EXPERIMENTAL
The
NEUROLOGY
Development
46, 445-451 (1975)
of Synapses in Kitten During Visual Deprivation B. G.
CRAGG
Department of Physiology, Clayton, Victoria, Received
Visual
Cortex
1
Monash University, 3168, Australia
September
16, 1974
The dependence of synaptic development on visual functioning has been tested by comparing a control group of 6 kittens with 5 kittens whose eyelids had been sutured bilaterally, and with 2 kittens whose optic nerves had been crushed. The operations were done during the first week after birth, and all the kittens then lived together in a small room. The brains were fixed for electron microscopy when the kittens were 45 days old. Synapses were counted in a depth scan of the visual cortex, and neuronal cell bodies were counted by light microscopy of frozen sections. The average neuronal densities were 28 or 41% higher in the sutured or crushed groups than in the controls, while average neuronal diameters were 8% or 12% less. The average count of synapses in a fixed area of tissue was decreased at all cortical depths in both deprived groups, but the decrease was statistically significant only between 1.0 and 1.4 mm below the pia, where it reached 22%. The estimated density of synapses (or number in a column of cortex of unit volume) was lower, but the difference was not significant, and the average length of synaptic apposition was also unchanged. Synaptic density was divided by neuronal density to estimate the average number of synapses associated with one neuron, and this was significantly depressed by about 30% in both deprived groups. Thus, complete blindness had little more effect on the developing visual cortex than loss of pattern vision, and both forms of deprivation led to reduced neuronal connectivity at a time when synaptic development has reached a plateau in sighted kittens
INTRODUCTION The majority of synapsesin kitten visual cortex develops between 8 days and 5 wk after birth (8, 9). Durin, m the latter half of this period visual acuity improves and visually guided behavior develops (23). It is therefore of interest to know how much of synaptic development is dependent on functioning. In rats, unilateral deprivation produced by monocular eye1 Aided by N. H. and M. R. C. of Australia. 44.5 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
446
ES. G.
CRAGG
lid closure 14 days after birth resulted in the mean density of synapses at 8 wk being 20% less in the deprived visual cortex than on the contralateral side (IO). The volume of visual cortex was reduced in the same circumstances, and the density of cells increased ( 11) . In the cat it is necessary to close the eyes bilaterally to deprive the cortex of pattern vision. The intensity of retinal illumination is reduced about ten thousand times (24). Spontaneous discharge in optic nerve fibers would be expected to continue, though the temporal distribution of impulses would be altered (20). The optic nerve can be crushed behind the eyeball in a simple atraumatic operation, and this must reduce the optic input to zero. These two forms of binocular deprivation have been applied to two groups of kittens, and the development of synapses and neurons has been compared with a control group of sighted kittens. The spontaneous behaviour of the blind kittens in the small room in which all the kittens grew up was so little affected, that it was difficult to distinguish them from the controls. When picked up however, they clung with their claws more tenaciously than sighted kittens. MATERIALS
AND
METHODS
In 7 kittens the eyelids were sutured together or the optic nerves crushed under ether anesthesia during the first week after birth. For the latter operation, a small opening was made in the dorsomedial aspect of the conjunctiva and a pair of curved forceps was passed round the back of the eyeball to crush the optic stalk. Disappearance of the pupillary reflex indicated the completeness of nerve interruption. The operated animals were injected with penicillin daily for 1 wk. All the kittens were anesthetized with pentobarbital sodium and fixed by intracardiac perfusion when 45 days old. The part of the visual cortex studied was on the medial surface of the marginal gyrus just anterior to the representation of the center of vision and as far dorsal as the flat surface of the gyrus extended. A thin plate was cut of the full depth of the cortex and processed for electron microscopy, while the remainder of the brain was frozen and sectioned at 20 pm to count neurons in the region immediately ahead of the sample taken for electron microscopy. The neuronal density was calculated (1) after counting and measuring the neuronal cell bodies in scans of the depth of the visual cortex in six successive frozen sections. The number of synaptic appositions was counted in the final screen of the electron microscope at a fixed magnification of 18,000 which was measured with a carbon replica of a diffraction grating. Twenty fields were counted in each grid square at random positions obtained by flicking the specimen controls. About 1200 synapses were counted altogether in a scan of the depth of the cortex. The lengths of a random sample of 2-300 synaptic appositions were mea-
SYNAPSES
IN
DEPRIVED
CORTEX
447
sured in order to calculate (2) the density of synapses. Full details of the counting and measuring procedures have been published elsewhere (9). The tissue in the frozen sections at counting and in the plastic sections used for electron microscopy was found to be not shrunk from the sizes measured when the blocks were dissected out of the perfused brains. RESULTS Nezwonal Density. The average density of neurons in each of the seven deprived kittens was higher than in any of the controls (Table 1) and the mean values were significantly different (P < 0.001). When the neuronal densities at different depths were plotted, the distributions in the control and deprived kittens were substantially parallel, and there did not seem to be a concentration of the effect at any one depth. The diameters of the neurons in the visual cortex were significantly less than in the sighted kittens (Table 1) for both forms of visual deprivation (P < 0.01). The two kittens with crushed optic nerves were compared with the five kittens with eyelid suture, and the neuronal density was significantly higher in the former, while neuronal diameter was not significantly different. The kittens with crushed optic nerves showed extreme atrophy of the optic chiasm and tracts. In the lateral geniculate nucleus the mean cell area in the binocular part of lamina A was 29% less than in the sighted controls (P < 0.02), whereas the cell area in the sutured kittens was only 95% less than in the controls, and this difference was not significant (I am indebted to Miss Kum Wan for these measurements). The thickness of the visual cortex is difficult to define accurately because of the individual variations in gyral curvatures, but it was estimated in all the slides available, and found to be decreasedby 9% in the eyelid sutured group (P < 0.01) and by 10% in the cats with crushed optic nerves (P < 0.05). Tl lus, deprivation of vision has reduced the growth of the neuropil that progressively separates the neuronal perikarya and reduces neuronal density during development. Synaptic Density. The average density of synapseswas slightly lower in the seven deprived kittens than in the controls (Table 1), but these differences were not significant. However, the average count of synapsesper screen area was higher in the controls than in the eyelid sutured kittens, and this difference was significant (P < 0.05). The distributions of the synaptic count in the depth of the cortex were examined, and the differences reached the significance level of P < 0.05 in the part of the cortex between 1.0 and 1.4 mm below the pia only (layers 4 and 5). where the control kittens had a higher area density of synapses than either of the deprived groups. The average lengths of the synaptic appositions were the samein all three groups.
15.7
Mean
15.5 14.3
14.9
1 2
Mean
Crushed
15.3 15.6 16.4 16.2 14.8
1 2 3 4 5
Sutured
17.0
Mean
Size, I.cm
TABLE
1
5.3
5.2 5.3
4.8
4.7 5.2 4.7 4.4 4.8
3.73
3.7 3.6 3.9 3.9 3.7 3.6
Density X 107/cm3
.26
.26 .25
.26
.25 .25 .24 .27 .27
.26
.26 .25 .24 .27 .29 .27
Length Irm
7.2 7.6 7.4
.212
7.1
6.2 6.2 8.9 7.7 6.3
7.9
8.6 8.2 8.9 8.1 7.2 6.7
Density X 10n/cm3
.214 .209
.205
.179 .180 .242 .225 .200
.23.5
.248 .233 .244 .240 .231 .211
Count/$
Synapse
14,000
13,800 14,300
14,960
13,200 11,900 19,000 17,500 13,100
21,250
23,200 22,700 22,800 20,700 19,000 18,700
Neuron
Synapses
IN THE VISUAL CORTEX OF SIX CONTROL KITTENS, FIVE KITTENS WITH BILATERAL KITTENS WITH BILATERALLY CRUSHED OPTIC NERVES, ALL 4.5 DAYS OLD
Neuron
DENSITIES AND Two
17.2 17.3 15.7 16.9 17.4 17.5
AND SYNAPTIC
1 2 3 4 5 6
Controls
Cat
NEURONAL
SUTURE,
1483
1364 1602
1498
1.504 1.510 1490 1516 1469
1652
1782 1569 1668 1633 1619 1641
Thickness pm
EYELID
F s
n
y
I$ co
SYNAPSES
Synaptic the average significantly groups (P the neurons
IN
DEPRIVED
CORTEX
449
density was divided by neuronal density (Table 1) to estimate number of synapses associated with one neuron, and this was higher in the control kittens than in either of the deprived < 0.01). Thus, visual deprivation reduced the connectivity of in developing visual cortex by about 3070 at 45 days after birth. DISCUSSION
It is not surprising that neuronal density changed more than synaptic density, for much of the volume of the neuropil consists of axons and dendrites engaged in making synapses, and the axon terminals alone may account for 30% of the volume of neuropil (3). Thus, deprivation may retard the growth of neuropil and so cause a higher neuronal density, but the density of synapses is little changed in what neuropil does develop. The removal of spontaneous retinal discharge by optic nerve crush resulted in a significantly greater change than did eyelid closure in only one of the parameters measured: neuronal density. In the mouse is has been found that eye removal stops the growth of neuropil and neuronal diameter, while rearing in darkness allows a slow rate of neuropil development (17). Thus, in the cat, too, the difference between the effects of the two forms of deprivation may increase with survival time. Binocular lid closure resulted in a non-significant reduction in lateral geniculate nucleus cell area as has been found by other authors (4, IS), in contrast with the marked cell shrinkage (3040%) produced by monocular lid closure (4, 24, 25). Bilateral optic nerve crush reduced lateral geniculate nucleus cell area by 29% in the growing kittens, although in older cats, tract section has little effect on the lateral geniculate nucleus, for at least 120 days (12). Neither form of deprivation is likely to affect the cortex by a mechanism requiring lateral geniculate nucleus atrophy, for in the squirrel with monocular lid closure the neuronal density is higher in both the monocular and binocular parts of the visual cortex, whereas lateral geniculate nucleus cell shrinkage is found in the binocular segment only (16). It has been shown that the amomit of protein exported from the cell body into the neuronal processes increases with electrical stimulation of the neuron (18). It is likely then that the two forms of visual deprivation affect the growth of neuropil mainly by reducing the signalling activity of the neurons. In the visual cortex, extrinsic connections account for a small proportion of the synapsespresent (5, 7, 14) and are confined to particular depths, whereas the affects of deprivation are widespread. Thus, it must be mainly the signalling activity of cortical neurons that controls the growth of cortical neuropil. The reduced neuronal connectivity in the deprived cats would be espetted to affect the functioning of the visual system. When eyelids are re-
450
B.
‘3.
CRAGG
opened after binocular closure the cat may be behaviorally blind for several days (4, 21, 25). Single units in the visual cortex (4, 19) and in the lateral geniculate nucleus (22) show deficits in binocular properties and in responsiveness. Moreover, the influence of the visual cortex on the superior colliculus is lost so that the cat ignores objects in the contralateral field of each eye (21). It was suggested (21) that the visual cortex may remain nonfunctional for visually guided behavior, although it may take part in discrimination learning. The reversibility of these behavioral effects is uncertain, and two cats trained on visual tasks for long periods learned slowly (4). As regards structural recovery, a rapid concentration of the synaptic vesicles in axon terminals in response to brief visual input after dark-rearing has been found in cat visual cortex (13) and in rat retina (6). Whether the other structural changes produced by binocular deprivation in the cat could be reversed by visual experience is not yet known. REFERENCES 1. ABERCROMBIE,
Anat.
M. 1946. Estimation
of nuclear population
from microtome
sections.
Rec. 94 : 239-247.
2.
ANKER,
R. L., and B. G. CRACK 1974. Estimation of the number of synapses in a volume of nervous tissue from counts in thin sections by electron microscopy.
3.
ARMSTRONG-JAMES,
J. Neurocytol.
3: 725-735.
M., and R. JOHNSON. 1970. Quantitative studies of post-natal changes in rat superficial motor cerebral cortex. 2. Zellforsch. 110: 559-568. 4. CIIOW, K. L., and D. L. STEWART. 1972. Reversal of structural and functional effect of long term visual deprivation in cats. Exp. Nenrol. 34: 409-433. 5. COLONNIER, M., and S. ROSSIGNOL. 1969. Heterogeneity of the cerebral cortex, pp. 26-40. In “Basic mechanisms of the epilepsies.” H. Jasper, A. Ward and A. Pope [Eds]. Little Brown & Co., Boston. 6. CRACG, B. G. 1969. Structural changes in naive retinal synapses detectable within minutes of first exposure to daylight.‘Brain Res. 15: 79-96. 7. CRAGG, B. G. 1971. The fate of axon terminals, in visual cortex during transsynaptic atrophy of the l$eral geniculate nucleus: Brain Res. 34: U-60. 8. CRAGG, B. G. 1972. The developnient’ of synapses ‘in cat visual cortex. Invest. Ophthalmol. 11: 377-385. 9. CRAGG, B. G. 1975. The development of synapses in the visual system of the cat. J. Camp. Neurol. (in press). 10. FIFKOVA, E. 1970. The effect of monocular deprivation on the synaptic contacts of the visual cortex. J. Neztrobiol. 1 : 285-294. 11. FIFKOVA, E. 1970. The effect of unilateral deprivation of visual centers in rats. J. Comb. Neural. 140: 431-438. 12. GAREY, L. J., R. A. FISKEN and T. P. S. POWELL. 1973. Effects of experimental deafferentation on cells in the lateral geniculate nucleus of the cat. Brain Res. 52 : 363-369. 13. GAREY, L. J., and J. D. PETTIGREW. 1974. Ultrastructural changes in kitten visual cortex after environmental modification. Brain Res. 66: 165-172. 14. GAREY, L. J., and T. P. S. POWELL. 1971. An experimental study of the termination of the lateral geniculo-cortical pathway in the cat and monkey. Proc. Roy. Sot. Loltdon Ser. B 179 : 41-63.
SYNAPSES
IN
DEPRIVED
CORTEX
451
15. GUILLERY, R. W. 1973. The effect of lid suture upon the growth of cells in the dorsal lateral geniculate nucleus of kittens. /. Colrrp. Neural. 148: 417-422. 16. GUILLERY, R. W., and J. H. KAAS. 1974. The effects of monocular lid suture upon the development of the visual cortex in squirrels (Sciureus carolinensis). J. Cotllp. Neural. 154 : 443-452. 17. GYLLENSTEN, L., T. MALMFORS and M.-L. NORRLIN-GRETTVE. 1967. Visual and non-visual factors in the centripetal stimulation of postnatal growth of the visual centers in mice. J. Conzp. Nearol. 131 : 549-558. 18. Lux, H. D., P. SCHUBERT, G. W. KREUTZBERG and A. GLOBC-S. 1970. Excitation and axonal flow : autoradiographic study on motoneurones intracellularly injected with a 3H-amino acid. Exb. Brain Res. 10: 197-204. 19. PETTIGREII;, J. D. 1974. The effect of visual experience on the development of stimulus specificity by kitten cortical neurons. J. Physiol. 237: 49-74. 20. SANDERSON, A. C., W. M. KOZAK and T. W. CALVERT. 1973. Distribution coding in the visual pathway. Biopkys. J. 13 : 218-244. 21. SHERMAN, S. M. 1973. Visual field defects in monocularly and binocularly deprived cats. Brain Res. 49 : 25-45. 22. SHERMAN, S. M., and SANDERSON, K. J. 1972. Binocular interaction on the cells of the dorsal lateral geniculate nucleus of visually deprived cats. Brain Res. 37: 126131. 23. WARKENTIN, J., and K. V. SMITH. 1937. The development of visual acuity in the cat. J. Genet. Psychol. 50: 371-399. 24. WIESEL, T. N., and D. H. HUBEL. 1963. Effects of visual deprivation on morphology and physiology of cells in the cat’s lateral geniculate body. J. Neurophysiol. 26 : 978-993. 25. WIESEL, T. N., and D. H. HUBEL. 1965. Comparison of the effects of unilateral and bilateral eye closure on cortical responses in kittens. J. Neurophpsiol. 28: 1029-1040.
The
NEUROLOGY
Development
46, 445-451 (1975)
of Synapses in Kitten During Visual Deprivation B. G.
CRAGG
Department of Physiology, Clayton, Victoria, Received
Visual
Cortex
1
Monash University, 3168, Australia
September
16, 1974
The dependence of synaptic development on visual functioning has been tested by comparing a control group of 6 kittens with 5 kittens whose eyelids had been sutured bilaterally, and with 2 kittens whose optic nerves had been crushed. The operations were done during the first week after birth, and all the kittens then lived together in a small room. The brains were fixed for electron microscopy when the kittens were 45 days old. Synapses were counted in a depth scan of the visual cortex, and neuronal cell bodies were counted by light microscopy of frozen sections. The average neuronal densities were 28 or 41% higher in the sutured or crushed groups than in the controls, while average neuronal diameters were 8% or 12% less. The average count of synapses in a fixed area of tissue was decreased at all cortical depths in both deprived groups, but the decrease was statistically significant only between 1.0 and 1.4 mm below the pia, where it reached 22%. The estimated density of synapses (or number in a column of cortex of unit volume) was lower, but the difference was not significant, and the average length of synaptic apposition was also unchanged. Synaptic density was divided by neuronal density to estimate the average number of synapses associated with one neuron, and this was significantly depressed by about 30% in both deprived groups. Thus, complete blindness had little more effect on the developing visual cortex than loss of pattern vision, and both forms of deprivation led to reduced neuronal connectivity at a time when synaptic development has reached a plateau in sighted kittens
INTRODUCTION The majority of synapsesin kitten visual cortex develops between 8 days and 5 wk after birth (8, 9). Durin, m the latter half of this period visual acuity improves and visually guided behavior develops (23). It is therefore of interest to know how much of synaptic development is dependent on functioning. In rats, unilateral deprivation produced by monocular eye1 Aided by N. H. and M. R. C. of Australia. 44.5 Copyright All rights
0 1975 by Academic Press, Inc. of reproduction in any form reserved.
446
ES. G.
CRAGG
lid closure 14 days after birth resulted in the mean density of synapses at 8 wk being 20% less in the deprived visual cortex than on the contralateral side (IO). The volume of visual cortex was reduced in the same circumstances, and the density of cells increased ( 11) . In the cat it is necessary to close the eyes bilaterally to deprive the cortex of pattern vision. The intensity of retinal illumination is reduced about ten thousand times (24). Spontaneous discharge in optic nerve fibers would be expected to continue, though the temporal distribution of impulses would be altered (20). The optic nerve can be crushed behind the eyeball in a simple atraumatic operation, and this must reduce the optic input to zero. These two forms of binocular deprivation have been applied to two groups of kittens, and the development of synapses and neurons has been compared with a control group of sighted kittens. The spontaneous behaviour of the blind kittens in the small room in which all the kittens grew up was so little affected, that it was difficult to distinguish them from the controls. When picked up however, they clung with their claws more tenaciously than sighted kittens. MATERIALS
AND
METHODS
In 7 kittens the eyelids were sutured together or the optic nerves crushed under ether anesthesia during the first week after birth. For the latter operation, a small opening was made in the dorsomedial aspect of the conjunctiva and a pair of curved forceps was passed round the back of the eyeball to crush the optic stalk. Disappearance of the pupillary reflex indicated the completeness of nerve interruption. The operated animals were injected with penicillin daily for 1 wk. All the kittens were anesthetized with pentobarbital sodium and fixed by intracardiac perfusion when 45 days old. The part of the visual cortex studied was on the medial surface of the marginal gyrus just anterior to the representation of the center of vision and as far dorsal as the flat surface of the gyrus extended. A thin plate was cut of the full depth of the cortex and processed for electron microscopy, while the remainder of the brain was frozen and sectioned at 20 pm to count neurons in the region immediately ahead of the sample taken for electron microscopy. The neuronal density was calculated (1) after counting and measuring the neuronal cell bodies in scans of the depth of the visual cortex in six successive frozen sections. The number of synaptic appositions was counted in the final screen of the electron microscope at a fixed magnification of 18,000 which was measured with a carbon replica of a diffraction grating. Twenty fields were counted in each grid square at random positions obtained by flicking the specimen controls. About 1200 synapses were counted altogether in a scan of the depth of the cortex. The lengths of a random sample of 2-300 synaptic appositions were mea-
SYNAPSES
IN
DEPRIVED
CORTEX
447
sured in order to calculate (2) the density of synapses. Full details of the counting and measuring procedures have been published elsewhere (9). The tissue in the frozen sections at counting and in the plastic sections used for electron microscopy was found to be not shrunk from the sizes measured when the blocks were dissected out of the perfused brains. RESULTS Nezwonal Density. The average density of neurons in each of the seven deprived kittens was higher than in any of the controls (Table 1) and the mean values were significantly different (P < 0.001). When the neuronal densities at different depths were plotted, the distributions in the control and deprived kittens were substantially parallel, and there did not seem to be a concentration of the effect at any one depth. The diameters of the neurons in the visual cortex were significantly less than in the sighted kittens (Table 1) for both forms of visual deprivation (P < 0.01). The two kittens with crushed optic nerves were compared with the five kittens with eyelid suture, and the neuronal density was significantly higher in the former, while neuronal diameter was not significantly different. The kittens with crushed optic nerves showed extreme atrophy of the optic chiasm and tracts. In the lateral geniculate nucleus the mean cell area in the binocular part of lamina A was 29% less than in the sighted controls (P < 0.02), whereas the cell area in the sutured kittens was only 95% less than in the controls, and this difference was not significant (I am indebted to Miss Kum Wan for these measurements). The thickness of the visual cortex is difficult to define accurately because of the individual variations in gyral curvatures, but it was estimated in all the slides available, and found to be decreasedby 9% in the eyelid sutured group (P < 0.01) and by 10% in the cats with crushed optic nerves (P < 0.05). Tl lus, deprivation of vision has reduced the growth of the neuropil that progressively separates the neuronal perikarya and reduces neuronal density during development. Synaptic Density. The average density of synapseswas slightly lower in the seven deprived kittens than in the controls (Table 1), but these differences were not significant. However, the average count of synapsesper screen area was higher in the controls than in the eyelid sutured kittens, and this difference was significant (P < 0.05). The distributions of the synaptic count in the depth of the cortex were examined, and the differences reached the significance level of P < 0.05 in the part of the cortex between 1.0 and 1.4 mm below the pia only (layers 4 and 5). where the control kittens had a higher area density of synapses than either of the deprived groups. The average lengths of the synaptic appositions were the samein all three groups.
15.7
Mean
15.5 14.3
14.9
1 2
Mean
Crushed
15.3 15.6 16.4 16.2 14.8
1 2 3 4 5
Sutured
17.0
Mean
Size, I.cm
TABLE
1
5.3
5.2 5.3
4.8
4.7 5.2 4.7 4.4 4.8
3.73
3.7 3.6 3.9 3.9 3.7 3.6
Density X 107/cm3
.26
.26 .25
.26
.25 .25 .24 .27 .27
.26
.26 .25 .24 .27 .29 .27
Length Irm
7.2 7.6 7.4
.212
7.1
6.2 6.2 8.9 7.7 6.3
7.9
8.6 8.2 8.9 8.1 7.2 6.7
Density X 10n/cm3
.214 .209
.205
.179 .180 .242 .225 .200
.23.5
.248 .233 .244 .240 .231 .211
Count/$
Synapse
14,000
13,800 14,300
14,960
13,200 11,900 19,000 17,500 13,100
21,250
23,200 22,700 22,800 20,700 19,000 18,700
Neuron
Synapses
IN THE VISUAL CORTEX OF SIX CONTROL KITTENS, FIVE KITTENS WITH BILATERAL KITTENS WITH BILATERALLY CRUSHED OPTIC NERVES, ALL 4.5 DAYS OLD
Neuron
DENSITIES AND Two
17.2 17.3 15.7 16.9 17.4 17.5
AND SYNAPTIC
1 2 3 4 5 6
Controls
Cat
NEURONAL
SUTURE,
1483
1364 1602
1498
1.504 1.510 1490 1516 1469
1652
1782 1569 1668 1633 1619 1641
Thickness pm
EYELID
F s
n
y
I$ co
SYNAPSES
Synaptic the average significantly groups (P the neurons
IN
DEPRIVED
CORTEX
449
density was divided by neuronal density (Table 1) to estimate number of synapses associated with one neuron, and this was higher in the control kittens than in either of the deprived < 0.01). Thus, visual deprivation reduced the connectivity of in developing visual cortex by about 3070 at 45 days after birth. DISCUSSION
It is not surprising that neuronal density changed more than synaptic density, for much of the volume of the neuropil consists of axons and dendrites engaged in making synapses, and the axon terminals alone may account for 30% of the volume of neuropil (3). Thus, deprivation may retard the growth of neuropil and so cause a higher neuronal density, but the density of synapses is little changed in what neuropil does develop. The removal of spontaneous retinal discharge by optic nerve crush resulted in a significantly greater change than did eyelid closure in only one of the parameters measured: neuronal density. In the mouse is has been found that eye removal stops the growth of neuropil and neuronal diameter, while rearing in darkness allows a slow rate of neuropil development (17). Thus, in the cat, too, the difference between the effects of the two forms of deprivation may increase with survival time. Binocular lid closure resulted in a non-significant reduction in lateral geniculate nucleus cell area as has been found by other authors (4, IS), in contrast with the marked cell shrinkage (3040%) produced by monocular lid closure (4, 24, 25). Bilateral optic nerve crush reduced lateral geniculate nucleus cell area by 29% in the growing kittens, although in older cats, tract section has little effect on the lateral geniculate nucleus, for at least 120 days (12). Neither form of deprivation is likely to affect the cortex by a mechanism requiring lateral geniculate nucleus atrophy, for in the squirrel with monocular lid closure the neuronal density is higher in both the monocular and binocular parts of the visual cortex, whereas lateral geniculate nucleus cell shrinkage is found in the binocular segment only (16). It has been shown that the amomit of protein exported from the cell body into the neuronal processes increases with electrical stimulation of the neuron (18). It is likely then that the two forms of visual deprivation affect the growth of neuropil mainly by reducing the signalling activity of the neurons. In the visual cortex, extrinsic connections account for a small proportion of the synapsespresent (5, 7, 14) and are confined to particular depths, whereas the affects of deprivation are widespread. Thus, it must be mainly the signalling activity of cortical neurons that controls the growth of cortical neuropil. The reduced neuronal connectivity in the deprived cats would be espetted to affect the functioning of the visual system. When eyelids are re-
450
B.
‘3.
CRAGG
opened after binocular closure the cat may be behaviorally blind for several days (4, 21, 25). Single units in the visual cortex (4, 19) and in the lateral geniculate nucleus (22) show deficits in binocular properties and in responsiveness. Moreover, the influence of the visual cortex on the superior colliculus is lost so that the cat ignores objects in the contralateral field of each eye (21). It was suggested (21) that the visual cortex may remain nonfunctional for visually guided behavior, although it may take part in discrimination learning. The reversibility of these behavioral effects is uncertain, and two cats trained on visual tasks for long periods learned slowly (4). As regards structural recovery, a rapid concentration of the synaptic vesicles in axon terminals in response to brief visual input after dark-rearing has been found in cat visual cortex (13) and in rat retina (6). Whether the other structural changes produced by binocular deprivation in the cat could be reversed by visual experience is not yet known. REFERENCES 1. ABERCROMBIE,
Anat.
M. 1946. Estimation
of nuclear population
from microtome
sections.
Rec. 94 : 239-247.
2.
ANKER,
R. L., and B. G. CRACK 1974. Estimation of the number of synapses in a volume of nervous tissue from counts in thin sections by electron microscopy.
3.
ARMSTRONG-JAMES,
J. Neurocytol.
3: 725-735.
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