Anat. Embryol. 149, 1 13 (1976) 9 by Springer-Verlag 1976
Postnatal Growth of the Dorsal Lateral Geniculate Nucleus of the Cat H e l l m u t Elgeti, R i c a r d a Elgeti, a n d K u r t Flcischhauer
*
Anatomisches Institut der Universit~t Bonn Received January 9, 1976
Summary. The postnatal growth of the dorsal part of the lateral geniculate nucleus (LGNd) is studied in paraffin sections through the brains of 32 eats of known age. The changes in shape and position of the LGNd are described and it is shown that i~s volume increases from about 3.4 mm3 at bir~h to about 26.4 mm3 in the adult cat. When ~his value is corrected for shrinkage, the volume of the LGNd in the adult cat turns out to be about 44 mm 3. Tim detailed measurements reveal that during the second and third week of postnatal life tllere is a particularly steep increase in volume and that the final values are already reached at around the 40th day. Concomitant with the increase in volume there is a decrease of the number of cells per unit volume of grey matter. In the binocular segment of lamina A the number of cells decreases h'om about 470 per (0.i ram) ~ at birth to between 95 and 130 per (0.1 ram) a in the adult cat. Separate measurements of nerve cells and neuroglial cells indicate that the absolute number of nerve cells remains fairly constant during postnatal life, whereas between the second and sixth week a great number of neuroglial cells are newly formed. Key words: Postnatal development - - Cat - - Visual system - - Lateral geniculate nucleus.
Introduction I n recent years n u m e r o u s i n v e s t i g a t i o n s have been carried out to s t u d y the effects of visual d e p r i v a t i o n o n various s t r u c t u r e s of t h e visual s y s t e m i n t h e cat. However, t h e r e is still a lack of i n f o r m a t i o n on t h e n o r m a l d e v e l o p m e n t of various c o m p o n e n t s of this system, of which the dorsal p a r t of t h e lateral geniculate nucleus (LGNd) is a p a r t i c u l a r l y i m p o r t a n t structure. I n t h e m o n o c u l a r a n d b i n o c u l a r segments of l a m i n a A of this nucleus Garey, F i s k e n a n d Powell (1973a, b) have studied the growth of t h e n e r v e cells i n 6 k i t t e n s a n d 5 a d u l t cats, a n d i n t h e same region Cragg (1975) has r e c e n t l y c o u n t e d t h e n u m b e r of synapses. Both i n v e s t i g a t i o n s show t h a t t h e d e v e l o p m e n t i n t h e L G N d precedes t h a t of the cortex b y a few days a n d is p a r t i c u l a r l y r a p i d d u r i n g t h e t h i r d a n d fourth week of p o s t n a t a l life. However, so far there is no systematic i n f o r m a t i o n o n the p o s t n a t a l growth of t h e size of t h e L G N d , on changes of its position a n d shape a n d on changes i n cell density. To fill this gap, t h e v o l u m e a n d shape of the L G N d have b e e n s y s t e m a t i c a l l y s t u d i e d i n a series of 27 cats b e t w e e n b i r t h a n d the eighth m o n t h of p o s t n a t a l life, a n d i n 5 a d u l t animals. I n addition, i n 27 of these 32 eats the n u m b e r of cells has b e e n d e t e r m i n e d i n t h e b i n o c u l a r segment of l a m i n a A (cf. Guillery a n d Stelzner, 1970), which is easy to find e v e n i n the b r a i n of a n e w b o r n k i t t e n . * Dedicated to Professor W. Bargmann, Kiel, on the occasion of his seventieth birthday 1 Anat.Embryol.
2
H. Elgeti et M. Material and Methods
:For this study the brains of 32 cats were used. Most of the animMs were born a n d reared in the institute, a n d the age of all animals was exactly known except for K 13 and K 15. These two cats were adult animals and one, K 13, was known to have given b i r t h to at least one litter of young. The age of the cats a n d the plane of sectioning of the brains are given in Table 1. The animMs were anaesthetized with pentobarbitone sodium a n d fixed by perfusion from the aorta with a plasma expander (Periston | or Macrodex | followed b y Bouin's solution. To avoid postmortem damage to nerve cells (cf. Cammermeyer, 1962), some hours were allowed to elapse before the skull was opened a n d the brain t a k e n out. The brains were postfixed in Bouin's solution for 1-3 days ~nd, either in toto or without brMnstem a n d cerebellum, dehydrated and embedded in paraffin. Serial sections were cut with the mierotome set at 8 ~zm or, for some series, a t 9 or 10 tzm. Later, the actual average thickness of the sections was determined b y measuring, with the help of a n oil immersion, the true thickness in a t least 10 regions of 8 sections in each series. The mean values obtained from these measurements were used a n d are termed " t r u e thickness." I n each series every 10th and l l t h slide was stained with chrome-hematoxylin-phloxin (Gomori, 194f). Adjacent slides were stained with Luxol-fast-blue (Klfiver-Barrera, 1953) followed by a periodic acid-Schiff (PAS) reaction and counterstaining with Ehrlieh's hematoxylin. Other sections were treated with Luxol-fast-btue followed b y Goldner's triehrome (cf. Fleisehhauer, 1960) or with a silver impregnation according to Bodian in the modification of Luna (1964).
Determination o/ Toti~l Volume. Since pilot studies h a d shown t h a t the outline of the L G N d is equally well traceable in all sections irrespective of the various stMnings used in this study, sections from each series were selected a t regular intervMs of between 90 and 330 ~zm, b u t mostly 200 izm. The sections were projected on to a screen, and a t a linear magnification of 36 • the outline of the L G N d was drawn. I n all eats, the nucleus interlaminaris medialis bordering the optic t r a c t was included, whereas the nerve fibres around
Table 1 Cat
Age
Plane Hemisphere
Volume mm 3
Nuclei Nuclei (total) (neuronal) in (0.1 mm) ~ in (0.1 mm) 3 (IV[4- 2slg ) (M 4- 2sM)
Nuclei (glial) in (0.1 mm) 3 (M ~ 2s5i )
K 75
1d
f f
right left
3.48 3,36
483.9 4- 11.4
423.44- 10.5
60.5:t: 4.3
2
K 182
1d
s
right
3.55
453.7 i 39.3
410.5=t=38.6
43.24- 7.0
3
E 23
2d
f f
right left
3.25 2.93
4
K 192
3d
f f
right left
4.49 4.48
389.44- 25.4
334.4•
55.0JC 5.4
5
K 17
4d
f f
right left
3.07 2.78
6
K 77
7d
s
right
4.02
349.44- 10.6
296.04- 9.3
53.44- 5.1
7
K 193
8d
s
right
5.96
314.6 ~: 7,9
264.34- 7.8
50.2i
8
K 195
8d
f f
right left
4.87 4.67
402.9 4- 40.7
332.84-40.6
70.1::c: 4.1
9
K 45
11 d
f f
right left
4.98 4.64
315.8 4- 19.3
2 6 0 . 8 i 18.7
55.0=t= 4.9
1
1.4
Postnatal Growth of Lateral Geniculate i~lucleus Table 1 (continued)
Cat
Age
Plane Hemisphere
Volume mms
Nuclei (totM) in (0.1 ram) 3
:Nuclei Nuclei (neuronal) (glial) in (0.1 ram) ~ in (0.1 ram) 3
(M :~ 2%p
(M • 2sM)
(M • 2s~)
10
K 189
11 d
s
left
8.I7
2 9 4 . 4 ~ 18,3
233.9-{= 17.8
60.5•
11
K 50
16 d
s
right
5.93
2 9 4 . 1 i 13.9
2 3 1 . 4 ~ 12.9
62.7-~ 5.2
12
K 51
16 d
f f
right left
5.01 5.63
285.9::k36.3
224.2s
61.8~
13
K 22
17 d
f f
right left
8.89 8.83
230.1~
9.4
183.7~
8.9
46.4~: 3.1
14
K 5
20 d
f f
right left
11.04 10.71
15
K 25
27 d
f f
right left
14.89 15.33
156.5~
8.5
112.0•
8.0
44.5~_ 2.9
16
K 61
30 d
f f
right left
24.01 22.66
127.0::k 5.4
93.4•
4.2
33.6--
3.5
17
K 88
33 d
s
Ieft
11,96
181.9i10.7
135.7•
8.6
46.2~
6.3
18
K 44
38 d
f f
right left
15.75 15.71
173.15=22.7
130.2 ~: 22.2
42.9•
5.1
19
K 60
40 d
f f
right left
22.86 23.27
129.0•
6.7
89.3~
6.4
39.7~: 1.8
20
K 78
43 d
f f
right left
18.80 19.71
151.8~
9.1
101.9•
8.5
49.9:~ 3.1
21
K 87
43 d
s
left
15.86
2 3 9 . 4 ~ 17.8
1 5 2 . 0 ~ 14.6
8 7 . 3 i 10,2
22
E 32
48 d
f f
right left
23.01 22.25
155.5:~ 16.1
9 9 . 8 s 15.6
55.7•
23
K 37
84 d
f f
right left
27.14 26.77
ti7.8~t0.~
73.9=[- 8.9
43,8-L 5.2
24
K 12
5m
s
left
19.01
1 7 2 . 5 i 15.1
9 9 . 2 • 11.6
73.3•
9.7
25
K 104
5.5 m s
left
18.73
178.2:~ 9.0
97.9~: 6.3
80.3•
6,4
26
K 52
6.5 m f f
right left
23.45 23.01
27
K 26
8m
f f
right left
30.53 30.23
120.6i
5.7
79.0•
3.6
41.6~
4.4
28
K 16
1.5 y
s
left
33.42
129.8~
6.8
66.0~
5.9
63.8~
3.3
29
K 13
adult
f f
right left
25.78 25.64
96.6•
9.0
56.6~
7.7
40.0~: 4.7
30
K 15
adult
f f
right left
20.22 17.62
97.9:::E 7,3
60.2•
5.1
37.8•
5.3
31
K 1
11 y
f f
right left
25.06 24.93
66.6~: 6.8
50.9•
6.1
32
K 27
15 y
f f
right left
19.59 20.12
117.4~
9.1
4.2
8.0
4.1
4
H. Elgeti et al.
the LGNd were excluded. The area so drawn was measured with a planimeter (Fa. Ott, Type 144 L) calibrated to an accuracy of between 0.2 and 0.3 %. From the values thus obtained the volume (V) of the LGNd was calculated by using the formula
F=ZIFxd where F is the area of the selected sections of the LGNd measured in m m 2 and corrected for the magnification factor, and d the spacing of the sections including the thickness of the section used for making the measurement. Since during planimetry only a small error (less t h a n 0.3 %) is introduced, the accuracy of the volume determinations largely depends on the number of sections drawn and measured for each LGNd, which in cats of different age is a structure of roughly similar shape but vastly differing size. Comparable accuracy of volume determinations would best be achieved by using a similar, sufficiently large number of sections for each LGNd. With a spacing of between 100 and 200 ~m, a LGNd of larger animals will yield about 15 to 20 sections; and this was the usual number of sections evaluated. I n some instances, however, the spacing had to be wider ~or technical reasons or the LGNd was so small t h a t fewer t h a n t5 sections could be used. I n one animal (K 75), only 7 sections were evaluated. In order to find out whether the volumes determined in such cases are still sufficiently accurate, the following test was carried out. F r o m a complete series of frontal sections (K 26) as well as from a series of sagittal sections (K 189) through the LGNd, 4 series each of 7 equally spaced sections were taken. From each of these series as well as from a series consisting of all 28 sections, the volume of the LGNd was determined. I n the one cat (K 26) the values obtained from the 4 series consisting of 7 sections varied between 28.67 and 32.58 m m 3 and thus differed only 6.6% from the value (30.53 m m a) obtained from the series consisting of 28 sections. I n this case the standard error of the mean for the series consisting of 7 sections was calculated to be -4-3 %. By means of the Student-t-distritution (Sachs, 1968), the 95 % confidence intervall was determined to be • 9.0%. I n the other cat (K 189), the volumes determined from the 4 series of 7 sections differed even less ( • 3.1%) from the volume determined from the series consisting of 28 sections. With a standard error of the mean of =c 1.4%, the 95% area of confidence turns out to be • 4.2%. These two examples indicate t h a t even if only 7 sections can be evaluated, the volume of the L G N d is still determined with a margin of error of less t h a n 10% and thus sufficiently accurate. Since in the great majority of cats more t h a n 15 sections could be used for the volume determination of each LGNd, the values given in this paper are less influenced by the methods of measurement than by possible variations in shrinkage which of necessity affect every quantitative investigation carried out in material embedded in paraffin and which cannot be accurately determined for every brain. Determination o/ Cell Density. In 27 cats measurements were made in the binocular segment of lamina A of one LGNd. Great care was taken to keep to the same region in all cats. For this purpose the middle third of the LGNd was determined in every series of serial sections, and 8 closely spaced sections were selected. The measurements were made in sections stained with chrome-hematoxylin-phloxin. The nuclear counts were made with an ocular grid at a magnification of 800 • (oil immersion). At this magnification the side length of the field covered by the grid was 62.5 ~m and the area 3,906.25 ~m ~. Cell nuclei lying on the left or upper rim of the field were included, structures on the right or lower rim not counted. In each of the 8 sections of a LGNd 50 fields were counted, i.e. a total of 400 fields per LGNd. In order to find out whether such a sample is of sufficient size to give accurate resu|ts, the following test w~s made. From the LGNd of an adult cat and t h a t of a 43 day old cat additional sets of 8 sections were selected and evaluated in the same manner. I n both animals the results obtained from the additional 400 fields were found to be almost identical with those from the first 400 fields or with the mean of all 800 fields. Therefore it was concluded t h a t a value obtained from measurements of 400 fields of lamina A is accurate. The number of nerve cells per unit volume was determined by counting the nucleoli, i.e. only those of the light, large nuclei with loose chromatin t h a t contained the nuelcolus which in the ehrome-hematoxylin-phloxin sections is stained red and therefore easy to discriminate from similarly shaped ehromatin condensations. Since the nucleoli are small in
Postnatal Growth of Lateral Genieul~te Nucleus
5
relation to the thickness of the histological section, no correction factor was used Ior calculating the number of nuclei per unit volume of tissue. In young cats, there is a certain number of cells with more than one nucleolus. In such cases only one nucleolus was counted. The error resulting from the possibility that in some of these cells the nucleoli may lie in different sections as well as that due to split nucleoli must be very small and has been neglected. The number of glial cells was determined by counting the nuclei, which differ from those of the nerve cells in shape, size and chromatin structure and are usually lacking a distinct nucleolus. Since in relation to the thickness of the histological sections the glial cell nuclei are considerably Iarger than the nueleoli of nerve cells, the values obtained have to be corrected before the number of cells in a tissue volume of (0.1 ram) 3 can be calculated. This correction was carried out according to Haug (1967) by using the formula of Flodcrus (1944)
N
n• a@2r--2lc
where N is the real number of structures present, a the true thickness of the histological section, n the number of structures counted, 2/c the smallest structure still detectable (1 ~m, see Haug, 1967) and 2r the average diameter of the structures under consideration.--The nuclei of the endothelial and perivascular cells were not counted. The total number of ceils per unit volume was arrived at by adding the values for nerve cells and glial cells obtained in the manner described above. Nomenclature. With respect to the lateral geniculate nucleus of the cat a number of terminologies which in some respects are at variance have been used in the literature (cf. Hayhow, 1958; Guiilery, I970). In ~he presen~ study, the nomenclature of Thum~ (1928) is used throughouL, bu~ in gecordance with Szents (1973) the terms lamina A, A1 and B are substituted for pars dorsalis A, Aa and B. For measuring the volume, the dorsal part of the geniculate nucleus (LGNd) was delineated so as to comprise the medial interlaminar nucleus, whereas the posterior nucleus of the thalamus is not included (cf. Fig. 1 in Kinston, Vadas and Bishop, 1969). The layer of nerve fibres which in adult eats surrounds the LGNd cannot be delineated in young animals and is therefore excluded from the measurements.
Results Overall Growth o/the LGNd. I n t h e n e w b o r n cat t h e lateral geniculate nucleus s t a n d s out as a s t r u c t u r e of great cellular density. I n sagittal sections it has t h e form of a slightly curved capital "'S" ~ n d its l a m i n a e can a l r e a d y be discerned at this early stage (Fig. 1 a). I n the m i d d l e two thirds of t h e L G N d the d e n s e l y p o p u l a t e d l a m i n a e A a n d A 1 are seen to be separated b y t h e lightly s t a i n e d N. i n t e r l a m i n a r i s centralis; a n d t h e border betweerL A 1 a n d B is a p p a r e n t from t h e difference i n cellular d e n s i t y b e t w e e n the two laminae. N e a r t h e edges of t h e L G N d , the i n t e r l a m i n a r nuclei disappear a n d t h e cell rich l a m i n a e A, A 1 a n d B merge into one structure. D u r i n g t h e first few days after b i r t h there is little or no growth, b u t i n the second week a phase of rapid growth sets in. A r o u n d the 20th d a y t h e volume of t h e L G N d has a l r e a d y tripled. As shown in Figure 2, the phase of fast growth continues to a b o u t the 40th day. B y t h e n the v o l u m e approaches t h e final values of b e t w e e n 20 a n d 30 ram s which are f o u n d i n most exiult cats. The m e a s u r e m e n t s show t h a t in cats of similar age there are considerable i n d i v i d u a l differences in the size of the L G N d , b u t t h a t i n e v e r y cat in which b o t h L G N d ' s could be m e a s u r e d t h e values o b t a i n e d from the right a n d left side are almost identical. The growth of t h e L G N d is a c c o m p a n i e d b y changes i n t h e shape of the nucleus a n d b y a r o t a t i o n of its long axis from a more vertical into a horizontal
6
I-I. Elgeti et al.
Fig. l a--d. Sagittal sections through the middle of the LGNd of cats aged 1 day (a), 11 days (b), 43 days (c) and 1.5 years (d). Chrome-hematoxylin-phloxin. 10 •
position. This is best seen b y c o m p a r i n g s a g i t t a l sections t h r o u g h t h e m i d d l e of t h e L G N d such as shown in F i g u r e s l a - d . I n t h e s e sections t h e d o r s a l t i p of t h e L G N d is seen to r e m a i n in a n u p w a r d position a n d to r e t a i n its t o p o g r a p h i c a l
Postnatal Growth of Lateral Geniculate Nucleus
7
position with respect to the fimbria hippoeampi. Therefore, the dorsal tip can be taken as a fixed point in relation to which the growth changes can be described. In Figure 3 the outlines of the 4 LGNd's of Figure 1 have been superimposed in order to show that the long axis is rotated upward and that during growth the nucleus extends predominantly in the anterior direction. I t seems likely that the
8
tI. Elgeti et al. VOLUME
35
.ram 3
30
25
20 9
9
15
log I
I
I
I
I
I
I
I
2
'~
8
I':
30
90
240
5;0
AGE DAYS
:Fig 2 Volume of t h e L G N d in ea~s of various age
IS [] ~ el [] o c.. . . . . . . . . . . . . . . ,~ - -
~
~ ~ t", m~mr, 9 .,..~.~.~....~o
9
!:'..::~'~~'.'1~ I ':":'~'::':':::':i::':l'lrlJet't'~J "-:~'..:~::
.... '. 11 '. ; ~ i '. "1 '. ~ ~ ', ', ' ;~ ,, ', '. 1 '. ; ........... ................. .~
r~ i i i: ,. ,,,,,,;; :,,,.,,,, ==,,; =,,=,, ,, ,,; ;,, !!-ii~u~ I ~ . ' .~'.,~,'~ =-
Fig.
3. To illustrate t h e p o s t n a t a l changes in volume a n d shape, t h e outlines of t h e lateral genieulate nuclei s h o w n in ~igure 1 have b e e n superimposed. ~ 1 day; ~ 11 d a y s ; g . ~ 45 d a y s ; ~ 1.5 years
Postnatal Growth of Lateral Geniculate Nucleus
9
NUCLEI in (O,Irnm)3
500
0
400
D
0
300
0
@
g
g
200
9
9
100
log AGE I
I
J
J
i
i
J
2
l,
8
1/.
30
90
2~0
I 5l*0
0 A u
Fig. 4. Postnatal changes in the total number of cell nuclei per unit volume as determined in the binocular segment of lamina A of the LGNd
characteristic postnatal changes in the shape and position of the LGNd are due to a differential growth of the various parts of the laminae as well as of the interlaminar layers, but this question has not been studied in detail. Changes in Cell Density. The growth of the LGNd is accompanied by a considerable decrease in cell density which affects Lamina A and A 1 as well as lamina B. This is evident when Figures 1 a - d are compared. The decrease in cell density was studied quantitatively in the binocular segment of lamina A, which can be discerned in cats of all ages, and in the sagittal as well as in the frontal planes of sectioning. As shown in Figure 4, the decrease in total cell density commences right after birth and is measurable before the overall growth in volume becomes detectable. In lamina A, the number of cells decreases from about 470/(0.1 mm) 3 at birth to about 150 at the beginning of the second month; later there is little further change of cell density and in adult animals the values obtained are between 95 and 130 cells/(0.1 ram) 3.
10
It. Elgeti et al. NUCLEI in (0.1rn rn) 3
500
~00
3:00
o
200
o
o
o
o
oo
tOO
oo
o% o
9
9
o
9 ~
o 9
o
9
9
log I
I
1
I
I
i
I
I
2
L
g
1~.
30
90
2L0
5 "0
AGE DAYS
Fig. 5. Postnatal changes in the number of cell nuclei of nerve cells (ooo) and neurogliM cells (...) per unit volume ms determined in the binocular segment of lamina A of the LGNd
W h e n t h e m e a s u r e m e n t s of t h e n u m b e r of n e r v e cells a n d neuroglial cells per u n i t v o l u m e are d r a w n s e p a r a t e l y (Fig. 5), it becomes e v i d e n t t h a t t h e s t e e p decrease of t h e t o t a l n u m b e r of t h e cells p e r u n i t v o l u m e is a l m o s t e n t i r e l y due to a decrease in t h e n u m b e r of n e r v e cells whereas t h e n u m b e r of gtiM cells r e m a i n s f a i r l y c o n s t a n t . This conclusion c a n safely be d r a w n f r o m F i g u r e 5, a l t h o u g h it was f o u n d t h a t in e h r o m e h e m a t o x y l i n s t a i n e d p a r a f f i n sections t h r o u g h t h e L G N d of cats with a p o s t n a t a l age of between 4 a n d 6 weeks i t m a y occasionally be difficult to d i s c r i m i n a t e between the nucleus of a n e r v e cell a n d t h a t of a large glial cell. Discussion T h e r e seems to be no l i t e r a t u r e on t h e v o l u m e g r o w t h of t h e L G N d of cats, a n d no v a l u e for t h e v o l u m e of this nucleus in t h e a d u l t cat was found. H o w e v e r , t h e p r o j e c t i o n m a p s of S a n d e r s o n (1971), which h a d been o b t a i n e d b y fitting
Postnatal Growth of Lateral Geniculate Nucleus
Ii
together the values of 40 cats, enabled us to calculate a value with which to compare our own measurements in the adult animals. For this comperison planimetric measurements of the 9 figures of coronal sections given in the paper of Sanderson were made. From the values thus obtained (F1, F 2 . . . Fg) the volume was calculated by using the formula
V=
IF1
F2
(vl)2 + ~ + . . .
2'9]
+ ~
xd
in which " v " is a factor for the different magnifications of the figures of Sanderson and " d " a factor pertaining to the spacing of the sections. The value arrived at is 42.5 m m ~. Since all measurements in the paper of Sanderson are expressed in terms of Horsley-Clark coordinates, no correction needs to be made for shrinkage. Our own results, on the other hand, are expressed in absolute values as obtained by measuring paraffin sections, and have therefore to be corrected for shrinkage which according to Stowell (1941) and Stephan (1960) makes up as much as 40%. The uncorrected measurements for the volume of the L G N d in grown-up animals (No. 28-32 in Table 1) v a r y between 17.62 m m 3 and 33.42 m m 3 with a mean of 26.42 m m 3. If this mean value is corrected for 40 % shrinkage, the true volume of the L G N d in the adult oat turns out to be about 44 m m ~, which is in good agreement with the value of 42.5 m m 3 arrived at by calculating the volume from the figures published by Sanderson (1971). The postnatal changes in shape and the rotation of the axis of the L G N d indicate t h a t the various components of this nucleus have a different rate of growth. These differences have not been analysed systematically, but our preliminary observations suggest t h a t the highly ordered pattern of the growing dendritic trees as well as the ordered arrangement of the myelinating nerve fibres must be held responsible for the changes in shape and position. If the postnatal increase in volume is compared with the concomitant decrease in the number of nerve cells per unit volume in lamina A, the decrease in the number of nerve cells is seen to begin before a clear-cut increase in the size of the LGNd becomes detectable. It is most likely that this discrepancy is due to the fact that only one region of grey matter was investigated for cell numbers and that the changes observed here need not be immediately reflected in the size of the entire structure. It is improbable that the discrepancy is due to a degeneration of nerve cells, because no indication for such degeneration was found either in the quantitative measurements or in the histological appearance of the sections which were carefully studied at high magnification. The finding that during growth of the LGNd the decrease in the total number of cells per unit volume is mainly due to a decrease in the number of nerve cells per unit volume whereas the density of the glial cells remains fairly constant, suggests that new glial cells are formed after birth. If an extrapolation is made from the measurements in lamina A, a rough indication of the absolute number of nerve cells and glial cells can be obtained by multiplying the number of cells per unit volume found in lamina A with the total volume of the LGNd. The values obtained by such an extrapolation have to be interpreted with the greatest caution. However, they clearly indicate that during postnatal development the absolute number of nerve cells remains fairly constant whereas between the
12
H. Elgeti et al.
second and sixth week the absolute number of glial cells rises steeply. Since in Kliiver stained sections the first myelinated fibres in the LGNd were detected on the 16th day of postnatal life, this finding is in accordance with the fact that in white matter, too, a great number of glial cells are newly formed shortly before and during the period of myelination (for lit. see Fleischhauer, 1968). The measurements of the postnatal increase in volume of the LGNd and of the concomitant decrease in cell density in the binocular segment of lamina A are in good agreement with the measurements of pcrikaryal growth (Garey, Fisken and Powell, 1973) and increase in the number of synapses (Cragg, 1975). However, the comparison also shows that with a larger number of eats such as used in the present study, the existence of considerable individual differences between the values to be obtained at any given age becomes obvious. The existence of these large individual differences must be kept in mind when measurements of changes during postnatal growth are interpreted and results of various authors compared. For such studies mean values based on a sufficient number of animals in each age group are required. However, when studied in the same animal and with the same method, comparisons between different regions of the brain are less prone to error because such comparisons are not influenced by the individual differences mentioned above. I t has been shown by Cragg (1975) that the increase in the number of synapses in the LGNd precedes that in the visual cortex of the same animals by a few days. Another even more striking time-lag between the development of the LGNd and that of telencephalic structures is revealed when the growth of the LGNd is compared with that of the corpus callosum which had previously been studied in the same eats used for the present investigation (Fleischhauer und Sehliiter, 1970). The comparison shows that the increase in volume of the L G N d begins earlier than that of the corpus callosum, rises more steeply and terminates at about the fourtieth day, whereas the growth of the corpus callosum continues into adult life.
Adcnowledgement. This work was supported by a grant of the Bundesministerium ffir Forschung und Technologie.
References Cammcrmcyer, J.: An evaluation of the significance of the "dark" neuron. Ergebn. Anat. Entwickl.-Gesch. 86, 1-61 (1962) Cr~gg, B. G. : The development of synapses in the visual system of the cat. J. comp. Neurol. 168, 147-166 (1975) Fleischhauer, K. : Fluorescenzmikroskopische Untersnchungen an der Faserglia. Z. Zel]forsch. 51,467-496 (1960) Fleischhauer, K. : Postnatale Entwicklung der Neuroglia. Acta neuropath. (Berl.), Suppl. IV, 20-32 (1968) Fleischhauer, K., Schlfiter, G. : Uber das postnatale Wachstum des Corpus callosum der Katze (Fells domestica). Z. Anat. Entwiekl.-Gesch. 132, 228-239 (1970) Floderus, S. : Untersuchungen fiber den Bau der menschlichen Hypophyse mit besonderer Berficksichtigung der mikromorphologischen Verh~ltnisse. Act~ path. microbiol, scand., Suppl. 537 (1944) Garey, L. J., Fisken, g.A., Powell, T. P. S.: Observations on the growth of ceils in the lateral geniculatc nucleus of the cat. Brain Res. 52, 359-362 (1973a)
Postnatal Growth of Lateral Geniculate Nucleus
13
Garey, L. J., Fisken, R. A., Powell, T. P. S. : Effects of experimental deafferentation on cells in the lateral geniculate nucleus of the cat. Brain Res. 52, 363-369 (1973b) Gomori, G. : Observations with differential stains on human islets of Langerhans. Amer. J. Path. 17, 395-406 (1941) Guillery, R . W . : The laminar distribution of retinal fibers in the dorsal lateral geniculate mteleus of the cat: a new interpretation. J. eomp. Neurol. 138, 339 368 (1970) Guillery, R . W . , Stelzner, D. J.: The differential effects of unilateral lid suture upon the monocular and binocular segments of the dorsal lateral geniculate nucleus in the cat. J. comp. Neurol. 1119, 413422 (1970) I-Iaug, H. : Probleme und Methoden der Strukturzg~hlung in Schnittprgparaten. In: Weibel, E . R . , Elias, H. (eds.), Quantitative methods in morphology. Berlin-Heidelberg-New York: Springer 1967 Hayhow, W. R. : The cytoarehiteeture of the lateral geniculate body in the cat in relation to the distribution of the crossed and uncrossed optic fibers. J. comp. Neurol. 110, 1-64 (1958) Kinston, W. J., Vadas, M.A., Bishop, P. 0.: Multiple projection of the visual field to the medial portion of the dorsal lateral geniculate nucleus and the adjacent nuclei of the thalamus of the cat. J. comp. Neurol. 136, 295-316 (1969) Kliiver, H., Barrera, E.: A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol. 12, 400-403 (1953) Luna, L. G.: Further studies of Bodian's technique. Amer. J. Technol. 30, 355-362 (1964) Sachs, L.: Statistische Auswertungsmethoden. Berlin-Heidelberg-New York: Springer 1968 Sanderson, K. J. : The projection of the visual field to the lateral genieulate and medial interlaminar nuclei in the cat. J. comp. Neurol. 143, 101-118 (1971) Stephan, H.: ~ethodisehe Studien fiber den quantitativen Vergleich architektonischer Struktureinheiten des Gehirns. Z. wiss. Zool. 164, 143-172 (1960) Stowell, R . E . : Effects on tissue volume of various methods of fixation dehydration and embedding. Stain Technol. 16, 67 83 (1941) Szents J.: Neuronal and synaptic architecture of the lateral geniculate nucleus. In: Autrum, H. et al. (eds.), Handbook of sensory physiology, vol. VII/3, part B, p. 141-176. Berlin-Heidelberg-New York: Springer 1973 Thuma, B. D. : Studies on the diencephalon of the cat. I. The cytoarchitecture of the corpus geniculatum laterale. J. comp. Neurol. 46, 173-I97 (1928) Prof. Dr. K. Fleisehhauer Anatomisehes [nstitut Nussallee 1O D-5300 Bonn Federal Republic of Germany
Postnatal Growth of the Dorsal Lateral Geniculate Nucleus of the Cat H e l l m u t Elgeti, R i c a r d a Elgeti, a n d K u r t Flcischhauer
*
Anatomisches Institut der Universit~t Bonn Received January 9, 1976
Summary. The postnatal growth of the dorsal part of the lateral geniculate nucleus (LGNd) is studied in paraffin sections through the brains of 32 eats of known age. The changes in shape and position of the LGNd are described and it is shown that i~s volume increases from about 3.4 mm3 at bir~h to about 26.4 mm3 in the adult cat. When ~his value is corrected for shrinkage, the volume of the LGNd in the adult cat turns out to be about 44 mm 3. Tim detailed measurements reveal that during the second and third week of postnatal life tllere is a particularly steep increase in volume and that the final values are already reached at around the 40th day. Concomitant with the increase in volume there is a decrease of the number of cells per unit volume of grey matter. In the binocular segment of lamina A the number of cells decreases h'om about 470 per (0.i ram) ~ at birth to between 95 and 130 per (0.1 ram) a in the adult cat. Separate measurements of nerve cells and neuroglial cells indicate that the absolute number of nerve cells remains fairly constant during postnatal life, whereas between the second and sixth week a great number of neuroglial cells are newly formed. Key words: Postnatal development - - Cat - - Visual system - - Lateral geniculate nucleus.
Introduction I n recent years n u m e r o u s i n v e s t i g a t i o n s have been carried out to s t u d y the effects of visual d e p r i v a t i o n o n various s t r u c t u r e s of t h e visual s y s t e m i n t h e cat. However, t h e r e is still a lack of i n f o r m a t i o n on t h e n o r m a l d e v e l o p m e n t of various c o m p o n e n t s of this system, of which the dorsal p a r t of t h e lateral geniculate nucleus (LGNd) is a p a r t i c u l a r l y i m p o r t a n t structure. I n t h e m o n o c u l a r a n d b i n o c u l a r segments of l a m i n a A of this nucleus Garey, F i s k e n a n d Powell (1973a, b) have studied the growth of t h e n e r v e cells i n 6 k i t t e n s a n d 5 a d u l t cats, a n d i n t h e same region Cragg (1975) has r e c e n t l y c o u n t e d t h e n u m b e r of synapses. Both i n v e s t i g a t i o n s show t h a t t h e d e v e l o p m e n t i n t h e L G N d precedes t h a t of the cortex b y a few days a n d is p a r t i c u l a r l y r a p i d d u r i n g t h e t h i r d a n d fourth week of p o s t n a t a l life. However, so far there is no systematic i n f o r m a t i o n o n the p o s t n a t a l growth of t h e size of t h e L G N d , on changes of its position a n d shape a n d on changes i n cell density. To fill this gap, t h e v o l u m e a n d shape of the L G N d have b e e n s y s t e m a t i c a l l y s t u d i e d i n a series of 27 cats b e t w e e n b i r t h a n d the eighth m o n t h of p o s t n a t a l life, a n d i n 5 a d u l t animals. I n addition, i n 27 of these 32 eats the n u m b e r of cells has b e e n d e t e r m i n e d i n t h e b i n o c u l a r segment of l a m i n a A (cf. Guillery a n d Stelzner, 1970), which is easy to find e v e n i n the b r a i n of a n e w b o r n k i t t e n . * Dedicated to Professor W. Bargmann, Kiel, on the occasion of his seventieth birthday 1 Anat.Embryol.
2
H. Elgeti et M. Material and Methods
:For this study the brains of 32 cats were used. Most of the animMs were born a n d reared in the institute, a n d the age of all animals was exactly known except for K 13 and K 15. These two cats were adult animals and one, K 13, was known to have given b i r t h to at least one litter of young. The age of the cats a n d the plane of sectioning of the brains are given in Table 1. The animMs were anaesthetized with pentobarbitone sodium a n d fixed by perfusion from the aorta with a plasma expander (Periston | or Macrodex | followed b y Bouin's solution. To avoid postmortem damage to nerve cells (cf. Cammermeyer, 1962), some hours were allowed to elapse before the skull was opened a n d the brain t a k e n out. The brains were postfixed in Bouin's solution for 1-3 days ~nd, either in toto or without brMnstem a n d cerebellum, dehydrated and embedded in paraffin. Serial sections were cut with the mierotome set at 8 ~zm or, for some series, a t 9 or 10 tzm. Later, the actual average thickness of the sections was determined b y measuring, with the help of a n oil immersion, the true thickness in a t least 10 regions of 8 sections in each series. The mean values obtained from these measurements were used a n d are termed " t r u e thickness." I n each series every 10th and l l t h slide was stained with chrome-hematoxylin-phloxin (Gomori, 194f). Adjacent slides were stained with Luxol-fast-blue (Klfiver-Barrera, 1953) followed by a periodic acid-Schiff (PAS) reaction and counterstaining with Ehrlieh's hematoxylin. Other sections were treated with Luxol-fast-btue followed b y Goldner's triehrome (cf. Fleisehhauer, 1960) or with a silver impregnation according to Bodian in the modification of Luna (1964).
Determination o/ Toti~l Volume. Since pilot studies h a d shown t h a t the outline of the L G N d is equally well traceable in all sections irrespective of the various stMnings used in this study, sections from each series were selected a t regular intervMs of between 90 and 330 ~zm, b u t mostly 200 izm. The sections were projected on to a screen, and a t a linear magnification of 36 • the outline of the L G N d was drawn. I n all eats, the nucleus interlaminaris medialis bordering the optic t r a c t was included, whereas the nerve fibres around
Table 1 Cat
Age
Plane Hemisphere
Volume mm 3
Nuclei Nuclei (total) (neuronal) in (0.1 mm) ~ in (0.1 mm) 3 (IV[4- 2slg ) (M 4- 2sM)
Nuclei (glial) in (0.1 mm) 3 (M ~ 2s5i )
K 75
1d
f f
right left
3.48 3,36
483.9 4- 11.4
423.44- 10.5
60.5:t: 4.3
2
K 182
1d
s
right
3.55
453.7 i 39.3
410.5=t=38.6
43.24- 7.0
3
E 23
2d
f f
right left
3.25 2.93
4
K 192
3d
f f
right left
4.49 4.48
389.44- 25.4
334.4•
55.0JC 5.4
5
K 17
4d
f f
right left
3.07 2.78
6
K 77
7d
s
right
4.02
349.44- 10.6
296.04- 9.3
53.44- 5.1
7
K 193
8d
s
right
5.96
314.6 ~: 7,9
264.34- 7.8
50.2i
8
K 195
8d
f f
right left
4.87 4.67
402.9 4- 40.7
332.84-40.6
70.1::c: 4.1
9
K 45
11 d
f f
right left
4.98 4.64
315.8 4- 19.3
2 6 0 . 8 i 18.7
55.0=t= 4.9
1
1.4
Postnatal Growth of Lateral Geniculate i~lucleus Table 1 (continued)
Cat
Age
Plane Hemisphere
Volume mms
Nuclei (totM) in (0.1 ram) 3
:Nuclei Nuclei (neuronal) (glial) in (0.1 ram) ~ in (0.1 ram) 3
(M :~ 2%p
(M • 2sM)
(M • 2s~)
10
K 189
11 d
s
left
8.I7
2 9 4 . 4 ~ 18,3
233.9-{= 17.8
60.5•
11
K 50
16 d
s
right
5.93
2 9 4 . 1 i 13.9
2 3 1 . 4 ~ 12.9
62.7-~ 5.2
12
K 51
16 d
f f
right left
5.01 5.63
285.9::k36.3
224.2s
61.8~
13
K 22
17 d
f f
right left
8.89 8.83
230.1~
9.4
183.7~
8.9
46.4~: 3.1
14
K 5
20 d
f f
right left
11.04 10.71
15
K 25
27 d
f f
right left
14.89 15.33
156.5~
8.5
112.0•
8.0
44.5~_ 2.9
16
K 61
30 d
f f
right left
24.01 22.66
127.0::k 5.4
93.4•
4.2
33.6--
3.5
17
K 88
33 d
s
Ieft
11,96
181.9i10.7
135.7•
8.6
46.2~
6.3
18
K 44
38 d
f f
right left
15.75 15.71
173.15=22.7
130.2 ~: 22.2
42.9•
5.1
19
K 60
40 d
f f
right left
22.86 23.27
129.0•
6.7
89.3~
6.4
39.7~: 1.8
20
K 78
43 d
f f
right left
18.80 19.71
151.8~
9.1
101.9•
8.5
49.9:~ 3.1
21
K 87
43 d
s
left
15.86
2 3 9 . 4 ~ 17.8
1 5 2 . 0 ~ 14.6
8 7 . 3 i 10,2
22
E 32
48 d
f f
right left
23.01 22.25
155.5:~ 16.1
9 9 . 8 s 15.6
55.7•
23
K 37
84 d
f f
right left
27.14 26.77
ti7.8~t0.~
73.9=[- 8.9
43,8-L 5.2
24
K 12
5m
s
left
19.01
1 7 2 . 5 i 15.1
9 9 . 2 • 11.6
73.3•
9.7
25
K 104
5.5 m s
left
18.73
178.2:~ 9.0
97.9~: 6.3
80.3•
6,4
26
K 52
6.5 m f f
right left
23.45 23.01
27
K 26
8m
f f
right left
30.53 30.23
120.6i
5.7
79.0•
3.6
41.6~
4.4
28
K 16
1.5 y
s
left
33.42
129.8~
6.8
66.0~
5.9
63.8~
3.3
29
K 13
adult
f f
right left
25.78 25.64
96.6•
9.0
56.6~
7.7
40.0~: 4.7
30
K 15
adult
f f
right left
20.22 17.62
97.9:::E 7,3
60.2•
5.1
37.8•
5.3
31
K 1
11 y
f f
right left
25.06 24.93
66.6~: 6.8
50.9•
6.1
32
K 27
15 y
f f
right left
19.59 20.12
117.4~
9.1
4.2
8.0
4.1
4
H. Elgeti et al.
the LGNd were excluded. The area so drawn was measured with a planimeter (Fa. Ott, Type 144 L) calibrated to an accuracy of between 0.2 and 0.3 %. From the values thus obtained the volume (V) of the LGNd was calculated by using the formula
F=ZIFxd where F is the area of the selected sections of the LGNd measured in m m 2 and corrected for the magnification factor, and d the spacing of the sections including the thickness of the section used for making the measurement. Since during planimetry only a small error (less t h a n 0.3 %) is introduced, the accuracy of the volume determinations largely depends on the number of sections drawn and measured for each LGNd, which in cats of different age is a structure of roughly similar shape but vastly differing size. Comparable accuracy of volume determinations would best be achieved by using a similar, sufficiently large number of sections for each LGNd. With a spacing of between 100 and 200 ~m, a LGNd of larger animals will yield about 15 to 20 sections; and this was the usual number of sections evaluated. I n some instances, however, the spacing had to be wider ~or technical reasons or the LGNd was so small t h a t fewer t h a n t5 sections could be used. I n one animal (K 75), only 7 sections were evaluated. In order to find out whether the volumes determined in such cases are still sufficiently accurate, the following test was carried out. F r o m a complete series of frontal sections (K 26) as well as from a series of sagittal sections (K 189) through the LGNd, 4 series each of 7 equally spaced sections were taken. From each of these series as well as from a series consisting of all 28 sections, the volume of the LGNd was determined. I n the one cat (K 26) the values obtained from the 4 series consisting of 7 sections varied between 28.67 and 32.58 m m 3 and thus differed only 6.6% from the value (30.53 m m a) obtained from the series consisting of 28 sections. I n this case the standard error of the mean for the series consisting of 7 sections was calculated to be -4-3 %. By means of the Student-t-distritution (Sachs, 1968), the 95 % confidence intervall was determined to be • 9.0%. I n the other cat (K 189), the volumes determined from the 4 series of 7 sections differed even less ( • 3.1%) from the volume determined from the series consisting of 28 sections. With a standard error of the mean of =c 1.4%, the 95% area of confidence turns out to be • 4.2%. These two examples indicate t h a t even if only 7 sections can be evaluated, the volume of the L G N d is still determined with a margin of error of less t h a n 10% and thus sufficiently accurate. Since in the great majority of cats more t h a n 15 sections could be used for the volume determination of each LGNd, the values given in this paper are less influenced by the methods of measurement than by possible variations in shrinkage which of necessity affect every quantitative investigation carried out in material embedded in paraffin and which cannot be accurately determined for every brain. Determination o/ Cell Density. In 27 cats measurements were made in the binocular segment of lamina A of one LGNd. Great care was taken to keep to the same region in all cats. For this purpose the middle third of the LGNd was determined in every series of serial sections, and 8 closely spaced sections were selected. The measurements were made in sections stained with chrome-hematoxylin-phloxin. The nuclear counts were made with an ocular grid at a magnification of 800 • (oil immersion). At this magnification the side length of the field covered by the grid was 62.5 ~m and the area 3,906.25 ~m ~. Cell nuclei lying on the left or upper rim of the field were included, structures on the right or lower rim not counted. In each of the 8 sections of a LGNd 50 fields were counted, i.e. a total of 400 fields per LGNd. In order to find out whether such a sample is of sufficient size to give accurate resu|ts, the following test w~s made. From the LGNd of an adult cat and t h a t of a 43 day old cat additional sets of 8 sections were selected and evaluated in the same manner. I n both animals the results obtained from the additional 400 fields were found to be almost identical with those from the first 400 fields or with the mean of all 800 fields. Therefore it was concluded t h a t a value obtained from measurements of 400 fields of lamina A is accurate. The number of nerve cells per unit volume was determined by counting the nucleoli, i.e. only those of the light, large nuclei with loose chromatin t h a t contained the nuelcolus which in the ehrome-hematoxylin-phloxin sections is stained red and therefore easy to discriminate from similarly shaped ehromatin condensations. Since the nucleoli are small in
Postnatal Growth of Lateral Genieul~te Nucleus
5
relation to the thickness of the histological section, no correction factor was used Ior calculating the number of nuclei per unit volume of tissue. In young cats, there is a certain number of cells with more than one nucleolus. In such cases only one nucleolus was counted. The error resulting from the possibility that in some of these cells the nucleoli may lie in different sections as well as that due to split nucleoli must be very small and has been neglected. The number of glial cells was determined by counting the nuclei, which differ from those of the nerve cells in shape, size and chromatin structure and are usually lacking a distinct nucleolus. Since in relation to the thickness of the histological sections the glial cell nuclei are considerably Iarger than the nueleoli of nerve cells, the values obtained have to be corrected before the number of cells in a tissue volume of (0.1 ram) 3 can be calculated. This correction was carried out according to Haug (1967) by using the formula of Flodcrus (1944)
N
n• a@2r--2lc
where N is the real number of structures present, a the true thickness of the histological section, n the number of structures counted, 2/c the smallest structure still detectable (1 ~m, see Haug, 1967) and 2r the average diameter of the structures under consideration.--The nuclei of the endothelial and perivascular cells were not counted. The total number of ceils per unit volume was arrived at by adding the values for nerve cells and glial cells obtained in the manner described above. Nomenclature. With respect to the lateral geniculate nucleus of the cat a number of terminologies which in some respects are at variance have been used in the literature (cf. Hayhow, 1958; Guiilery, I970). In ~he presen~ study, the nomenclature of Thum~ (1928) is used throughouL, bu~ in gecordance with Szents (1973) the terms lamina A, A1 and B are substituted for pars dorsalis A, Aa and B. For measuring the volume, the dorsal part of the geniculate nucleus (LGNd) was delineated so as to comprise the medial interlaminar nucleus, whereas the posterior nucleus of the thalamus is not included (cf. Fig. 1 in Kinston, Vadas and Bishop, 1969). The layer of nerve fibres which in adult eats surrounds the LGNd cannot be delineated in young animals and is therefore excluded from the measurements.
Results Overall Growth o/the LGNd. I n t h e n e w b o r n cat t h e lateral geniculate nucleus s t a n d s out as a s t r u c t u r e of great cellular density. I n sagittal sections it has t h e form of a slightly curved capital "'S" ~ n d its l a m i n a e can a l r e a d y be discerned at this early stage (Fig. 1 a). I n the m i d d l e two thirds of t h e L G N d the d e n s e l y p o p u l a t e d l a m i n a e A a n d A 1 are seen to be separated b y t h e lightly s t a i n e d N. i n t e r l a m i n a r i s centralis; a n d t h e border betweerL A 1 a n d B is a p p a r e n t from t h e difference i n cellular d e n s i t y b e t w e e n the two laminae. N e a r t h e edges of t h e L G N d , the i n t e r l a m i n a r nuclei disappear a n d t h e cell rich l a m i n a e A, A 1 a n d B merge into one structure. D u r i n g t h e first few days after b i r t h there is little or no growth, b u t i n the second week a phase of rapid growth sets in. A r o u n d the 20th d a y t h e volume of t h e L G N d has a l r e a d y tripled. As shown in Figure 2, the phase of fast growth continues to a b o u t the 40th day. B y t h e n the v o l u m e approaches t h e final values of b e t w e e n 20 a n d 30 ram s which are f o u n d i n most exiult cats. The m e a s u r e m e n t s show t h a t in cats of similar age there are considerable i n d i v i d u a l differences in the size of the L G N d , b u t t h a t i n e v e r y cat in which b o t h L G N d ' s could be m e a s u r e d t h e values o b t a i n e d from the right a n d left side are almost identical. The growth of t h e L G N d is a c c o m p a n i e d b y changes i n t h e shape of the nucleus a n d b y a r o t a t i o n of its long axis from a more vertical into a horizontal
6
I-I. Elgeti et al.
Fig. l a--d. Sagittal sections through the middle of the LGNd of cats aged 1 day (a), 11 days (b), 43 days (c) and 1.5 years (d). Chrome-hematoxylin-phloxin. 10 •
position. This is best seen b y c o m p a r i n g s a g i t t a l sections t h r o u g h t h e m i d d l e of t h e L G N d such as shown in F i g u r e s l a - d . I n t h e s e sections t h e d o r s a l t i p of t h e L G N d is seen to r e m a i n in a n u p w a r d position a n d to r e t a i n its t o p o g r a p h i c a l
Postnatal Growth of Lateral Geniculate Nucleus
7
position with respect to the fimbria hippoeampi. Therefore, the dorsal tip can be taken as a fixed point in relation to which the growth changes can be described. In Figure 3 the outlines of the 4 LGNd's of Figure 1 have been superimposed in order to show that the long axis is rotated upward and that during growth the nucleus extends predominantly in the anterior direction. I t seems likely that the
8
tI. Elgeti et al. VOLUME
35
.ram 3
30
25
20 9
9
15
log I
I
I
I
I
I
I
I
2
'~
8
I':
30
90
240
5;0
AGE DAYS
:Fig 2 Volume of t h e L G N d in ea~s of various age
IS [] ~ el [] o c.. . . . . . . . . . . . . . . ,~ - -
~
~ ~ t", m~mr, 9 .,..~.~.~....~o
9
!:'..::~'~~'.'1~ I ':":'~'::':':::':i::':l'lrlJet't'~J "-:~'..:~::
.... '. 11 '. ; ~ i '. "1 '. ~ ~ ', ', ' ;~ ,, ', '. 1 '. ; ........... ................. .~
r~ i i i: ,. ,,,,,,;; :,,,.,,,, ==,,; =,,=,, ,, ,,; ;,, !!-ii~u~ I ~ . ' .~'.,~,'~ =-
Fig.
3. To illustrate t h e p o s t n a t a l changes in volume a n d shape, t h e outlines of t h e lateral genieulate nuclei s h o w n in ~igure 1 have b e e n superimposed. ~ 1 day; ~ 11 d a y s ; g . ~ 45 d a y s ; ~ 1.5 years
Postnatal Growth of Lateral Geniculate Nucleus
9
NUCLEI in (O,Irnm)3
500
0
400
D
0
300
0
@
g
g
200
9
9
100
log AGE I
I
J
J
i
i
J
2
l,
8
1/.
30
90
2~0
I 5l*0
0 A u
Fig. 4. Postnatal changes in the total number of cell nuclei per unit volume as determined in the binocular segment of lamina A of the LGNd
characteristic postnatal changes in the shape and position of the LGNd are due to a differential growth of the various parts of the laminae as well as of the interlaminar layers, but this question has not been studied in detail. Changes in Cell Density. The growth of the LGNd is accompanied by a considerable decrease in cell density which affects Lamina A and A 1 as well as lamina B. This is evident when Figures 1 a - d are compared. The decrease in cell density was studied quantitatively in the binocular segment of lamina A, which can be discerned in cats of all ages, and in the sagittal as well as in the frontal planes of sectioning. As shown in Figure 4, the decrease in total cell density commences right after birth and is measurable before the overall growth in volume becomes detectable. In lamina A, the number of cells decreases from about 470/(0.1 mm) 3 at birth to about 150 at the beginning of the second month; later there is little further change of cell density and in adult animals the values obtained are between 95 and 130 cells/(0.1 ram) 3.
10
It. Elgeti et al. NUCLEI in (0.1rn rn) 3
500
~00
3:00
o
200
o
o
o
o
oo
tOO
oo
o% o
9
9
o
9 ~
o 9
o
9
9
log I
I
1
I
I
i
I
I
2
L
g
1~.
30
90
2L0
5 "0
AGE DAYS
Fig. 5. Postnatal changes in the number of cell nuclei of nerve cells (ooo) and neurogliM cells (...) per unit volume ms determined in the binocular segment of lamina A of the LGNd
W h e n t h e m e a s u r e m e n t s of t h e n u m b e r of n e r v e cells a n d neuroglial cells per u n i t v o l u m e are d r a w n s e p a r a t e l y (Fig. 5), it becomes e v i d e n t t h a t t h e s t e e p decrease of t h e t o t a l n u m b e r of t h e cells p e r u n i t v o l u m e is a l m o s t e n t i r e l y due to a decrease in t h e n u m b e r of n e r v e cells whereas t h e n u m b e r of gtiM cells r e m a i n s f a i r l y c o n s t a n t . This conclusion c a n safely be d r a w n f r o m F i g u r e 5, a l t h o u g h it was f o u n d t h a t in e h r o m e h e m a t o x y l i n s t a i n e d p a r a f f i n sections t h r o u g h t h e L G N d of cats with a p o s t n a t a l age of between 4 a n d 6 weeks i t m a y occasionally be difficult to d i s c r i m i n a t e between the nucleus of a n e r v e cell a n d t h a t of a large glial cell. Discussion T h e r e seems to be no l i t e r a t u r e on t h e v o l u m e g r o w t h of t h e L G N d of cats, a n d no v a l u e for t h e v o l u m e of this nucleus in t h e a d u l t cat was found. H o w e v e r , t h e p r o j e c t i o n m a p s of S a n d e r s o n (1971), which h a d been o b t a i n e d b y fitting
Postnatal Growth of Lateral Geniculate Nucleus
Ii
together the values of 40 cats, enabled us to calculate a value with which to compare our own measurements in the adult animals. For this comperison planimetric measurements of the 9 figures of coronal sections given in the paper of Sanderson were made. From the values thus obtained (F1, F 2 . . . Fg) the volume was calculated by using the formula
V=
IF1
F2
(vl)2 + ~ + . . .
2'9]
+ ~
xd
in which " v " is a factor for the different magnifications of the figures of Sanderson and " d " a factor pertaining to the spacing of the sections. The value arrived at is 42.5 m m ~. Since all measurements in the paper of Sanderson are expressed in terms of Horsley-Clark coordinates, no correction needs to be made for shrinkage. Our own results, on the other hand, are expressed in absolute values as obtained by measuring paraffin sections, and have therefore to be corrected for shrinkage which according to Stowell (1941) and Stephan (1960) makes up as much as 40%. The uncorrected measurements for the volume of the L G N d in grown-up animals (No. 28-32 in Table 1) v a r y between 17.62 m m 3 and 33.42 m m 3 with a mean of 26.42 m m 3. If this mean value is corrected for 40 % shrinkage, the true volume of the L G N d in the adult oat turns out to be about 44 m m ~, which is in good agreement with the value of 42.5 m m 3 arrived at by calculating the volume from the figures published by Sanderson (1971). The postnatal changes in shape and the rotation of the axis of the L G N d indicate t h a t the various components of this nucleus have a different rate of growth. These differences have not been analysed systematically, but our preliminary observations suggest t h a t the highly ordered pattern of the growing dendritic trees as well as the ordered arrangement of the myelinating nerve fibres must be held responsible for the changes in shape and position. If the postnatal increase in volume is compared with the concomitant decrease in the number of nerve cells per unit volume in lamina A, the decrease in the number of nerve cells is seen to begin before a clear-cut increase in the size of the LGNd becomes detectable. It is most likely that this discrepancy is due to the fact that only one region of grey matter was investigated for cell numbers and that the changes observed here need not be immediately reflected in the size of the entire structure. It is improbable that the discrepancy is due to a degeneration of nerve cells, because no indication for such degeneration was found either in the quantitative measurements or in the histological appearance of the sections which were carefully studied at high magnification. The finding that during growth of the LGNd the decrease in the total number of cells per unit volume is mainly due to a decrease in the number of nerve cells per unit volume whereas the density of the glial cells remains fairly constant, suggests that new glial cells are formed after birth. If an extrapolation is made from the measurements in lamina A, a rough indication of the absolute number of nerve cells and glial cells can be obtained by multiplying the number of cells per unit volume found in lamina A with the total volume of the LGNd. The values obtained by such an extrapolation have to be interpreted with the greatest caution. However, they clearly indicate that during postnatal development the absolute number of nerve cells remains fairly constant whereas between the
12
H. Elgeti et al.
second and sixth week the absolute number of glial cells rises steeply. Since in Kliiver stained sections the first myelinated fibres in the LGNd were detected on the 16th day of postnatal life, this finding is in accordance with the fact that in white matter, too, a great number of glial cells are newly formed shortly before and during the period of myelination (for lit. see Fleischhauer, 1968). The measurements of the postnatal increase in volume of the LGNd and of the concomitant decrease in cell density in the binocular segment of lamina A are in good agreement with the measurements of pcrikaryal growth (Garey, Fisken and Powell, 1973) and increase in the number of synapses (Cragg, 1975). However, the comparison also shows that with a larger number of eats such as used in the present study, the existence of considerable individual differences between the values to be obtained at any given age becomes obvious. The existence of these large individual differences must be kept in mind when measurements of changes during postnatal growth are interpreted and results of various authors compared. For such studies mean values based on a sufficient number of animals in each age group are required. However, when studied in the same animal and with the same method, comparisons between different regions of the brain are less prone to error because such comparisons are not influenced by the individual differences mentioned above. I t has been shown by Cragg (1975) that the increase in the number of synapses in the LGNd precedes that in the visual cortex of the same animals by a few days. Another even more striking time-lag between the development of the LGNd and that of telencephalic structures is revealed when the growth of the LGNd is compared with that of the corpus callosum which had previously been studied in the same eats used for the present investigation (Fleischhauer und Sehliiter, 1970). The comparison shows that the increase in volume of the L G N d begins earlier than that of the corpus callosum, rises more steeply and terminates at about the fourtieth day, whereas the growth of the corpus callosum continues into adult life.
Adcnowledgement. This work was supported by a grant of the Bundesministerium ffir Forschung und Technologie.
References Cammcrmcyer, J.: An evaluation of the significance of the "dark" neuron. Ergebn. Anat. Entwickl.-Gesch. 86, 1-61 (1962) Cr~gg, B. G. : The development of synapses in the visual system of the cat. J. comp. Neurol. 168, 147-166 (1975) Fleischhauer, K. : Fluorescenzmikroskopische Untersnchungen an der Faserglia. Z. Zel]forsch. 51,467-496 (1960) Fleischhauer, K. : Postnatale Entwicklung der Neuroglia. Acta neuropath. (Berl.), Suppl. IV, 20-32 (1968) Fleischhauer, K., Schlfiter, G. : Uber das postnatale Wachstum des Corpus callosum der Katze (Fells domestica). Z. Anat. Entwiekl.-Gesch. 132, 228-239 (1970) Floderus, S. : Untersuchungen fiber den Bau der menschlichen Hypophyse mit besonderer Berficksichtigung der mikromorphologischen Verh~ltnisse. Act~ path. microbiol, scand., Suppl. 537 (1944) Garey, L. J., Fisken, g.A., Powell, T. P. S.: Observations on the growth of ceils in the lateral geniculatc nucleus of the cat. Brain Res. 52, 359-362 (1973a)
Postnatal Growth of Lateral Geniculate Nucleus
13
Garey, L. J., Fisken, R. A., Powell, T. P. S. : Effects of experimental deafferentation on cells in the lateral geniculate nucleus of the cat. Brain Res. 52, 363-369 (1973b) Gomori, G. : Observations with differential stains on human islets of Langerhans. Amer. J. Path. 17, 395-406 (1941) Guillery, R . W . : The laminar distribution of retinal fibers in the dorsal lateral geniculate mteleus of the cat: a new interpretation. J. eomp. Neurol. 138, 339 368 (1970) Guillery, R . W . , Stelzner, D. J.: The differential effects of unilateral lid suture upon the monocular and binocular segments of the dorsal lateral geniculate nucleus in the cat. J. comp. Neurol. 1119, 413422 (1970) I-Iaug, H. : Probleme und Methoden der Strukturzg~hlung in Schnittprgparaten. In: Weibel, E . R . , Elias, H. (eds.), Quantitative methods in morphology. Berlin-Heidelberg-New York: Springer 1967 Hayhow, W. R. : The cytoarehiteeture of the lateral geniculate body in the cat in relation to the distribution of the crossed and uncrossed optic fibers. J. comp. Neurol. 110, 1-64 (1958) Kinston, W. J., Vadas, M.A., Bishop, P. 0.: Multiple projection of the visual field to the medial portion of the dorsal lateral geniculate nucleus and the adjacent nuclei of the thalamus of the cat. J. comp. Neurol. 136, 295-316 (1969) Kliiver, H., Barrera, E.: A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol. 12, 400-403 (1953) Luna, L. G.: Further studies of Bodian's technique. Amer. J. Technol. 30, 355-362 (1964) Sachs, L.: Statistische Auswertungsmethoden. Berlin-Heidelberg-New York: Springer 1968 Sanderson, K. J. : The projection of the visual field to the lateral genieulate and medial interlaminar nuclei in the cat. J. comp. Neurol. 143, 101-118 (1971) Stephan, H.: ~ethodisehe Studien fiber den quantitativen Vergleich architektonischer Struktureinheiten des Gehirns. Z. wiss. Zool. 164, 143-172 (1960) Stowell, R . E . : Effects on tissue volume of various methods of fixation dehydration and embedding. Stain Technol. 16, 67 83 (1941) Szents J.: Neuronal and synaptic architecture of the lateral geniculate nucleus. In: Autrum, H. et al. (eds.), Handbook of sensory physiology, vol. VII/3, part B, p. 141-176. Berlin-Heidelberg-New York: Springer 1973 Thuma, B. D. : Studies on the diencephalon of the cat. I. The cytoarchitecture of the corpus geniculatum laterale. J. comp. Neurol. 46, 173-I97 (1928) Prof. Dr. K. Fleisehhauer Anatomisehes [nstitut Nussallee 1O D-5300 Bonn Federal Republic of Germany
