The effect of dark rearing and its recovery on synaptic terminals in the visual cortex of rabbits. A quantitative electron microscopic study

Brain Research, 78 (1974) 263-278

263

~:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

T H E EFFECT OF D A R K R E A R I N G A N D ITS RECOVERY ON SYNAPTIC T E R M I N A L S IN T H E VISUAL CORTEX OF RABBITS. A QUANTITATIVE ELECTRON MICROSCOPIC STUDY

G. V R E N S E N AND D. DE G R O O T

Department of Electron Microscopy, Mental Hospital "Endegeest', Oegstgeest (The Netherlands') (Accepted May 9th, 1974)

SUMMARY

The effect of dark rearing and its recovery on the visual and motor cortex of rabbits is studied. The number of synaptic contact zones per area (NA per 1000 sq.yn), their surface area (Sv in sq.#m/1000 cu./~m) and mean length (L in #m) and the number of synaptic vesicles per terminal (Nves) are studied, using specific staining techniques and stereological principles. Dark rearing for 7 months from birth does not affect NA, Sv and L, indicating that the formation of interneuronal connections is quantitatively normal in the absence of light. In the control and deprived animals a right-left dominance with respect to NA is observed in the motor cortex and the medial part of the visual cortex. The disappearance of this right-left dominance in deprived animals allowed to recover for 1 year, must be further investigated. No change in the synaptic density as a function of depth below the pial surface is observed. An obvious decrease in Nves is observed in the visual cortex after dark rearing. This decrease persists after recovery for 1 year. In the motor cortex there is no evident decrease. These results are related to recent studies regarding the significance of synaptic vesicles in the process of transmission of nerve impulses. It is hypothesized that light deprivation diminishes irreversibly the ability of synaptic terminals to synthesize and/or store transmitter substances. Electrophysiological and behavioral studies were performed on the same animals 46,61,71-79. Concomitant with the changes in synaptic vesicles, a decrease in visual evoked response (VER) and visual acuity (VA) is observed. The decrease in VA is persistent for at least 5 months whereas VER recovered to normal after 1-2 months.

264

G. VRENSEN AND D. DE GROOT

INTRODUCTION

A great number of biochemical, morphological and electrophysiotogical studies have been devoted to the effect of light deprivation on the visual system in a variety of mammals. Light deprivation is achieved by lid suture (monocularly and binocularly), eye enucleation or absolute or partial dark rearing. The visual centers investigated are the retina t0,25-27,54,55,57,68, the lateral geniculate nucleus *6,21,a'~,a6`60. the visual cortex (area striata, a r e a 17) 10,11,13,15.17,21.24,31 34,36,37,40,41,49,50,63,67.69.80.81.85.88.90 and the superior colliculus2S,47, 4s. Among the parameters estimated, protein metabolism a.11.aT-4°-41, free amino acid contentZl,s7, 66, activity of acetylcholinesterase ll49,50,6a, visual evoked response (V ER) x°.t3.31-s5-ss.90, electroretinogram (ERG)10.~1. neuron density la.25.9-s-3'.3a.aa.36.s~.'~s-63.s0 sL dendritic branching pattern ae :~t.6:l. spine density 32.33,63,67,s°,sl, synaptic density and diversity ta-1s-24--'~6.47 ls.~(~.se.s3, and synaptic vesicles s'4 are the most common. It is apparent from these studies that the effects of light deprivation are complex and that the mechanisms involved are still poorly understood. The complexity of the picture must partly be ascribed to the use of different species of mammals with their varying visual systems (degree of decussation, interhemispheric commissures) and the different approaches for obtaining light deprivation. Moreover. the correlation of biochemical, ultrastructural and electrophysiological data in this field is handicapped by the absence of a common basc at the molecular level. A most relevant aspect of these studies is the correlation of the structural functional alterations with changes in the visually guided behavior as studied by many investigators (e.g. see refs. 13. 63-65. 71-79, 85-88). The elucidation of brain mechanisms subserving behavior must be considered as a final goal of such studies. The present investigation intends to describe quantitative changes at the synaptic level as a result of long lasting dark rearing and recovery. Visual and motor cortices of rabbits submitted to dark rearing for 7 months and dark rearing for 7 months followed by recovery for l year were studied. Behavioral and electrophysiological measurements were carried out on the same or identically treated animals and are described by Van H o f eta/. 46,61,71-79. Synaptic density (NA), surface area of synaptic contact zones (Sv) and number of synaptic vesicles (Nves) are used for quantitative characterization. NA and Sv delineate the functional intactness of the wiring diagram whereas Nve~ informs about the functional intactness of the synaptic terminals. A critical discussion on the genetic and environmental factors influencing the organization of the visual system in more complex situations of retinal input restriction or modification is given by Van H o f 77.

MATERIAL AND METHODS

Dutch belted rabbits, raised in the Dept. of Physiology, Erasmus University, Rotterdam (Head: Prof. Dr: M. W. Van Hof), were used in this study. The animals belonged to a large group of rabbits used simultaneously for behavioral and electro,

265

DARK REARING, SYNAPTIC DENSITY AND VESICLES

physiological studies. Three groups of animals were raised under different environmental conditions: (1) control group; raised under normal lighting conditions; (2) deprived group; raised in complete darkness for 7 months from birth oi1; (3) deprived-recovered group; also raised in complete darkness for 7 months but thereafter in normal lighting conditions for 12 months. Prior to perfusion the control and deprived-recovered animals were kept in the darkroom for 24 h. The animals were fixed under urethane anesthesia by transcardial perfusion with glutaraldehyde-formaldehyde mixtures according to Peters ~9. After removal from the skull the brains were stored in cacodylate buffer. Small samples (2 mm x 2 mm extending from pia to white matter) were taken from different parts of the visual and the motor cortex in both the left and right hemisphere (Fig. 1). From these blocks of tissue thin slices (about 75 #m) were cut on a Vibratome (Cambridge Instruments). Adjacent slices were postfixed c.q. poststained with either: ( I ) OsO4, according to Palade 56, followed by section staining with lead citrate ~', (2) ethanolic phosphotungstic acid (E-PTA) according to Bloom and Aghajanianl,8, 9, with the modifications described previously by the present authorsS~, sa, or (3) osmic-zinc iodide (OZI) according to Kawana e t al. 4~ with the modifications and a counterstaining of synaptic contact zones as described previously 84. The slices were dehydrated in a graded series of ethanols, and embedded in Epon 812. Sections were cut on a Reichert OmU3 ultramicrotome using glass knives.

Lateral Medial

Fig 1

Fig. I. Cortical samples used in this study. Modified after Monnier and Gangloff53and ThompsonTM.

266

G. VRENSEN A N D D. I)E G R O O I

Starting directly under the pia (0 #m) ribbons of sections were cut at regular intervals of 100/zm parallel to the pial surface. The sections were examined in a Phi/ips EM300 electron microscope at different standard magnifications and photographed on Kodak F G P 35 mm film. The exact final magnification was tested in each set of micrographs by including a grating replica (2160 lines/mm) photographically handled identicallw

Quantitative evaluation As is critically investigated by the present authors 8',,83 the E-PTA material is very suitable for the quantitative characterization of the contact zones of synapt~c terminals. By simple countings the number of contact zones per area (NA/1000 sq./~m), their surface area per volume (Sv in sq.#m/1000 cu./zm) and their mean length (L m #m) can be obtained. These parameters are estimated for the different cortical samples either throughout the whole depth up to 1500 um or for the upper zones up to 200 # m below the pial surface. At each height level 10 random micrographs were analysed. The OZI-procedure, when optimally applied 84, enables the quantification of synaptic vesicles. We have counted the number of vesicles per synapttc profile in the micrograph. This relative measure, which is sufficient for a comparative study, can be transformed to an absolute measure by integration with the quantitative paratneters for synaptic contact zones 84. In a pilot experiment this measure was estimated throughout the whole depth of the cortex up to 1500 #m. No specific depth distribution was found and so for most of the measurements we have restricted these countings to 0, 100 and 200/zm below the pial surface, At each height level all synaptic terminals exhibiting a synaptic contact zone were analysed in 10 random picture~ .~.e. about 20-25 terminals per height level. RESULTS

The results of the quantitative characterization of synapses in the 3 groups of rabbits investigated are summarized in Tables l - I l l and Figs. 2 and 3*.

Synaptic contact zones The results of the quantitative characterization of synaptic contact zones will be described under three separate headings.

(1) Local distribution In preliminary experiments we encountered a striking difference m synaptic density in 2 samples of the visual cortex taken at different sites. Therefore we performed a pilot experiment on the local distribution of synaptic contact zones in superficial layers (0, 100 and 200 .urn) (Table 1). One of the most remarkable conclusions

* The spread in NA and Sv at each level (not given in the Tables) is very constant from animal to animal; S.E.M. about 10% of the mean. This indicates that we are dealing with synaptic populations with identical distribution.

115.6 106.5 189.1

± 9.0 (2) -- 3.4 (2) -- 21.3"* (2)

0,257 0.301 0.270

L

0.254 0.270 0.264

214.4=28.6 198.07! 3.6 200.5 = 11.4

NA

Right

230.9 :~ 6.1 216.3 = 24.2 193.6--21.3

(2) (2) (2)

(2) (2) (2)

0,234 0.247 0.287

L

0.266 0.255 0.265

236.9 _~ 32.2 220.5 = 3.2 203.7::!: 4.3

(2) (2) (2)

0.247 0.251 0,263

L

232.8 ± 12.5 (2) 249.4 ± 17.6 (2) 203.1 ± 16.1 (2)

NA

Lateral

0.236 0.236 0,284

L

* Mean, standard error of the mean and n u m b e r of rabbits. NA and L are estimated at 0, 100 and 200 Mm below pial surface f r o m 10 r a n d o m micrographs at each height level. ** Difference highly significant as compared with control and deprived animals (Student test).

Control Deprived Deprived recovered

N,4

Left

B. Motor cortex (see Fig,. 1)

132.4" ± 2.8 (2) 128.2 ! 9.1 (2) 216.9 = 11.4"* (2)

N;I

Na

?4,4

L

Medial

Lateral

Medial L

Right

Left

Visual cortex (see Fig. 1)

Control Deprived Deprived-recovered

A.

QUANTITATIVECHARACTERIZATIONOF SYNAPTIC CONTACT ZONES IN DIFFERENT SAMPLESOF ]'HE VISUALAND MOTOR CORTEX OF CONTROL, DEPRIVED AND DePRIVED--RECOVEREDRABmTS

TABLE 1

I',o -,-d

..<

,.< > 7'

Z >

Z

>

268

G. VRENSEN A N D D , DE G R O O t Control 40

Deprived 80

120

Depth 0

2oo NA

160

4o

80

~ ____j

2o0 NA

Depth 0

.OX

D

©

-=

©

o

x

x

o

500

O

=

•

500

O"

0

0

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0

o

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x

0

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0

1000

1000

D

•

O

0

D"

°0

-

0

0

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1500

OX

:i9 2

1500

0

:ig 3

Figs. 2 and 3. Depth distribution of the density of synpatic contact zones (NA) in left medial samples of the visual cortex of 3 ( . , × , 0) deprived and 3 ( . , - . 0/ control rabbits. Values at each height level are the mean of 10 random pictures. (Depth in/~m. NA per 1000 sq. t~m.1

to be drawn from this experiment was the existence of a right-left d o m i n a n c e m the rabbit cortex. In the control and deprived animals a right-left d o m i n a n c e (1.8) is observed for the m o t o r cortex and the medial areas o f the visual cortex. The lateral areas of the visual cortex on the contrary s h o w no statistical differences. The differences hold true for NA and Sv (not indicated in the Table). N o significant changes in the m e a n length are observed. N o such right-left d o m i n a n c e is observed in the deprived-recovered animals. This peculiar observation will be discussed later. Also noticeable is the g o o d agreement in NA and L o f the different groups o f animals and the small interindividual variation.

(2) Depth distribution On account of theoretical considerations it can be inferred that changes due to light deprivation are restricted to specific cortical laminae e.g. lamina IV which receives the main afferent input. Therefore we studied synaptic density (NA, Sv and L) at different levels up to 1500 # m below the pial surface in control and deprived animals, The results for NA are c o m p i l e d in the Figs. 2 and 3. Synaptic density reaches a high level directly below the piat surface, a level which is maintained u p to t2001300 ~m. At 1400 and 1500 # m synaptic density declines. In the visual cortex this decline is more pronounced than in the m o t o r cortex. In the depth region between

(2)

249.4±17.6 75.2 ± 6.4 0.236

control-deprived : n.s. control-depr/recov : n.s. deprived-depr/recov: n.s.

232.8"±12.5 69.7 ± 4.7 0.236

119.0 ± 9.0 41.4 ± 2.0 0.275

(3)

208.5 ± 10.7 54.3 z 3.4 0.264

control-deprived : n.s. control-depr/recov : significant P -< 0.01 deprived-depr/recov: significant P < 0.005

± 16.4 (3) ± 2.1

(3)

198.0±3.6 61.0 ± 2.7 0.247

111.1 = 5.0 (3) 41.5 ± 0.9 0.294

Deprived

(2)

200.5±11.4 67.7 ± 6.4 0.287

(2)

Deprived-recovered

183.1 ± 13.6 (3) 49.2 ± 2.9 0.269

Deprived-recovered

control-deprived : n.s. control-depr/recov : significant P ~< 0.005 deprived-depr/recov: significant P < 0.005

117.9 ± 5.7 (3) 39.0 ± 1.9 0.261

Control

Control

129.6 45.4 0.270

(2)

control-deprived : n.s. control-depr/recov : n.s. deprived-depr/recov: n.s.

214.4±28.6 65.4 ± 10.6 0.234

Deprived

* Mean, s t a n d a r d error o f the m e a n a n d n u m b e r o f rabbits. For the control a n d deprived a n i m a l s NA, Sv a n d L are estimated t h r o u g h o u t the whole depth up to 1500 l~m, for the deprived recovered a n i m a l s only at 0, 100 a n d 200/~m below the pial surface. At each height level 10 r a n d o m micrographs were analysed. The depth distribution of these m e a s u r e m e n t s for the control a n d deprived visual cortices are given in Figs. 2 a n d 3.

NMl000sq.ltm Svsq.l~m/1000cu.ltm L/~m

(2)

Deprived-reeovered

203.1 ~ 1 5 . 5 72.9 Z 3.5 0.284

Motor cortex Deprived

(2)

Visual cortex

B. Visual cortex, left medial; motor cortex, left (see Fig. l)

NA/1000sq./tm Sv sq./im/1000 CU.l~m L t~m

Control

Deprived-recovered

Control

Deprived

Motor cortex

Visual cortex

A. Visual cortex, right lateral," motor cortex, right (see Fig. 1)

Q U A N T I T A T I V E C O M P A R I S O N O F S Y N A P T I C C O N T A C T Z O N E S IN D I F F E R E N T S A M P L E S O F T H E V I S U A L A N D M O T O R C O R T I C E S OF C O N T R O L , D E P R I V E D A N D D E P R I V E D - RECOVERED RABBITS

T A B L E II

<

> Z

t~ Z

7, >

>

>

III

152 153 154 189

157 171 174 176 178

152 153 154 158 251

171 174 175 176 178

166 170 172 173 177

¢'~C0 |~et'ed

Deprived-.

P ~> 0 . 2 0

8.4 ~

48,2

7.2

4 5 . 9 . 5.7 30.1 3.6 58.9 -~ 6.8 37.1 4.1 69,4 5.4

r~ c o n t r o l - d e p r i v e d 2.30 0.025 ts c o n t r o l - d e p r , r e c o v 3 . 0 0 0.01 d e p r i v e d - d e p r r e c o v : n.s.

73.6 "*

5.5 c 8.9 5.4 8.6 6.1

P P

43.4

6.6

4.1 4.7 7.2 3.5 4.2

0.01 0.005

37,8 51.4 := 64.9 33.928.8

6.9

61.9

44.4

34.7 17.3 71.1 39.7 59.1

9.4

4.7 1.8 8.1 4.8 4.4

Deprived

t,~ c o n t r o l - d e p r ~ v e d 1.51 0. i0 t , c o n t r o l - d e p r , r e c o ~ - 2 A 6 0.05 d e p r i v e d - d e p r , r e c o v : n.s,

6.3 7.5 4.9 4.5 6.1

56.6 84.8 42.1 62.5 63.3

Control

Control

65.4 82.2 46.0 96.0 78.5

M o t o r cortex Deprived-"eco vered

0.30 -

4 5 . 4 ~ 11.5

39.9 5.4 2 5 , 9 ~_ 3.3 78.7 ± 7.7 37.2 :~ 3.9

Deprived

Visual cortex Deprived

0.64

3.6

52.4 t'r

4.2 5.2 4.0 4.6 5.1

~ _~ :t:

44.1 50.9 47.6 64.9 54.5

Control

M o t o r cortex

P P

42.8

(t.05 0.025

557

38.0 4.6 35.7 4.3 64.6 ~- 6.2 41.0 5.3 32.8 - 3.6

Depri~,edrecovered

DEPRIVED AND DEPRIVED--RECOVERED

micrographs were analysed. "~* I n t e r i n d i v i d u a l m e a n a n d s t a n d a ~ ' d e r r o r o f t h e m e a n .

°' M e a n a n d s t a n d a r d e r r o l o f t h e m e a n o f 6 0 70 p r e s y n a p t i c t e r m i n a l s at 0. 100 a n d 2 0 0 ; m l b e t o ~ the piaI s u r f a c e A t e a c h h e i g h t level l(1 r a l 3 d o m

Deprived

Control

Rabbit

B. Visual cortex, left medial: motor cortex, le#

3.3

3.6 3.5 4.9 3.1

0.005

• ~ -

~ee Fig, 1 }.

P

t7

4.40

38.1

36.6 33.3 47.7 34.7

Deprived

6 9 . 0 * * -~ 5.6**

:_ 5 . 1 ' - 5.8 _:_ 7.0 z 5.0 5.8

Control

Deprived

Control 58.7* 70.8 88.7 57.2 69.4

Visual cortex

Rabbit

A. Visual cortex, right lateral," motor cortex, right (see Fig. 1 )

RABBITS

N U M B E R OF VESICLES P E R S Y N A P T I C T E R M I N A L A T D I F F E R E N T S I T E S I N T H E V I S U A L A N D M O T O R C O R T E X O F C O N T R O L ,

TABLE

X

yr~

z

7

©

t~

DARK REARING~ SYNAPTIC DENSITY AND VESICLES

271

500 and 1000/tm a tendency of lower NA values is observed. This decrease is not significant, however. Surveying all the individual registrations we get the strong impression that remarkable variations exist in the horizontal plane. This could mean that depth distribution is confused by horizontal distribution. Further experiments concerning this horizontal distribution are in preparation. The present data show no changes in depth distribution between control and deprived animals neither in the visual nor in the motor cortex.

(3) The eff~ct of light deprivation Light deprivation for 7 months has no significant effect on the number of synaptic contact zones, their surface area and mean length in the visual and motor cortex of rabbits. This conclusion is based on measurements throughout the whole depth of the cortex up to 1500 #m (Table II and Figs. 2 and 3) at 2 different locations and on superficial measurements (0, 100, 200/zm) in 4 visual cortex and 2 motor cortex locations (Table I). The absence of any effect at different levels below the pial surface is critically discussed under (2). Light deprivation for 7 months followed by recovery for I year results in an increase (1.7) in the number of contact zones in the medial left region of the visual cortex. A similar increase is observed in the left motor cortex. This unexpected result must be interpreted very carefully. Either it really points to an increment in a specific region of the cortex or it is simply the result of the fact that samples are taken a little bit more laterally. The concomitant increase in the left medial part of the visual cortex and the left motor cortex pleads for the first view, the absence of this increase in other region smakes this conclusion at least conspicuous. Further experiments are in preparation to elucidate this.

Synaptic vesicles (1) Local distribution No significant differences are observed in the mean number of synaptic vesicles in the 2 areas of the visual and motor cortex investigated in the 3 groups of animals.

(2) Depth distribution in 3 animals, 1 of each group, we have studied the number of synaptic vesicles as a function of depth. No specific relation could be observed and so we decided to restrict the analysis of synaptic vesicles to the superficial layers (0, 100 and 200/~m).

(3) The effect of light deprivation The effect of light deprivation for 7 months on the number of synaptic vesicles in the visual cortex is obvious (Table III). From measurements at 2 different sites in 6 animals it can be concluded that there is a clear cut diminution of synaptic vesicles in comparison with the control group (0.55 and 0.65). This decrement is highly significant (Student test) despite the substantial interindividual spread. Recovery for 1 year after deprivation for 7 months does not result in an increase

272

G . VRENSEN A N D I). DE G R O O ]

of synaptic vesicles. The overall mean of 5 animals shows a similar difference with the control animals as do the deprived animals (0.59). This difference is also highly significant. In the motor cortex the situation is somewhat more complicated. No difference between control and deprived animals can be observed in the right cortex. In the left cortex there exists a tendency of difference between deprived, deprived-recove,ed and control animals, (0.10 - P > 0.05 and 0.05 P - 0 . 0 2 5 respectively). The differences between the experimental animals and control animals might be to() small to be detected with small groups of animals which show such considerable interindividual variation. DISCUSSION

Globus and Scheibe133 and Globus 3~ have reported on the effect of dark rearing in rabbits using Golgi preparations. N o definite changes in the number of neurons. the relative frequency of the different cell types, the length of basilar dendrites and the number of spines could be observed in the visual cortex. These observations ave strongly supported by the present electron microscopic registrations on synaptic contact zones. This support is the more significant because of the visualization of all synaptic contacts in an uttrastructural picture, unlike the visualization of one type of synapse in a limited number of cells in the Golgi preparations. On account of these studies it seems justified to conclude that dark rearing from birth has no significant effect on the adult neuronal connectivity. Whether dark rearing delays the synaptic development, as is observed by Valverde e t a / . 67,8°,81 in mice, must be further investigated. This conclusion does not imply, however, that the neuronal connections are functionally intact. Globus and Scheibel ~3 and Globus 32 describe a deformation of spines in the apical dendrites of pyramidal cells. Synaptic contact zones are highly organized with dense projections, intercleft line and postsynaptic band as the most relevant substructures. The ultrastructural organization of synaptic membranes and the functional significance of it are recently reviewed by Akert 2. Bloom 8 describes changes in these synaptic membrane substructures in the cerebellar cortex of rats during development. The effect of dark rearing on these structures m the visual cortex of rabbits will be studied, The unique position of the visual system of rabbits 30,43 in the group of mammals makes a comparison of the present results with those found in other species very troublesome. In mice, Valverde et al. 67,s°,sl observed an obvious decrease in number of apical spines of visual pyramids up to 40 days after the onset of dalk rearing at birth. At day 50 only a small difference, although still significant, was observed. No changes were present in the motor cortex. The decrement rapidly reversed when the animals were brought into light at day 20. It seems likely that dark rearing only delays the normal development of spines. Dark rearing has no effect on the number and relative frequency of the different types of synapses in the lateral geniculate nucleus of mice as is recently reported by Pysh and Talat K h a n 6°. tn rats. Cragg 11-1:~ observed changes in the visual cortex after dark rearing. These changes were distinct

DARK REARING, SYNAPTIC DENSITY AND VESICLES

273

and to some extent opposite in the upper and lower half of the visual cortex. Synaptic diameter was smaller in the upper layers, and larger in the lower half. The number of synapses was smaller in the lower half of the cortex. Fifkovfi24 describes a decrease of 20 °/o in the number of synapses and an increase in the size of synaptic contacts of 7.5 ~o in the superficial layers of the visual cortex of rats contralateral to the deprived eye. A decrease in the number of synapses in the lateral geniculate nucleus of rats after dark rearing is reported by Cragg 1~. Studies concerning changes in synaptic density after eye enucleation are omitted because of transsynaptic atrophy after this treatment. They do not register the effect of light deprivation but merely degeneration effects. Data on synaptic density within different zones and different hemispheres of the visual and motor cortex are not present in the literature. Our data points to a right left dominance in the motor cortex and a distinct zone of the visual cortex. This dominance is present in control and deprived animals. This finding is complicated by the peculiar situation after recovery in which this right-left dominance is abolished. The possible importance of this observation can only be evaluated after more detailed studies on local distribution and more systematic studies of recovery after light deprivation. Cragg 1'~ and Molliver and Van der Loos 51 observed a depth distribution of synapses with 2 inain peaks (0-200, and 500-1000/zm below the pial surface) in prenatal cats and newborn dogs. In human fetuses 52 only one peak (40-80/tin) is present. In adult cats a remarkably even distribution of synapses is observed by Cragg 19. This observation completely agrees with our observation in adult rabbits. We have to be careful, however, since small depth differences may be masked by differences in synaptic density in the horizontal plane. The most prominent result of the present study is the finding that dark rearing results in an obvious decrease in synaptic vesicles. This decrease is also present in animals recovered for 1 year after the deprivation period. This finding is not likely to be a technical artefact. In a previous paper s~ we have described a reliable OZIprocedure that results in the complete staining of synaptic vesicles. We have excluded the possibility that the observed decrement is simply the result of a short term dark period prior to fixation by using animals submitted to dark rearing for 24 h as controls. Moreover, no differences were observed between these controls and control animals perfused without previous dark rearing for 24 h. An accidental effect of anesthesia is not likely. Van Hof 72 has shown that urethane has little effect on visual evoked responses. From recent literature on synaptic transmission of nerve impulses a detailed picture of the substructures involved in this process emerges. Akerff recently reviewed the ultrastructural organization of synaptic membranes and changes in this 'presynaptic vesicular grid' associated with alterations in synaptic activity. A most crucial role in the process of transmission is played by specific chemical substances, the transmitters. These transmitters are thought to be stored in the presynaptic agranular and granular vesicles (e.g. see refs. 2, 5, 22, 89). There is good evidence that the transmitter is released into the synaptic cleft by exocytosis.

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Studies regarding the effect of stimulation of peripheral nerves ~.,-~:~.2°-~:~-as.:~9. 42.44.58 and of the cortex 3,e3 have shown that the number of synaptic vesicles in the terminals diminishes as the result of this treatment. Replenishment is observed after recovery. This process is energy dependent. Ceccarelli et al. 12 studied this process in great detail in the frog's neuromuscular junction. On account of their combined electrophysiological-ultrastructural studies also using horseradish peroxidase and dextran as tracer substances, they conclude: "(a) that synaptic vesicles fuse with. and re-form from, the membrane of the nerve terminal during and after stimulation, and / b) that the re-formed vesicles can store and release transmitter'. The release of transmitter by exocytosis is strongly endorsed by the work of Heuser and Reese ~s and of Holtzman e t al. 39. Whether the retrieval of vesicular membranes is due to endocvtosis of axolemma or to formation from intraterminal membranes is still in discussion. In thi~ framework the present results, of a definite decrease in synaptic vesicles in the visual cortex after dark rearing, probably have far reaching consequences. It can be hypothesized that dark rearing diminishes the ability of synapses to synthesize and or to store transmitter substances. The irreversibility of the decrease in vesicles after recovery could probably point to an induction process which is light mediated and irreversibly affected by long term dark rearing. It i~ quite clear that many further experiments are needed before this hypothesis can be fully accepted. Most of the knowledge regarding the structural-functional relationship m synaptic transmission is based on relatively simple peripheral systems, as briefly outlined above. Identical correlations for the cerebral cortex are scarce~ A correlation between the number and distribution of synaptic vesicles and stimulation, identical to that in peripheral nerves, is described by Fehdr e t al. 23 for the primary auditory cortex. The complex organization of the cerebral cortex with its many afferent, intracortical and efferent connections greatly hampers this structural-functional correlation, Therefore, the comparison of the present morphological results with the electrophysiological and behavioral data. scored on identically treated animals 46.~.71 79. will be discussed with great reserve. The effects of light deprivation on wsuat processing in rabbits has recently been reviewed by Van Hof w. The results of the electrophysiological and behavioral investigations can be summarized as foltow~: (1) dark rearing does not affect the ability to discriminate vertical against horizontal and 45 '~ v e r s u s 135'~ striations as well as the angular threshold of tilt discriminationV~.w.vs: (2) visual acuity is lower in dark reared animals, an alteration which lasts for at least 5 months after the deprivation period 7s' (3) the amplitudes of the visual evoked responses (VER) are markedly decreased after light deprivation for 7 months. This VER restores to normal by exposure to light for 1-2 months, at least for flash frequencies of l/sec46: (4) the electroretinogram (ERG) in the adults is not affected by dark rearing 6l. This finding is consistent with observations in cats and monkeys ~:'Since light deprivation does not affect the ERG. it must be assumed that the decrease in the VER is of geniculate and or cortical origin. Changes in the VER and in single cell responses are also found in other mammalsla,6'% The visual evoked

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response must be considered as the summation of postsynaptic potentials. A decrease of this V E R can thus be explained as either a disturbance o f the wiring diagram or as a functional inactivation of existing morphological intact synaptic contacts 7v. On account o f the present results the second explanation seems the more likely. It cannot be excluded, however, that besides a deterioration of the ability to store and synthesize transmitter a structural disturbance o f the vesicular grid also contributes to the dysfunction o f synaptic terminals. C h o w and Stewart ~3 have pointed out that the restoration of the V E R and the single cell response in cats is only partial. Both types of potentials fail to follow rapid rates of stimulation, (flash frequencies 2 l/sec). They consider this feature as fatigation. This fatigation may be consistent with our finding of an irreversibility of the diminution in synaptic vesicles. Whether the persistence of the lower visual acuity can also be explained by this irreversible effect can only be speculated about at present. ACKNOWLEDGEMENTS

We are grateful to Prof. Dr. M. W. van H o f (Dept. of Physiology, Erasmus University, Rotterdam) for his stimulating discussions. The technical assistance of Miss C. Passchier and Mr. W. Dijksma is greatly acknowledged, and we thank Miss S. Koning and Miss N. Diels for typing the manuscript. We thank Prof. Dr. W. Th. Daems (Dept. Electron Microscopy, University of Leiden) for placing at our disposal the Vibratome and calculator facilities. NOTE ADDED IN PROOF

Since completion of this paper Garey and Pettigrew (Brain Research, 66 (1974) 165-172) have reported changes in synaptic vesicle density in the visual cortex of kitten after environmental modification, and C h o w ( H a n d b o o k o f Sensory Physiology VII/3A, Ed. R. Jung, Springer Verlag, Berlin, 1973, 599-627) has reviewed neuronal changes in the visual system following visual deprivation.

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