MORPHOMETRY STUDY ON PRE- AND POST-HATCHING
NERVE CELL BODIES OF LUMBAR SPINAL GANGLIA OF
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
GALLUS DOMESTICUS
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
CLAUDIO A. FERRAZ DE CARVALHO * CIRO F. DA SILVA **
BONFIN A. SILVA JUNIOR *** M. VILMA DE ARAUJO ****
The migration of the spinal ganglion neuron mother cells in the chick
embryo, from the neural crest towards the lateral part of the neural tube,
occurs during the first 2 1/2 days of incubation
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2 8 , 5 9 , 6 0 , 6 1 .T
w odifferent regions
(ventrolateral and dorsomedial) can be described in the ganglion whose cells
present distinct morphological, as well as functional features
2 2. Similar aspects
are described in humans by McKinniss
3 4. But, as the smaller-sized cells of
the dorsomedial region present a higher growing rate than those of the
ventro-lateral region, no histological distinction between the two regions may be
observed after the 15th day of incubation. The ultrastructure of adult spinal
ganglion neurons has been studied extensively by different authors
1 , 1 0 , 2 4 , 4 2 , 5 0 , 5 1 ^as well as their differentiation in G.
domesticus
4 3 , 4 6 , 5 7: Up to the 5th day of
incubation the neuroblasts present scarce cytoplasm with few ribosomes and
scanty developed RER; later on they start to show all the features of mature
cells, i.e. well developed RER and Golgi complex, many mitochondria, and free
ribosomes. Environmental influences acting on the spinal ganglion neuron were
studied in embryos submitted to limb excision and grafts in order to analyse,
for example, the influence of the innervation field on the number of sensory
neurons
2 6.
The present paper reports a morphometric study looking for quantitative
modifications of the fine structure of these neurons at six different ages,
ranging from the 10th day of incubation to the 120th post-hatching day. W e
estimated the nuclear and cytoplasmic volumes and for the latter the relative
volumes of hyaloplasm (including free ribosomes) inside and outside the Nissl
bodies, RER, SER, mitochondria dense bodies and Golgi complex, the
surface--to-volume ratio, and total surface of the organelles.
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
M A T E R I A L A N D M E T H O D S
L u m b a r spinal g a n g l i a of
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
G. domesticus ( H y b r o ) on the 10th a n d 18th d a y s ofi n c u b a t i o n a n d 8th, 35th, 61st a n d 120th p o s t - h a t c h i n g d a y s w e r e u s e d . T h e g a n g l i a v/ere r e m o v e d f r o m the h a t c h e d c h i c k s i m m e d i a t e l y a f t e r e t h e r a n e s t h e s i a a n d f r o m the e m b r y o s a f t e r d e c a p i t a t i o n . G a n g l i a f r a g m e n t s w e r e f i x e d b y 2 . 0 % g l u t e r a l d e h y d e in O.IM c a c o d y l a t e b u f f e r ( p H 7.2; 480 m O s m ) até 4 . 0 C f o r 2h a n d p o s t f i x e d f o r l h b y
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
1 % o s m i u m t e t r o x i d e in 0.05M p h o s p h a t e b u f f e r w i t h 123 m g / m l s u c r o s e at<ioC. A f t e r a q u i c k r i n s e in p h o s p h a t e b u f f e r , the f r a g m e n t s w e r e d e h y d r a t e d with c t h a n o l p r o p y l e n e o x i d e a n d e m b e d d e d in a r a l d i t e .
T h e d i a m e t e r o f 200 nuclear t r a n s e c t i o n s ( f o r each a n i m a l ) o f spinal g a n g l i a n e u r o n s w e r e m e a s u r e d w i t h a filar m i c r o m e t r i c e y e p i e c e ( K p l x 8 , w i t h an oil i m m e r s i o n o b j e c t i v e xlOO) in O.S^m m e t h y l e n e b l u e — a z u r I I stained s e c t i o n s *. C o n s i d e r i n g the n e u r o n nuclei as h a v i n g a s p h e r i c a l s h a p e , the m e a n r a d i u s w a s e s t i m a t e d b y the B a c h
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2 m e t h o d . T h e c o r r e s p o n d i n g n u m e r i c a l p r o c e s s i n g s w e r e p e r f o r m e d b y a s u i t a b l yp r o g r a m m e d HP-9810A e l e c t r o n i c c a l c u l a t o r . T h e a b s o l u t e n u c l e a r v o l u m e s w e r e finally o b t a i n e d f r o m the f o r m u l a f o r the s p h e r e .
T h e relative v o l u m e o f c y t o p l a s m w a s evaluated in 100 m i c r o s c o p i c fields f o r each animal, c o u n t i n g p o i n t s l y i n g o n nuclei a n d c y t o p l a s m w i t h a Z e i s s 100point i n t e -g r a t i n -g ( K p l x 8 ) a n d oil i m m e r s i o n o b j e c t i v e (xlOO). T h e s e d a t a a n d t h o s e of t h e n u c l e a r v o l u m e e n a b l e d u s t o o b t a i n t h e a b s o l u t e c y t o p l a s m i c v o l u m e .
T h e u l t r a s t r u c t u r a l m o r p h o m e t r i c e v a l u a t i o n s w e r e c a r r i e d o u t u s i n g an 84-segment
t e x t s y s t e m ( b a s e d on W e i b e l et al
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
.rvs) s u p e r i m p o s e d in t w o o r t h o g o n a l d i r e c t i o n s o n20 e l e c t r o n m i c r o p h o t o g r a p h s ** (x21,000) f o r each animal, taken at r a n d o m o v e r the g a n g l i a f r a g m e n t s .
T h e d a t a o b t a i n e d b y c o u n t i n g o f p o i n t s on c y t o p l a s m i c c o n s t i t u e n t s ( R E R , S E R , m i t o c h o n d r i a , d e n s e b o d i e s , O o l g i c o m p l e x a n d h y a l o p l a s m i n s i d e a n d o u t s i d e the NissI b o d i e s ) a n d i n t e r s e c t i o n s w i t h c o n t o u r s o f t r a n s e c t e d o r g a n e l l e m e m b r a n e s e n a b l e d
us t o e s t i m a t e t h e relative v o l u m e ( V . ) o f t h e s e c o n s t i t u e n t s , t h e s u r f a c e - t o - v o l u m e
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
V I
ratio ( s / v ) and the total s u r f a c e (S ) o f the o r g a n e l l e s . . T h e f o l l o w i n g f o r m u l a s
1 i t o t
w e r e u s e d :
P . 4 . N 4 . N .
i i l V . = ; s . / v . « = ; S =
s
.. V ; S . = .V I 1 1 t o t V I V I
P Z . P Z . P
t i t
w e r e P . = differential c o u n t i n g o f p o i n t s ; P^ = total n u m b e r o f p o i n t s ; N . == n u m b e r o f i n t e r s e c t i o n s o v e r t r a n s e c t e d o r g a n e l l e m e m b r a n e s ; Z — e n l a r g e m e n t f a c t o r ; S^. ~ s u r f a c e d e n s i t y ; V = c y t o p l a s m i c a b s o l u t e v o l u m e .
T h e mean values of 3 s p e c i m e n s used f o r e a c h a g e a r e p r e s e n t e d in t a b l e 1.
* P e r f o r m e d in a U l t r a t o m e L K B u i t r a m i c r o t o m e , d o n a t e d b y the F A P E S P ( P r o -c e s s o 70/1445).
R E S U L T S
T h e estimated d a t a a r e p r e s e n t e d in T a b l e 1 a n d , g r a p h i c a l l y in f i g u r e s 1 a n d 2. T h e c h a n g e s of t h e h i s t o l o g i c a l c h a r a c t e r i s t i c s o b s e r v e d in o u r m a t e r i a l a r e s i m i l a r t o t h o s e o b s e r v e d b y H a m b u r g e r a n d L e v i - M o n t a l c i n i
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2 2 . On t h e 18th d a y o f i n c u b a t i o nthe t w o until then d i s t i n c t g r o u p s o f n e u r o n s a r e a l r e a d y i n t e r m i n g l e d , s h o w i n g d i f f e r e n t sizes a n d s t a i n i n g intensities ( F i g . 3 ) . A s o n e can s e e in t a b l e 1 and f i g u r e 1,
t h e m e a n cell v o l u m e i n c r e a s e s c o n s t a n t l y , p a r t i c u l a r l y b e t w e e n the 1st a n d the 2nd a g e s k e e p i n g the f o l l o w i n g p r o p o r t i o n s a m o n g t h e m s e l v e s : 1.0 : 13.5 : 12.5 : 24.1
: 23.8 : 30.2, r e s p e c t i v e l y on the 10th a n d 18th d a y s o f i n c u b a t i o n a n d o n t h e 8th, 35th, 61st, a n d 120th p o s t - h a t c h i n g d a y s . A p a r t i c u l a r l y intense i n c r e a s e b e t w e e n the 1st a n d the 2nd a g e s w a s o b s e r v e d f o r the r e l a t i v e a n d t h e a b s o l u t e c y t o p l a s m i c v o l u m e . T h e v a r i a t i o n s o f n u c l e a r v o l u m e o c c u r r e d a c c o r d i n g t o t h e f o l l o w i n g p r o p o r t i o n s : 1.0 : 3.5 : 2.4 : 3.5 : 3.0 : 2.6. T h e n u c l e a r c y t o p l a s m i c r a t i o d e c r e a s e d u n i f o r -m e l y : 16.3 : 3.1 : 2.2 : 1.7 : 1.4 : 1.0.
v a r i a t i o n s , d e c r e a s i n g s l i g h t l y o n t h e 120th d a y (8 ± 0.8 — 459 ± 76 — 473 ± 152 — 514 ± 134 — 518 ± 205 — 496 ± 62 ^ m S ) . O n t h e o t h e r h a n d , the a b s o l u t e v o
-l u m e o f S E R i n c r e a s e d d r a m a t i c a -l -l y (20 ± 5 — 213 ± 101 — 224 ± 125 — 579 ± 46 — 1,574 ± 218 — 1,478 ± 345 ^ m 3 ) . T h e relative v o l u m e o f G o l g l c o m p l e x
d e c r e a s e s f r o m t h e 2nd t o t h e 4th a g e , t h e n b e c o m i n g s t a b l e . T h e m i t o c h o n d r i a
( e x c e p t f o r t h e 120th d a y ) a n d d e n s e b o d i e s s h o w a p r o g r e s s i v e d e c r e a s e o f the s a m e p a r a m e t e r .
T h e b e h a v i o r o f t h e h y a l o p l a s m i n s i d e a n d o u t s i d e t h e Nissl b o d i e s w i t h r e s p e c t
t o relative v o l u m e is t h e o p p o s i t e ; w i t h t h e e x c e p t i o n o f t h e 10th d a y o f i n c u b a t i o n ,
t h e r e l a t i v e v o l u m e o f N i s s l h y a l o p l a s m d r o p s , w h i l e t h e a b s o l u t e v o l u m e o f t h i s
c o n s t i t u e n t g r o w s until t h e 35th d a y , d e c r e a s i n g s l i g h t l y a f t e r w a r d s (194 ± 4 — 4,575
± 346 — 3,987 ± 189 — 7,882 ± 754 — 7,544 ± 344 — 7,252 ± 1,228 ^ m 3 ) . T h e
s u r f a c e - t o - v o l u m e r a t i o d r o p s s h a r p l y f o r all o r g a n e l l e s f r o m the 10th t o the 18th
d a y o f i n c u b a t i o n . A f t e r h a t c h i n g , a t e n d e n c y t o i n c r e a s e w a s o b s e r v e d . T o t a l s u r f a c e
a l s o g r o w s s h a r p l y b e t w e e n t h e f i r s t t w o a g e s . I n t e n s e g r o w t h o f t o t a l s u r f a c e of
S E R o n the 61st a n d 120th d a y is o b s e r v e d , t o g e t h e r w i t h a s l i g h t i n c r e a s e o f
t h o s e o f d e n s e b o d i e s a n d n o n e o f R E R b e t w e e n the last t w o a g e s , in spite o f the
g r e a t i n c r e a s e o f the a b s o l u t e c y t o p l a s m i c v o l u m e . H o w e v e r , the c h a n g e s in total
s u r f a c e c a n b e b e t t e r u n d e r s t o o d in t e r m s o f v a r i a t i o n s o f t h e m e m b r a n e s u r f a c e t o
-- c y t o p l a s m i c v o l u m e r a t i o s ( SA 7 V ) ( t a b l e 2 ) . T h e s e s e q u e n c e s s h o w a d e c r e a s e o f
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
tot
m e m b r a n e / ^ m 3 o f c y t o p l a s m f r o m t h e 10th t o t h e 18th d a y s of i n c u b a t i o n . A f t e r
h a t c h i n g , the r a t i o i n c r e a s e s f o r m i t o c h o n d r i a , G o l g i c o m p l e x , a n d p a r t i c u l a r l y f o r
S E R , in t h e last t w o a g e s , w h i l e it d e c r e a s e s f o r d e n s e b o d i e s a n d alternates f o r R E R
Regarding neuron growth in the spinal ganglia of
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
G. domesticus,
our
findings are equivalent to those of Olivo et a l .
4 0; these authors found a
marked growth during the embryonic period which decreases immediately after
hatching, while increasing progressively even after 6 months. W e observed
significant growth in cell volume between the 1st and 2nd ages, with a slight
decrease on the 8th post-hatching day and continuous growth during all
other ages.
Except for the last age, the positive ratio between nuclear and cell body
sizes observed by H a t a i
2 3was confirmed by our findings. Based on L e v i ' s
3 0suggestions, Donaldson and N a g a s a k a
1 2and Ohta et a l .
3 9correlated cell size
and innervation field during the post-natal development, in order to explain
the continuous growth of the sensory neurons, months after hatching. It is
generally assumed that the control the periphery exerts on differentiating
ganglia is chemical in nature and is transmitted to the cell bodies by retrograde
axonal flow in peripheral nerve fibers
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
2 6 , 2 7 , 3 6 .Concerning the cytoplasmic constituents specifically, Fujita and F u j i t a
1 0,
Pannese
4 6»
4 7and T e n n y s o n
5 7studied the development of the various organelles
(RER, SER, Golgi complex) from the neuroblastic to the neuronal period,
re-porting a particularly intense increase in number of the mitochondria
4 7. Our
estimates regarding the organelle development of the cell body start during
the neuronal period. W e observed an increase in the relative volume of RER
and Golgi complex during the incubation periods, a decrease in those of SER
and dense bodies, and no modification in mitochondria.
According to Pannese et a i .
4 4»
4 5the acetylcholinesterase (AChE) activity
increases considerably in the pseudounipolar nerve cell when compared to the
primitive neuroblast, being parallel to the development of RER and the Golgi
complex. AChE-activity in sensory ganglia cells could be considered as one
of the most important events characterizing their biochemical differentiation. A
relationship between the increase in AChE and the development of RER would
be supported by our morphometric data and Giacobini et al.'s
2 0findings; they
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
pointed out an activity raise from the 6th to the 12th day of incubation, a
progressive decrease to the minimum value by the time of hatching, and a
slight increase in the following two days.
The opposite bahavior of the RER and SER concerning the relative volume
could be dependent on the specific role of this kind of cell. The proteins
synthesized by the neuron would be greatly used for the biochemical turnover
of cell body constituents and of the axon
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
I «zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
I1 T ^ D.On the other hand, the Nissl
bodies ergastoplasm differs cleary from that of the high-production
protein--exporting gland cells. Palay and P a l a d e
4 2and Peters et al.
4 8have reported
numerous free ribosomes among the ER-cysternae; the membrane-attached
ribosomes do not present a uniform distribution pattern along the cysternae
and it is not difficult to demonstrate a continuity of RER with SER in nerve
cells. More than 9 8 % of the proteins of nerve endings would be synthesized
by RER, reaching the nerve endings by fast axonal flow where SER
plays the most important role. The increase of relative volume and total surface
of SER in the cell body seems to emphasize this role.
As a consequence of the diffuse character and continuity with RER and
cell surface, the SER would be the ideal structure for a fast spread of substances
from the site of their synthesis or capture. According to different authors
the proteins, glycoproteins, or phospholipids would be transported by the SER
from the cell body to the nerve endings
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
6,8,9,14,15,17,18,33,53,56.
SER is involved
in the origin of microvesicles and the transport of substances to mitochondria
and axolemma (including the presynaptic plasma membrane
1 3 , 1 4 , 1 7 ) a nd from
the perikaryon towards the a x o n
1 4. SER is also an important pathway for
substance transport from the nerve endings towards the cell b o d y
3 7'
5 4. The
changes of relative volume of Golgi complex and RER are similar. The Golgi
complex of the nerve cell plays an important role in the synthesis of
glyco-p r o t e i n s
5'
2 5'
3 8.
4 1.
5 5. On the other hand changes of relative volume of
mitochon-dria are slight, when compared to the other constituents. Apparently the
mitochondria are distributed at random in the perikaryon among the Nissl
bodies in the so-called 'Plasmastrassen'
1or ' r o a d s '
7where they seem to move
4 9,
probably aided by microtubuli and microfilaments
3. These considerations
suggest uniform physiological role of mitochondria in different cell parts. Under
the designation 'dense bodies' we can consider different structures such as
the lysosomal complex, melanin (neuromelanin) and lipofuscin-containing
pigment bodies; however, they are related to one another and to mitochondria,
Golgi complex and R E R
4 8.
In sensory ganglia cells literature data refer to an age-associated increase
in lipofuscin b o d i e s
2 1'
3 5'
5 2and l y s o s o m e s
3 2and the presence of very scarce
melanin granules
4*
1 1. According to our findings the dense bodies in G.
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
do-mesticus showed a gradual and constant decrease of relative volume at least
for the period between the 10th day of incubation and the 120th post-hatching
S U M M A R Y
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
A cytomorphometric study was performed in lumbar spinal ganglia neurons
of
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Gallus domesticus
on the 10th and 18th incubation days and 8th, 35th, 61st,
and 120th post-hatching days. The absolute volume of nucleus and relative
volume of cytoplasm were respectively estimated by the B a c h
2caryometric
method and by point-counting volumetry, carried out in 0.5
ζψξωϖυτσρθπονµλκϕιηγφεδχβαΖΨΞΩςΥΤΣΡΘΠΟΝΜΛΚϑΙΗΓΦΕ∆ΧΒΑ
m
m thick araldite
sections. The relative volume, the surface-to-volume ratio and the total surface
of RER, SER, mitochondria, dense bodies, Golgi complex and the relative volume
of hyaloplasm inside and outside the Nissl bodies were estimated from
electronmicrographs by the Weibel et a l .
5 8method. T h e conclusions w e r e :
a) there was an increase of the cell volume and a decrease of the
nucleo-cytoplasmic ratio, particularly between the first two ages; b ) the relative
volumes of RER and SER change inversely with respect to each other: the
RER increases before hatching, decreasing progressively afterwards; the changes
of relative volume of dense bodies are similar to those of the RER, and the
mitochondria show relatively small variations concerning the same parameter;
c ) the relative volume of hyaloplasm inside the Nissl bodies decreases while
those outside increases; d ) the surface-to-volume ratio drops sharply for all
organelles from the 10th to the 18th day of incubation; after hatching, a
tendency to increase is observed; e) the membrane surface-to-cytoplasmic
volume ratio decreases for all organelles from the 10th to the 18th day of
incubation; after hatching, this ratio increases slightly for mitochondria and
Golgi complex, sharply for SER, dropping for dense bodies. The RER values
alternate regularly.
R E S U M O
Estudo morfométrico sobre corpos de células nervosas de gânglios espinais
lombares de
Gallus domesticus
antes e depois da eclosão.
Foi realizado estudo citomorfométrico em neurônios de gânglios espinais
lumbares de
Gallus domesticus
no 10º e 18º dias de incubação e no 8º, 35º, 61º
e 120º dias após a eclosão. Os volumes absoluto do núcleo e relativo do
citoplasma foram estimados, respectivamente, pelo método cariométrico de
B a c h
2e pela volumetria de contagem de pontos, em cortes de araldite de
0.5
m
m. O volume relativo, a relação superfície/volume e a superfície total do
RER e SER, mitocôndria, corpos densos, complexo de Golgi e volume relativo
de hialoplasma, no interior e fora dos corpos de Nissl, foram estimados a
partir de eletron-micrografias, pelo método de Weibel et a l .
5 8. As conclusões
diminui, enquanto o situado no interior, aumenta; d ) a relação
superfície/vo-lume decresce muito para todas as organelas, do 10º para o 18º dia de incuba¬
ç ã o ; após a eclosão foi observada tendência à elevação; e) a relação superfície
de membrana volume citoplasmático cai para todas as organelas, do 10º para
o 18º dia de incubação; após a eclosão, esta relação diminui levemente para
mitocôndrias e complexo de Golgi, fortemente para o SER, caindo, também,
para corpos densos. Os valores do RER alternam-se, regularmente.
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
R E F E R E N C E S
1. A N D R E S , K . H . — U n t e r s u c h u n g e n u e b e r d e n F e i n b a u v o n S p i n a l g a n g l i e n . Z. Z e l l f o r s c h . 55:1, 1961.
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