (0) and at which well-filled cells were classified (*). In each case the symbol is placed over the centre of the sampled area. The total extent of the sampled area varied with density of labelled cells, which itself depended partly on eccentricity, degeneration and whether the sample was nasal or temporal. The black circle indicates the optic disk. The dashed lines intersect at the centre of the fovea and indicate the horizontal and vertical meridians. The former was determined from the position of the fovea, optic disc and macular blood vessels. The latter was drawn at right angles to it, but was actually visible in the degenerated retinae as a result of the sharp change in the density of labelled cells and as portrayed in Fig. 4.

Cortical lesions No striate cortex remained in the left hemisphere of the two experimental monkeys. As shown in Fig. 2 the calcarine fissure was missing. A comparison of coronal sections from right and left hemispheres also shows that parts of area 18 immediately adjacent to area 17 in the lunate and inferior occipital sulci, and on the ventral surface of the occipital lobe were also missing. In both monkeys the entire dLGN was degenerated (see Fig. 2). It contained only occasional solitary neurons, as described by Yukie and Iwai.69 Their synaptic relationship to surviving retinal ganglion cells will be described in a separate report.

Table I. The numbers and overall percentage, of Pa, P/I and Py ganglion cells at several eccentricities in the nasal and temporal retina of one normal monkey (C2) and two unilaterally destriated monkeys in which the temporal retina of the left eye showed retrograde transneuronal degeneration

LSI was determined from the conversion factor of 223 pm/” as measured in the intact eye of rhesus monkeys by Perry and Cowey.” We measured the areas of outline drawings of the labelled cells with a digitizing tablet and microprocessor. Classification of labelled cells. Although cell body size correlates strongly with cell type it may not distinguish between Pp cells and the majority of Py cells, or between Pa cells and the rare large-bodied Py cells. We therefore classified cells as Pee, Pfi, or Py on the basis of their dendritic branching patterns in both the abnormal retinae and in one normal retina. In each retina at least four regions were chosen at roughly corresponding eccentricities and radial directions. The principal regions were 10 and 20” eccentric in nasal retina, and 20 and 30” eccentric in temporal retina. As normal cell density is higher nasally than temporally we chose different eccentricities in nasal and temporal retina in an attempt to compare regions that have similar cell densities in a normal retina. The regions are shown in Fig. 3. Some coincide with the regions used to measure cell body size. Their choice depended on the following considerations. A cell cannot be adequately classified unless its dendritic field is well-filled. Therefore, a majority of the cells had to have densely filled dendrites, as shown in Fig. 5. Classification is also unreliable if cell density and the proportion of cells labelled are both high, because it is impossible to disentangle overlapping dendrites and trace them from origin to termination. Areas were therefore selected in which labelled cells were well-filled but did not overlap extensively. The criteria for classifying P/I, Pa and Py cells have been described elsewhere.4),4h In each area sampled, densely labelled cells that could not be classified because their dendrites were not correspondingly well-filled or were too enmeshed with those

Eccentricity

Pa

PB

PY

5”N IO”N 20”N 30”N

3 8 6 28

27 50 59 120

0 0 3 22

Total

45 (13.8%)

256 (78.5%)

25 (7.7%)

5’T IO”T 20”T

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Total LS2

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30 54 32 116(46.1%) 79 61 32

0 2 24 26(10.3%)

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Total

27 (1 I .9%)

9”T 10”T 20”T

21 27 19

Total

67 (49.6%)

20”N 30”N

12 19

91 132

9 15

Total

31 (11.2%)

223 (80.2%)

24 (8.6%)

10”T 20”T

I1 7

Total

18 (10.3%)

172(76.2%)

6 16 5 27 (11.9%)

7 21 13

5 3 19

41 (30.4%)

27 (20%)

62 91 153 (87.4%)

0 4 4 (2.3%)

Cells were classified on the basis of their denritic pattern. The precise positions of the centres of the regions sampled are shown in Fig. 3. Eccentricity is given to the nearest degree and refers to the centre of the area sampled. N = nasal retina; T = temporal retina.

A. COWEK et al.

70

Fig. 4. A shows the positions at which we counted HRP-labclled cells in the left retina and the Nissl-stained “large” cells in the right retina of the unilaterally destriated monkeys. B shows the macular region of the left eye of a macaque monkey in which the left optic tract was sectioned a year before HRP was placed in the optic nerve. The vertical meridian is sharp. The few ganglion cells involved in the nasotemporal overlap would not encroach on the areas chosen in the other animals to make measurements in the upper retina and as close as 0.5 mm from the meridian (see Figs 3 and 4). C is a photomicrograph of the region to each side of the vertical meridian in which the measurements were made in the Nissl-stained right retina of LS 2. The transition at the vertical meridian is shown in greater detail in D. E and F are photomicrographs taken at comparable positions in the Nissl-stained retina of the monkey with optic tract section. Note the gross similarity between C and E and between D and F and how few ganglion cells are involved in the nasotemporal overlap in F. The remaining neurons in the degenerated half of F are amacrine cells. Scale bars: B = 500 pm; C and E=2OOnm; Dand F=50pm.

Extent of ganglion cell degeneration This was measured

in the Nissl-stained

right

retina.

In LSl and LS2 the degeneration in the ganglion cell layer of the nasal retina was so conspicuous that it could be seen with the naked eye. Figure 4C and D shows the border between the normal and degenerated regions in the central retina. It is almost as sharp as the border produced by section of one optic tract (Fig. 4E and F). Figure 5A shows the corresponding border in the other eye, in which ganglion cells were

labelled with HRP from the optic nerve. The results of ganglion cell counts are shown in Fig. 6. In the normal, temporal, hemiretina of the right eye ganglion cell density was similar in all the animals. In striking contrast, the density in all three directions in the degenerated nasal retina was greatly reduced. The area under each curve in Fig. 6 was measured and the area for the experimental animals was expressed as a proportion of the area for the normal animals for the same part of the retina. For the two unilaterally de-

Transneuronal

retinal degeneration

71

,

Fig. 5. A shows the left retina of monkey LS2 about 5 mm above the fovea. The two arrows indicate the line of the vertical retinal meridian. B and C are photomicrographs of HRP-labelled neurons in a region about 1mm (approx. 5”) nasal or temporal to the vertical meridian shown in A. As the plane of focus coincides with the cell bodies, most of the dendrites are out of focus. D and E are drawings of the neurons from the regions shown in B and C. In D, from the normal hemiretina, the great majority of labelled cells are P/I cells, recognizable by the small soma and spray of beaded dendrites that resembles a bunch of cherries. Cells marked by shading were too poorly labelled to be even tentatively classified. Note the low number and proportion of Pfi cells in the degenerated region drawn in E. Scale bars: A = 1 mm; B and C = 200 pm; D and E = 200 pm.

striated animals LSl and LS2 the respective proportions were: 0.95 and 0.94 for the horizontal meridian of the normal hemiretina, 0.33 and 0.26 for the horizontal meridian of the nasal degenerated hemiretina, 0.31 and 0.21,0.31 and 0.27 respectively for dorsal and ventral directions adjacent to the vertical meridian in the degenerated hemiretina. These proportions indicate a massive depletion in the degenerated hemiretina, its extent being similar in upper and lower retina and along the horizontal meridian. In both degenerated hemiretinae the ganglion cell depletion was greater at low eccentricities. For example, up to 85% of

the ganglion cells were missing at an eccentricity of 2.5 mm (1 I”), 50% at approx. 8 mm (35”) and perhaps even less in the far periphery. The reduction in central retina is very close to that reported in a single monkey by Cowey,X but the peripheral retina was more affected in the present animals. Dimensions

of identified cells in degenerated retina

The dimensions of assessed because there data on these cells and examined by applying

identified Py cells were not are no previous quantitative they are in any case far better HRP to the midbrain rather

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ECCENTRICITY (mm) Fig. 6. The relation between ganglion cell density and retinal eccentricity. The graphs show the mean of the results from four normal monkeys, together with the separate results from the right eye of the experimental monkeys LSI and LS2. The temporal retina was normal in all animals, the nasal retina was degenerated in LSI and LS2. The measurements in dorsal and ventral retina of LSI and LS2 were parallel

to the vertical meridian and 2 mm nasal to it.

than the optic nerve. Furthermore, their dendrites branch so sparsely that simply outlining their tips may give a misleading impression of the area they cover. Figure 7A and B shows the variation in cell body size with eccentricity for identified Pa and P/I cells respectively. The solid circles show both new and previously pub1ished46 data from ganglion cells in the temporal retina of several normal monkeys. The open circles show measurements made in the degenerated temporal retina of LSl and LS2. The cell bodies of surviving Pa and P/l cells are well within their normal range. Figure 7C and D shows similar measurements for dendritic field size. Again, Pfi cells in normal and degenerated retina are indistinguishable. Beyond 6 mm (approx.

25”) there is a suggestion that the dendritic fields of surviving Pee cells are larger than normal. This is consistent with the fact that the vast majority of the labelled surviving cells are in dorsal retina, where cell densities are normally lower than in ventral retina.46 As dendritic area varies inversely with cell density the slight difference is expected. The results shown in Fig. 7 indicate that any comparisons between the relative proportions of cells of different sizes in normal and degenerated retina made in an attempt to indicate whether cell classes are unequally involved in degeneration, are unlikely to be contaminated by changes in the mean somatic or dendritic size of a particular class in the degenerated

2

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ECCENTRICITY (mm)

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Fig. 7. The variation of cell body size (A and B) and dendritic field size (C and D) with eccentricity in the temporal retina. Per cells are shown in A and C, P/3 cells in B and D. In each graph the filled circles show new and previously measured data in normal retina (Perry et al.,“) and the unfilled circles show the results from the degenerated hemiretinae of LSI and LS2. Note the similarity between the dimensions of labelled neurons in normal and degenerated retinae.

ECCENTRICITY (mm)

,

.

D

.

CELL BODY AREA ( pm21 Fig. 8. Cell size histograms of HRP-labelled ganglion cells at eccentricities of 5, IO and 20 at the positions shown in Fig. 3. Cross-hatched area shows cells from nasal retina, which was normal in all monkeys; white area shows cells from temporal retina, which was degenerated in LSI and LS2. The statistical significance of the difference between each nasal and temporal sample is shown on each graph.

regions. In particular, the cell body size of surviving labelled Pr and Pp cells has not changed. Czll hod), six The great

uniformly

majority of cells in the areas chosen were and densely filled (see Fig. 5) and it was

a straightforward matter to draw their outline. The results of measuring the area of 200 labelled cell bodies at three nasal and three temporal positions in four retinae are shown in Fig. X. Note that thcrc arc relatively more large cells in the dcgeneratcd temporal retina than in the normal temporal retina. Cell sire

Transneuronal

retinal degeneration

varies even between identical positions in different normal retinae, probably because of differences in size of the eye and in ganglion cell density.& We therefore compared the normal and experimental animals with respect to the magnitude of the difference between the mean size in nasal and temporal retina. The percentage difference between nasal and temporal retina at matching eccentricities was greater in the experimental than the normal animals (t-test, df = 10, t = - 2.65, P < 0.025, 1 tailed). As surviving Pa and Pp cells are of normal size for their class, the increased nasotemporal difference in cell body size in abnormal retinae indicates that the transneuronal degeneration of retinal ganglion cells preferentially involves cells with small somata. N

ClassiJication of labelled cells A total of 1391 cells were classified. An additional 712 clearly labelled cells in the regions examined could not be classified, i.e. about one cell in three could not be identified even in regions chosen for excellent labelling. Furthermore, these figures exclude cells that were sparsely labelled, e.g. cells with only pale reaction product in the soma and no visible dendrites. They were rare in the regions chosen for analysis, but examples appear in Fig. 5. The results for the cells that could be classified are presented in Table 1. It can be seen that the proportions of the three cell types in the normal nasal hemiretinae of the two experimental animals and throughout the retina of the normal animals are very similar; they are close to our previous estimates of about 10% Pa, 80% P/J’ and 10% Py.43.46 In striking contrast, the proportion of Par cells rises and that of Pp cells falls in the degenerated temporal retina. For the proportions of the two classes to become roughly equal would require between 80 and 90% of the P/I cells to die. The total extent of ganglion cell loss shown in Fig. 6 is compatible with this conclusion. At eccentricities from 10 to 30”, where most of the measurements shown in Table 1 were taken, the overall reduction in the degenerated retinae in all three radial directions in both retinae was about 75%. If this depletion were confined to P/l cells their proportion would fall from 80 to 5% and they would then be outnumbered by Per cells. This in fact occurred in one of the two degenerated retinae. The change in the proportions of Pee, Pb and Py cells in the two degenerated hemiretinae was statistically assessed by comparing the overall mean proportion from four normal and two degenerated hemiretinae, as shown in Fig. 9. The difference was significant (x2 = 132.44, df = 2, P < 0.001). The Py cells require special comment. Their frequency was previously estimated43 by selectively labelling them from the mid-brain. When all cell types are labelled from the optic nerve rather than from their cerebral targets, the Py cells are much more difficult to identify with certainty because their thin and sparselybranched dendrites are concealed among the stouter and profuse dendrites of Per and Pfi cells. This was

T

-

c2 LSI LS2 Fig. 9. The overall proportions of identified PG(,PB and Py cells measured at the positions indicated by stars in Fig. 3. N = nasal retina, T = temporal retina.

confirmed in the temporal retina of the normal monkey C2 where only 2.3% of identified cells were in this class. The low and unchanged proportion of Py cells in the degenerated retina of LSl is therefore not surprising. However, 20% of cells were classified as Pr in the samples in the second and slightly better stained degenerated retina. This is at least double the highest estimates previously determined by selective labelling of Py cells, suggesting that Py cells do not degenerate and that their proportion consequentiahy increases. Degeneration

of Pu cells?

The data presented in Table 1 show that the transneuronal degeneration affects predominantly P/I cells. Does it also affect Per cells, albeit to a lesser degree? We made three further sets of measurements in an attempt to answer this question. First, we counted all labelled cells with a cell body greater than a given mean diameter in an area of 0.25 mm2 and about 0.5 mm nasal and temporal to the vertical meridian in the two experimental animals. The precise areas are shown in Fig. 4A. The centre of each region was 3.2 mm from the fovea in LS2 and 6.0 mm in LSI. The mean minimum diameter chosen was 15 pm for LS2 and 20 pm for LSl. At these eccentricities the vast majority of ganglion cells with somas larger than these values are Pa cells (Fig. 7 and Perry et aZ.46). As the two regions were so close in each retina and both lay within a larger region where the labelling was excellent there was no reason to suppose that differences in the number of labelled large cells could arise unless the numbers of large cells actually differed as a result of degeneration. The number of “large” labelled cells in LS2 was 87 nasally (normal) and 93 temporally (degenerated); in LSl it was 31 nasally (normal) and 23 temporally (degenerated). There is therefore no strong evidence that large cells, most of which will be Pa cells, do degenerate.

A. COWEY CI al.

16

In the second attempt to assess any degeneration of Pa cells we counted and measured “large” ganglion cells in an identical or very similar region of the other retina, in which the cell bodies of all neurons in the ganglion cell layer were stained by Cresyl Violet. In the right eye it is the nasal retina that is degenerated, as shown in Fig. 6. The numbers of neurons with a diameter greater than I5 pm at a mean eccentricity of 3.2 mm, and about 0.5 mm nasal or temporal to the vertical meridian in upper retina, were: LS2, 68 temporal (normal) and 60 nasal (degenerated); and LS I, 80 temporal (normal) and 49 nasal (degenerated). Although the numbers in the normal temporal retina are higher, there is no evidence of massive loss of presumed Pa ganglion cells. As in the left eye labelled with HRP, the difference is larger in LSl. Finally, we estimated the number of putative Pee cells in the degenerated retina of LS2 at an eccentricity where their number and proportions have been carefully assessed in a normal retina. It was not possible to repeat this with monkey LSl because this precise region was not well labelled. By first classifying cells on the basis of their dendritic form Perry and Silveira4’ showed that in a sample of 237 ganglion cells. 79% of cells with a cell body area greater than 100 pm’, at several positions from 0.8 to 1.2 mm from the centre of the fovea, are Pa-cells. Accordingly, we measured the area of the cell bodies of all HRP labelled cells in four regions, each 113 x 113 pm, 0.9-l .4 mm from the centre of the fovea in the degenerated temporal retina of LS2. Out of a total of 175 labelled somata in an area of 0.051 mm’. I53 were larger than IOO~~m’. If 79% (121) of these are Pee cells, and normal ganglion cell density44 at this mean eccentricity is 3 I x IO3/mm’, then surviving Pa cells in this region of the degenerated retina form 7.6% of the original total. This is within or just below the normal proportion of PCYcells in central retina.4h Even if some Pee cells have degenerated they still form at least 69% of surviving ganglion cells in this region as opposed to the normal 10% or so. DISCUSSION

Up to 85% of retinal ganglion cells die in the central I5 of the retina eight years after removing striate cortex on one side of the brain. More eccentrically the percentage loss is smaller, but still substanial. Measurements of cell soma size and classification of ganglion cells on the basis of the size and branching pattern of their dendrites indicates that the transneuronal depletion affects chiefly, and perhaps solely, the P/l cells. The selective vulnerability of P/? cells, the cause of transneuronal degeneration, and the implications for residual vision will be discussed, after first considering two technical matters. Tecltnical considtwtiuns To what extent could our results other than the death of neurons?

reflect

factors

Axonal diameter probably influences retrograde transport and consequently the extent to which cells are labelled. As P/I cells have much finer axons than Pee cells, any reduction in axonal diameter, caused by the cortical lesion and resulting degeneration in dLGN, may render P/J cells so difficult to label with retrogradely transported HRP that they appear to be selectively affected. However, the degenerated half of the Nissl-stained retina of the other eye, in which all ganglion cells are stained, showed that about 80% die (see Figs 4 and 6). As at least 80% of ganglion cells in central retina are Pfi cells these cells must be extensively involved. Furthermore the evidence that “large” cell bodies are not obviously reduced in number suggests that Pee cells are not greatly involved. In sparsely labelled regions of normal retinae the proportions of the three major cell types may depart substantially from those observed overall, and it would be possible to select a small region where Pfl cells were rarer than expected. This may reflect more than natural fluctuations in small samples, for example if the axons of different cell types are partially segregated into different fascicles in the optic nerve they may be differentially labelled, especially at the edge of the HRP deposit. We attempted to avoid this problem by analysing areas of the retinae that were within rather than at the edge of the well-labelled area. Furthermore, the samples that we selected in order to compare cell body size at an eccentricity of 20 and close to the vertical meridian (see Fig. 3) lay on the path followed by the optic axons in the arcuate bundle. It would be very difficult to place HRP just behind the eye and to label radically different proportions of cell types in these two regions.

Whereas P; cells can be labelled from the mid-brain there is no evidence that P[j cells and more than a tiny percentage of Per cells project to the mid-brain in macaque monkeys.4’.4h Their only known targets are the parvo- and magnocellular layers of the dLGN, respectively. but the dLGN shows rapid and almost complete retrograde degeneration after removal of striate cortex.” Up to 2000 large LGN neurons survive and many can be labelled by HRP injections into extra-striate visual cortex.h’ However. it is not yet known whether surviving dLGN cells in monkeys receive a direct retinal input from any class of ganglion cell. Following intraocular injection of tritiated amino acid both parvocellular and magnocellular divisions of the degenerated dLGN are labelled. the former sparsely but the latter apparently normally.67~hx This is consistent with a direct projection but does not demonstrate synaptic connections. Dineen et ~1.” identified retinal terminals in synaptic contact with post-synaptic elements in both degenerated parvoand magnocellular divisions but were unable to determine the nature of the postsynaptic structures. Some were axonal, i.e. the contacts were axoaxonal, but none was definitely a neuron projecting to extra-

Transneuronal retinal degeneration striate cortex. Where else might surviving ganglion cells project? The pulvinar, and accessory optic nuclei receive retinal input, but the type of ganglion cells that projects to these structures has not been identified in primates. Nor has the input to the pre-geniculate nucleus, which hypertrophies when the dLGN degenerates.13 In the cat, the direct projection from the degenerated A and C layers of the LGN to cortical area PMLS is enhanced when areas 17, 18 and 19 are all ablated at an early age.” Whether there is any expansion of the projection from dLGN to prestriate cortex following striate damage in adult monkeys is unknown. IV& do some retinal ganglion cellf show retrograde transneuronal degeneration? The usual answer to this question is that they lack any collaterals to other targets or that their collaterals are not sufficiently sustaining.8,37.6* Unfortunately the former is difficult to prove, and the latter is a truism. Cowey* suggested that some cells might escape degeneration by having a collateral to the superior colliculus, and Weller et ai.@suggested that these cells are physiologically Y-type and provide the input to the magnocellular layers. However, there is at present no evidence that either Peeor P@cells do have such a collateral in macaques, and branching patterns can therefore not be used to explain the selective loss of P/3 cells. An alternative reason for their vulnerability might lie in their smaller size. When the optic nerve is sectioned, all axons are equally pruned and differences in the time course of degeneration of different cell types cannot be explained by different branching patterns. After optic nerve section in the cat, the degeneration of /I cells precedes that of G( cell~.‘~ Size may therefore be important. However, if size alone were the determining factor, peripheral Pp cells, which are larger than central Pa cells, should resist degeneration. Although there is less degeneration in the retinal periphery the loss is still substantial, and must include Pp cells. The complementary prediction, namely that central Per cells should degenerate, was unfortunately not directly testable with our material. However, the number of large labelled cells, most of which are presumed to be Pa cells, close to the fovea in the degenerated hemiretina of LS2 was not greatly different from normal, indicating that at least a substantial proportion of even the smallest Pa cells survive. The question of why the P/l cells are selectively affected cannot yet be answered satisfactorily. It is also unclear why a small percentage of these cells, even close to the fovea where they are smallest, survives long-term removal of striate cortex, and whether they too would eventually die. One possible explanation for their survival is that they project to the colouropponent cells in the retinorecipient zone of the pulvinar,“’ and/or to the few surviving projection neurons in the parvocellular division of the dLGN, and/or to the pregeniculate nucleus. However, if the

77

same explanation were given for what might seem to be the ~rmanent survival of an even larger number of Pp cells at an earlier stage of degeneration it would presumably be wrong. It is therefore important to reveal the projection patterns of surviving cells directly, and to determine whether additional degeneration of P/I cells would occur with even longer periods after cortical damage. The relative invulnerability of PO: cells is unlikely to be explained by their projecting to surviving neurons in the dLGN because the latter are no more common in the magnocellular than the parvocellular layers. 69 However, if Pa cells in macaques prove to have much larger terminal arborizations in the dLGN than Pp cells, as is the case with the retinal axon arbors in the magnocellular and parvocellular layers in the prosimian Galago,* they may be better preserved because each has a greater probability of innervating an isolated surviving projection neuron in the magnocellular layers. There is a remaining puzzle about the Pczcells. Four years after unilateral removal of the striate cortex in a macaque monkey van Buren6’ reported that “‘The larger peripheral ganglion cells were nearly entirely absent in the degenerated retinal half” (p. 106). As large meant larger than 15 pm in diameter, many of these were presumably Pa cells, especially as about 25% linear shrinkage should be allowed for in the sectioned material. We did not find any evidence indicating substantial loss of Pa cells and have no convincing explanation for this discrepancy. Relation to residual Disuaffunctions The transneuronal retrograde degeneration of retinal ganglion cells imposes peripheral restrictions on the information that can be processed after damage to striate cortex. The selective and massive loss of Pb cells should impair resolution of fine spatial detail, as reflected in measures of acuity and the processing of form and texture, especially with respect to their high-frequency components. Form discrimination within the visual field defects of both monkeys and patients is undoubtedly difficult even when it can be demonstrated.36,39.66 And although large stimuli were used by Humphrey’* and Barbur et ai.’ their monkeys or human subjects showed no evidence of true form discrimination. Furthermore, Weiskrantz@ has shown that apparent form discrimination may be an artefact of the discrimination of the orientation of prominent contours that make up the forms, and the only direct measure of visual acuity in destriated monkeys revealed a reduction of at least two octaves.‘” Stereoacuity and vernier acuity, which require exquisitely fine judgements of relative spatial position, have not been measured in the visual defects of primates, but one would expect them to be greatly impaired. The ability to use information about the retinal disparity of images falling within field defects was examined by Richards,*’ who found little evidence that even large disparities could be registered, which is tantamount to saying that the disparity threshold is enormously in-

78

A. COWEY Pi crl.

creased. Removal of area 17 in cats, which also leads to transneuronal loss of retina beta cell~,~~produces at least a l5-fold increase in vernier acuity threshold even though simple form and pattern discrimination are unimpaired. 33 Precise positional information does not seem to be available in “blind” fields, and localization of isolated targets is impaired.63 These properties of “blindsight” and residual vision in primates are in accord with the results of placing lesions in the parvocellular portion of the dLGN.53 Within the resulting field defect, detection was only mildly impaired but the discrimination of retinal disparity, motion and texture were all severely affected except for stimuli of low spatial frequency. Similar changes follow the systemic administration of acrylamide monomer, which selectively damages the retinal cells whose axons project to the parvocellular layers of the dLGN.-” In tests of contrast sensitivity the monkeys were impaired when discriminating grating stimuli of high spatial and low temporal frequencies, as would be expected from the functional properties of the P/3 cells with their small receptive fields and limited temporal resolution. At high temporal frequencies the animals also have a greatly reduced sensitivity for chromatic but not for achromatic gratings.30 Pb cells have such small dendritic fields that their anatomical coverage factor is as little as two, i.e. one on-centre and one off-centre at any point on the retina.4h When their numbers are reduced by up to 85%, some points on the retina will not be sampled by a Pp cell and one might expect residual vision to show sharp local variations in the visual field. For example, discrimination of the hue of small coloured targets should be very variable. However, when mapped physiologically with small spots’* the diameter of the receptive field centre of colour opponent ganglion cells is about 50% larger than dendritic field diameter, presumably because of lateral interactions, and normal retinal coverage becomes five, Crook el f&“also using physiological measurements, suggest a coverage of 24. Every point on the retina may therefore still be covered by several colour-opponent receptive fields after retrograde transneuronal degeneration, and residual wavelength discrimination may be more spatially uniform than dendritic coverage predicts. The P/l system, with its projection to the parvocellular dLGN and from there to striate cortex, is the only known colour-opponent system. Consequently it is generally considered to underlie wavelength processing. Coiour vision should therefore be conspicuously impaired in “blindsi~ht”, and indeed hue disc~mination could not be demonstrated at all in several studies on monkeys!4,“,24.28.62 and human patients.‘.4’.63.64 AIthough there is other evidence for at least rudimentary hue discrimination, again in both monkeys22,36,5’ and man2.4.57 this evidence does not invalidate the general argument as only well-separated hues were tested and nothing is known about wavelength discrimination thresholds in “blindsight”. Even a 50-fold elevation in threshold could allow discrimination between, say,

deep red and blue-green. Why the spectral sensitivity of destriated monkeys was found to be scotopic rather than photopic even at photopic luminance levels26 is unclear on the basis of the present anatomical findings, as a greatly reduced but nevertheless still substantial population of Pp cells remains. It could be argued that the residual P/l cells and the apparently unscathed population of Per cells make no contribution to residual vision via their only known targets in the dLGN and the projection from the latter to extrastriate visual cortex. This possibility, coupled with the proposal that residual vision is mediated by the Py cells and their mid-brain projections, is attractive in that it suggests that “blindsight” is blind because it is non-cortical. However, there is evidence against this view. Monkeys with lesions of striate cortex retain good visual sensitivity within their field defects,y can localize small targets presented there,hs and can make saccadic eye movements to such targets.34 However, a preliminary report indicates that restricted lesions of the dLGN, within which all cortical projection neurons and retinogeniculate terminals will be destroyed, produce much denser field defects and destroy these abilities.” More direct evidence regarding the functional normality or abnormality of the surviving ganglion cells is not available for any of the three cell classes. Although the retinocollicular projection, made up of the apparently undegenerated class of Py cells, is involved in saccadic eye movements to targets presented in field defects,34 a long-standing striate cortex lesion may well alter its functional properties, which are poorly understood even in the normal animal. as a result of the elimination of the striate-collicular projection. Functional changes may also have occurred in the depleted population of P[J cells and in the apparently undiminished population of Pa cells that continue to provide some input to the degenerated dLGN or in the properties of surviving geniculate cells. After damage to areas 17. 18 and I9 in the cat, surviving neurons in the degenerated dLGN have abnormally large receptive field centres despite having normal spatial resolution and normal optimal spatial frequency? In summary, it is still not certain whether all the remaining pathways are functional years after damage to the striate cortex, what properties they have, what specific input they receive and which experimental tasks will differentially reveal their role in residual vision. What is known is that the ganglion cells of all three classes are still present, albeit in abnormal and roughly equal proportions, and that they are morphologicaIly normal.

AcknowLedgemenrs-This work was supported by MRC Grant G971/397/B. V.H.P. is a Wellcome Senior Research Fellow. P.S. held a European Science Foundation Visiting Fellowship, and also gratefully acknowledges the award of a travel grant from the von Karajan Neuroscience Research Trust. We are grateful to Dr B. E. Reese for many helpful comments on the manuscript.

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