the effect of unilateral deprivation on visual centers in rats

The Effect of Unilateral Deprivation on Visual Centers in Rats

E. FIFKOVA Institute of Physiology, Czeckodot.uk Academy o f Sciences, Prague, and T h e Max-Plnnck Institute f o r Brain Research, Frankfur t /Main

ABSTRACT The effect of unilateral visual deprivation by lid suture on visual centers was studied in rats. In animals with previous visual experience this caused a decrease in volume of the dorsal nucleus of the lateral geniculate body and nucleus lateralis pars posterior of 10% and 8% respectively, a decrease in the thickness of layers I1 to IV of the visual cortex of 8% and an increase in cell density in cortical layers I11 and IV of 12% and 10% respectively. These changes were of the same magnitude as in rats which had no previous visual experience. Quantitative changes in deprived visual centers were detectable ten days after lid suture; the process reached its maximum after 30 days. The num- ber of spines on the apical dendrites of pyramidal cells of layer V, observed in layer IV, was reduced by 17% after ten days of visual deprivation and by 28% after 30 days. Reopening of the eye for 10 and 30 days following two months of lid closure caused no recovery in the deprived visual centers.

In earlier papers the effect of unilateral monocular lid closure of two months dura- tion on the morphology of the visual cen- ters was described in rats without previous visual experience (Fifkova, '67, '68, '70; Fifkov5 and Hassler, '69). Such closure causes changes in volume, cell density and synaptic contacts in the lateral geniculate body and visual cortex on the side con- nected with the deprived eye. The aim of the present paper was to study the effect of unilateral deprivation in animals with previous visual experience, and the time course of the development of the anatomi- cal changes. The extent of the recovery after reopening the eye was also investi- gated.

MATERIAL AND METHODS

The number and age of the animals at the time of the operation, the length of the deprivation period and of the period after reopening the eye, together with the histological technique used in the different experimental groups, are summarized in table 1. The deprivation was achieved by lid suture of the right eye in 54 white rats (Lewis strain). The lid suturing and eventual lid opening were performed under

J. COMP. NEUR., 140: 431-438.

light ether anesthesia. With the exception of group HA, the rats were transferred three days after the operation from their usual cages into larger ones which allowed greater freedom of movement and en- hanced pattern vision (FifkovB and Hass- ler, '69).

After completion of the experiments the animals to be used for cell staining were sacrificed under allobarbital anesthesia by perfusion through the heart with 10% formol-saline. The brains were embedded in celloidin and cut serially a t 20 p ; every third section was stained with cresyl violet.

From cresyl violet stained sections rele- vant parts of the brain were drawn at X 40 magnification. The volumes of the dorsal nucleus of the lateral geniculate body (GLD) and of the nucleus lateralis pos- terior (Lp) were determined from these drawings by planimetry. The thickness of the visual cortex was determined in coronal sections of the entire area 17 spaced 120 p apart. Starting from the medial limit of the area the thickness of the cortex was measured in each section at 0.5 mm inter- vals. The mean value of about 70 mea- surements in right and left hemispheres were compared. Because of lack of sharp

431

432 E. FIFKOVA

Period - Histological

Age at Number the day

of eye Of eye After eye technique animals closure closure reopening

Group of

I 6 60

A 6 14 7 14 J 14

B C

D 6 14

E 5 14

9 14 10 14

I1

I11 6

cytoarchitectonic boundaries between dif- ferent cortical layers in the rat's primary visual area, the thickness of the supra- granular complex (11-IV layers) and that of layers V and VI were measured sepa- rately. Cell counts were carried out in layers I11 and IV on tissue volumes of 297,680 p3 and 11,858 respectively. Cells in thirty of these tissue volumes of both layers were determined in each hemisphere and the mean computed.

For the impregnation procedure of Ram6n-Moliner ('58) the anesthetized ani- mals were killed by decapitation, the brains were quickly removed and the occipital half of the hemisphere was placed in the im- pregnation solution. The blocks of tissue were embedded in celloidin and cut at 80 p, perpendicular to the cortical surface. Ap- proximately equally impregnated pyra- midal cells corresponding to class I (Globus and Scheibel, '67) of layer V within the boundaries of the visual cortex were chosen for spine counts. The shafts of the apical dendrites were divided in 50 long segments on which the number of spines were determined. All counts were carried out with a Zeiss oil immersion objective ( X 100) and a 12.5 x ocular supplied with a graticule. Altogether 574 segments were evaluated, 327 on the control side corresponding to the non-deprived eye and 247 on the experimental side.

All measurements and counts were done without knowledge as to whether the de- prived or control side was being measured. After counts were completed the side iden- tification was done.

90 - Nissl

3 - Nissl 10 - Nissl 30 - Nissl

Ram6n- 10 - Moliner

Ram6n- 30 - Moliner

60 10 Nissl 60 30 Nissl ~-

RESULTS

Group 1 The rats of this group were allowed nor-

mal vision for six weeks before the lids of the right eye were sutured. They sur- vived the operation for three months (ta- ble 1). The total volume of the GLD. of its rostral and caudal part and of the Lp supplied by the lid sutured eye was ex- pressed as a percentage of these values on the opposite side (table 2 ) . The volumes on the side connected to the deprived eye were all significantly smaller. The thick- ness of layers 11-IV of the primary visual cortex connected with the lid sutured eye was significantly decreased, whereas that of layers V and VI was not changed. Cell counts revealed an increased densitv per unit volume in layers I11 and IV (table 2).

Group I1 The rats in this group had their lids

sutured just before the time of normal eye opening (14 days). The animals survived this operation for 3, 10 and 30 days (table 1). Some were used for cell staining (group IIA, B, C ) , some for the Ram6n-Moliner technique (group IID, E). No right-left vol- ume difference could be observed either in GLD or Lp in rats after three days of lid closure. After ten days of deprivation a vol- ume decrease was found in the deprived GLD. No right-left difference could be ob- served in the Lp at that time. The usual criterion (cell size) used for distinguishing the rostral (large cells) and caudal part (small cells) of the lateral geniculate body could not be used in the animals of group

TA

BL

E 2

Differen

cr of m

ean valu

es between

Tight and left visual centers in perccritages

Subcortex V

olume

T

R

C

Group

GL

D

- -~

~~~ ~~~

I -1

0.4

6+

1.1

8'

-7.6

52

2.0

6

-11.31-C3.15

p < 0

.001

p < 0.02 p < 0.02

Cortex

-

LP

Th

ickn

ess C

ell den

sity

11-IV

v-VI

I11 IV

~

- 8.23 2

1.95 - 8.33 i

1.28 - 1

.48

2 2.10

+ 11

.75

2 1.98

+ 10

.20

t 1.75 p < 0.005

p < 0.005

ns p

< 0.005 p

< 0.005

I1 A

-2.43%

1.72

_-

-

- 0.10 2

1.48 - 0.92 2

5.86 -2.05

-C 3.17 -+ 2.38 -C

2.90 + 2.35 %

2.42 11s 2

ns ns

ns ns

ns

B

-11

.04

L1

.91

-

-

- 6

.37

k4

.10

- 5.58 i

2.00 - 1.38 f2

.80

+

6.8

22

1.61 + 7.25"

1.34 p

< 0.001 ns

p < 0.02

ns p

< 0.0

05

p

< 0.005 C

-15.1O

C3.47

-3.5

4t4

.00

-25.8024.80

-112.482525 -9

.40

23

.22

-3

.26

t1.6

6

$1

3.6

32

0.6

3

+12.20-C

O.30

p < 0.02

ns p < 0.02

ns p

< 0.05 ns

p < 0.01

p < 0.005

I11 A

-10.96-C

l.95 -6.61+

4.98 -10.23+

2.84 - 5.33 -1- 2.28

- 9.63 -C 2.05

- 0.56 L

0.54 + 12.68 2

0.20 + 10.85 t

1.17 p

< 0.005 n

s p < 0.01

p < 0.05

p < 0.005

ns p

< 0.001 p

< 0.001 B

-7.2

5e0

.99

-4

.50

23

.85

-1

2.0

62

1.77

-4.71 2 1.82

- 6.22+

1.20 -2.20"

0.20 + 1

1.0

6k

1.39 + 9.50-C

1.38 p

< 0.001 ns

p < 0.01 p

< 0.025 p

< 0.0

05

p

< 0.001 p

< 0.0

05

p

< 0.005 1 Standard d

eviation.

2 Not sign

ificant.

Valu

es of th

e visual cen

ter of the right sid

e correspon

din

g to the op

en eye w

ere taken

a

s 1

00

%.

GL

D. L

ateral genicu

late body dorsal part.

T. T

otal. R; R

ostral. C

, Cau

dal.

Lp, N

ucleu

s lateralis pars p

osterior,

434 E. FIFKOVA

I IA and B because at thesc ages both ele- ments are mixed together in a considerable part of the nucleus. The 30 day deprivation period caused significant volume changes in the caudal part of the GLD but not in the Lp. Figure 1 shows the growth of the GLD and Lp from the seventeenth day of life (14 days before the lid closure + 3 days after) to the seventy-fourth day (14 days before lid closure + 60 days after). The nondeprived side shows a continuous growth whereas after ten days of depriva- tion there is a decrease in volume of the GLD which, however, is not statistically significant. The nucleus grows again in the period between the tenth and the sixtieth day of deprivation.

In animals killed three days after lid closure no significant right-left differenccs were noted in measuring the thickness OP cell layers in the visual cortex. After ten days of deprivation by contrast layers 11-IV were found significantly narrower in the deprived visual cortex than on the opposite side, a difference that had become some- what greater after 30 days of deprivation (table 2). In addition, an increased cell density in layers 111 and IV was observed in case of ten days standing and after 30 davs of demivation this deviation from nor- m i l had* become considerably (fig. 3).

30

200

70

40

11 0

rl

3 NU

b G

greater

h IS mLP

Fig. 1 Differences in mean values of volumes (vertical axis) of the lateral geniculate body (G) and the nucleus lateralis pars posterior (Lp) at different time periods after lid suturing (3 , 10, 30, 60 days). Black columns indicate structures connected with the deprived eye (the 60 day values are taken from the paper by Fifkova and Hassler, '69).

The definitive thickness of layers 11-IV and V-VI are on the control side almost established at the seventeenth day of life (fig. 2 ) . However, in layers 111 and I V the cell density decreases from the seventeenth day of Iife to the forty-fourth day (fig. 3 ) . No further changes in cell density appear to take place after this age.

The group of rats used for the Ramon- Moliner technique survived the lid suturing for 10 and 30 days. The number of spines on the apical dendrites of the layer V pyramidal cells of the deprived and normal visual cortices were compared in layer IV. In figure 4 an apical dendrite from the normal ( A ) and the deprived (B) visual cortex after 30 days of lid suture are shown. The spines seem sparser on the deprived than on the control side. In table 3 the number and percentage difference for each 50 p segment are given. The mean quantitative deficit for the entire measured length of the dendrites was lower in cases of ten day deprivation (IID) than in the case of 30 days standing ( I I E ) , 17 and 28% respectively.

In previous studies, the number of spines has been found to increase on the normal side up until the forty-fourth day (Fifkov5, '68). No similar increase was observed on the deprived side. The dif- ference at these ages is significant (p < 0.05 j table 3.

Group III The lids were sutured in this group at

an age of 14 days, before eye opening.

3 ri

20

10

0

3 10 30 -lV v-VI

60 Fig. 2 Differences in the mean values of

thickncss of cortical layers 11-IV and V-VI at dif- ferent time periods after lid suturing. Details as in figure 1.

VISUAL CENTERS AFTER UNILATERAL DEPRIVATION 435

After two months of eye closure the lids were separated and the animals were al- lowed to see for 10 (IIIA) and 30 (IIIB) days (table 1 ) . The results are summarized

4;>

XI

2 0~

" 0.

C. I 3 1 ll', 10

3C 60 Fig. 3 Differences in the mean values of the

cell density of cortical layers I11 and IV at dif- ferent time periods after lid suturing, Same as figure 1.

in table 2. In both groups the total volume of the lateral geniculate body was still sig- nificantly smaller on the deprived than on the control side. While no statistically sig- nificant left-right difference was found be- tween the rostra1 parts of the GLD, such a difference was found for the caudal part. The nucleus lateralis pars posterior like- wise was significantly smaller on the de- prived side. The thickness of layers 11-IV was less on the deprived side and the cell density in layers I11 and IV on this side was significantly increased.

DISCUSSION

According to the findings here reported previous visual experience for the time and at the period of the rats' life falling within the scope of the present experiments does not diminish the effect of unilateral depri- vation on the visual centers. Changes of

Fig. 4 Shafts of apical dendrites of layer V pyramids, photographed in layer IV, of the visual cortex connected with the normal eye (A) and with the deprived eye ( B ) .

436 E. FIFKOVA

1 I the same magnitude were observed in the lateral geniculate body and visual cortex of animals which had been allowed unob- structed vision for two months before the eye was closed as in rats who never had visual experience. Similar results were noted by Gyllensten et al. ('65) who ex- amined the visual centers of mice kept in the dark either from birth or from the fourth month of age. Changes in size and density of synaptic terminals were found by Cragg ('69) in the lateral geniculate body of rats kept in the dark from four weeks of age forward. In the experiments of Rosenzweig et al. ('69), variations in the richness of the environment affected the thickness of the cerebral cortex irre- spective of the age at which rats were ex- posed to such variations. However, Burke and Hayhow ('68) found that in cats raised in the dark over a period of two years after initial exposure to diurnal light conditions, the responses of the lateral ge- niculate neurons to physiological stimuli were no different from those of controls who were never deprived of light; neither were histological changes observed in such animals. Wiesel and Hubel ('63). Hubel and Wiesel ('70) and Dews and Wiesel ('70) found that the lid suturing in adult cat was without any effect. In kittens the extent of damage was related to a period of susceptibility in which the visual sys- tem starts to react to deprivation at the age of one month, reaches the maximum in the second and disappears around the third month. Until now there has been no evi- dence as to whether or not there is a period of similar duration in rats. It seems likely from these observations that species differ- ences exist, and that these are possibly re- lated to the extent to which plasticity is retained during and after maturation.

In the present experiment reopening of the eye following two months of lid closure caused no recovery in the deprived visual centers. The anatomical changes found in cases of 10 and 30 days of reopening the eye were entirely comparable to those seen in animals killed immediately after two months of deprivation. In earlier experi- ments decreased population density (14% ) of geniculate neurons has been found after a similar deprivation period (FifkovA and Hassler, '69). Wiesel and Hubel ('65)

m ci d

+I f

2 r-

m

t- 3

4 m

+ I t. : 2 4

m

Y.

8 1

i l

m m i a $1 m

'" m

m

* 0 m m

3 0 $: V

Lr) 3

ci m cq lx

0 c!

$ 1 m 3

lx

a

0.1

0 0 0.1

2 1 $12 a m m LD

Lr) m t i c

tl * 3 3 *

m

VISUAL CENTERS AFTER UNILATERAL DEPRIVATION 437

found that there was only a very limited capacity for recovery from the effects of visual deprivation in kittens. After 3-15 months of exposure to light no morphologi- cal improvement in the lateral geniculate was seen and many cortical neurons were found which gave abnormal respon, ces to visual stimuli. Results obtained with the light microscope do not, however, exclude the possibility of a recovery process oc- curring in the ultrastructure. Cragg ('69) described in the lateral geniculate body of rats raised in the dark (till weaning) and then exposed to daylight, an increase in density of geniculate synapses. The same author ('67) observed an increase in the size of axon terminals in the upper half of the visual cortex of dark-raised rats as early as three hours after the light ex- posure. In retinal receptors three minutes of light exposure were sufficient to produce detectable changes (Cragg, '69).

In the present experiments eye closure for three days was not long enough to have significant effect upon volume and cell density of the lateral geniculate body and visual cortex. Differences detectable in the light microscope were observed only after a ten day deprivation period. Larger right- left differences were observed in these structures in case of 30 day lid closure; earlier findings (Fifkovh and Hassler, '69) suggest that such diff'erences reach their maximum at about that time. The increase in volume of the GLD and the Lp during the deprivation period indicates that the growing process, although impaired, is not completely halted by the deprivation.

The volume measurements and cell- density count gave negative results after three days of eye closure. Since the im- pregnation technique (Golgi-Cox modifica- tion) used gives no satisfactory results in very young animals (less than 15 days, Ram6n-Moliner, '58) the shortest depriva- tion period chosen for spine counts were performed earliest in 24 day old animals, i.e., ten days after lid closure. A signifi- cantly smaller number of spines (17% ) was observed at that time on the shaft of apical dendrites of layer V pyramids pass- ing through layer IV on the deprived than on the control side. A larger difference (28% ) was found after 30 days of depri- vation. Extension of the deprivation period

by an additional month had earlier been found to have no added effect upon the number of spines (Fifkov5, '68). Ruiz- Marcos and Valverde ('69) observed in young, dark-raised mice a greater loss of spines than was found in the present ex- periments. By contrast, Globus and Schei- be1 ('67) found in dark-raised rabbits the spines pathologically changed but not de- creased in number.

Whether the asymmetry resulted from size reduction of the experimental side or size increase of the control one or both can follow from the comparison of absolute values in lid-suture and unoperated ani- mals. The preliminary results seem to in- dicate that the asymmetrical changes are due to the reduction on the experimental side (Fifkovh and Hassler, '69).

As the unilateral eye closure took place in young animals the question whether the described asymmetrical changes are due to growth inhibition induced by deprivation or to deprivation itself should be con- sidered. The observation that the same changes in volume and cell density were observed whether lids were sutured on the fifteenth or sixtieth day of postnatal life argues in favor of the latter alternative.

ACKNOWLEDGMENT

The author expresses her gratitude to Professor A. Van Harreveld for his help in preparation of the manuscript.

LITERATURE CITED

Burke, W., and W. R. Hayhow 1968 Disuse in the lateral geniculate nucleus in the cat. J , Physiol. (Lond.), 194: 495-519.

Cragg, B. G. 1967 Changes in the visual cortex on first exposure of rats to light. Nature, 215: 251-253.

1969 The effects of vision and dark- rearing on the size and the density of synapses in the lateral geniculate nucleus measured by electron microscopy. Brain Res., 13: 53-67.

1969 Structural changes in naive re- t inal synapses detectable within minutes of first exposure to daylight. Brain Res., 15: 79-96.

Dews, P. B., and T. N. Wiesel 1970 Conse- quence of monocular deprivation on visual be- haviour in kittens. J. Physiol., 206: 437-455.

Fifkov5, E. 1967 The influence of unilateral visual deprivation on optic centers. Brain Res., 6: 763-766.

1968 Changes in the visual cortex of rats after unilateral deprivation. Nature, 220: 379-381.

438 E. FIFKOVA

1970 The effect of monocular depriva- tion on the synaptic contacts of the visual cor- tex. J. Neurobiol., 1: 285-294.

FifkovB, E., and R. Hassler 1969 Quantitative morphological changes in visual centers in rats after unilateral deprivation. J. Comp. Neur.,

Globus, A, , and A. B. Scheibel 1967 The effect of visual deprivation on cortical neurons: A Golgi study. Exp. Neurol., 19: 331-345.

Gyllensten, L., T. Malmfors and M. L. Norrlin 1965 Effect of visual deprivation on the optic centers of growing and adult mice. J. Comp. Neur., 124: 149-160.

Hubel, D. H., and T. N. Wiesel 1970 The period of susceptibility to the physiological ef- fects of unilateral eye closure in kittens. J. Physiol., 206: 419-436.

135: 167-178.

Ramon-Moliner, E. 1958 A tungstate modifica- tion of the Golgi-Cox method. Stain Technol..

Rosenzweig, M. R., E. L. Bennett, M. C. Diamond. W. W. Su-Yu, R. W. Slagle and E. Saffran 1969 Influence of environmental complexity and visual stimulation on development of occi- pital cortex in rat. Brain Res., 14: 427-445.

Ruiz-Marcos, A., and F. Valverde 1969 The temporal evolution of the distribution of den- dritic spines in the visual cortex of normal and dark raised mice. Exp. Brain Rcs., 8: 284-294.

Wiesel, T. N., and D. H. Hubel 1963 Effects of visual deprivation on morphology and physio- logy of cells in the cat’s lateral geniculate body. J. Neurophysiol., 26: 978-993.

Wiesel, T. N., and D. H. Hubel 1965 Extent of recovery from the effects of visual depriva- tion in kittens. J. Neurophysiol., 28: 1060- 1072.

33: 19-29.