INTRODUCTION
The demonstration in 1987 of the formation of Nitric Oxide (NO)
by an enzyme in the vascular endothelial cells opened up what can now
be considered a new area of the biological research (Bredt, 1996; Prast
and Philippu, 2001; Wang et al., 1994). Nitric oxide is an important
molecule because of its role as a universal modulator of neuronal function
at the synaptic level, mainly in the central nervous system. In the mammalian
organism, NO is synthesized in several types of cells, such as neurons,
INTRODUCTION
The demonstration in 1987 of the formation of Nitric Oxide (NO) by an enzyme in the vascular endothelial cells opened up what can now be considered a new area of the biological research (Bredt, 1996; Prast and Philippu, 2001; Wang et al., 1994). Nitric oxide is an important molecule because of its role as a universal modulator of neuronal function at the synaptic level, mainly in the central nervous system. In the mammalian organism, NO is synthesized in several types of cells, such as neurons, endothelial cells and macrophages by a family of three isoenzymes termed nitric oxide synthases or NOS (Lopez-Costa et al., 1997). nNOS is present in neurons. It is constitutively expressed and its activity is regulated by Ca2+. Another constitutive and Ca2+-dependent type of NOS is present in endothelial cells (eNOS) of the vasculature.
Nitric oxide synthase can produce nitric oxide with the aid of NADPH in response to changes in intracellular free calcium by deimidating arginine to citrulline. The selective coexistence of citrulline-like immunoreactivity in NADPH-diaphorase positive neurons is thus consistent with NADPH diaphorase being a nitric oxide synthase (Geurges, 1999). Thus the extensive literature on the histochemistry of NADPH diaphorase should be reexamined in light of the fact that this simple histochemical technique allows the cellular localization of nitric oxide synthase in the nervous system (Dawson et al., 1991; Bruce, 1991).
In 1961, Thomas and Pearse first described the presence of large, isolated neurons containing outstanding high NADPH-diaphorase activity which were scattered throughout the cerebral cortex and basal ganglia (Schober et al., 1994).
The aim of the present study was to clarify if the rat optic vesicle cells using the NADPH-d as an indicator to the NOS-histochemistry (Rangarajan et al., 1999). Effect of NOS expression on optic vesicle development is very important on assessment abnormalities.
MATERIALS AND METHODS
In this study, 11 young adult female Spargue-Dawley rats were used. The rats
were mated and the gestational age was estimated according to the day of vaginal
plaque formation, to mark the first day of life. Fetuses aged 8-18 days of gestation
were obtained, decapitated and washed in solution consisting of 0.1 M Phosphate
Buffer Saline (PBS) in pH = 7.4. The heads were transformed into fixative for
24 h; The fixative contained fresh solution of 4% paraformaldehyde in 0.1 M
PBS, pH = 7.4, at 4deg; C. Subsequently the heads were kept in solution consisting
of 15% sucrose in PBS for 1 h and then in 30% sucrose at 4deg; C for 2 h. They
were then frozen and transverse and longitudinal sections to 10 μ M were
cut on a cryostat. Sections were collected on gelatin-coated slides. They were
later rinsed in 0.1 M PBS (pH = 8.0) for 10 min. NADPH-diaphorase histochemistry
was performed by incubating the sections in a solution consisting of 30 μ
M malic acid, 1 μ M manganese hydrochloride, 0.2 μ M nitro blue tetrazolium
chloride, 1 μ M NADPH, 0.5% Triton X-100 in buffer of Tris-HCl in pH 7.4,
at 37deg; C, for 90 min in a dark place. Sections were mounted in entellan and
photographs were taken.
RESULTS
At E13, the diencephalic vesicle is composed of only two layers: the ventricular zone, where all neurons are generated and the marginal zone, where the first-generated neurons are setting (Fig. 1).
NADPH-d histochemistry was not observed in the diencephalic vesicle at this stage in either transverse or longitudinal sections.
At E14, the diencephalic vesicle is greatly enlarged and increased in thickness, mainly due to growth of the ventricular zone (Fig. 2). The marginal zone is also thicker. An incipient intermediate zone between the ventricular zone and marginal zone is observed. The whole marginal zone of the diencephalic vesicle was populated by NOS-positive cells. There were two chief types of NADPH-d histochemistry cells (Fig. 3), large cells in the middle part of marginal zone with long tangentially oriented processes, which showed the morphological characteristics of Cajal-Retzius cells and externally situated round cells with processes oriented upward.
At E16, the diencephalic vesicle has four layers: the ventricular zone, which occupies roughly one-third of the thickness, the intermediate zone, with easily visible fibers coursing along it, marginal zone and the cortical plate and more prominent in the lateral than in the dorsal aspect of the diencephalic vesicle.
At E18, NADPH-d reactivity is observed at low magnification showed
five clearly defined layers (Fig. 4).
|
Fig. 1: |
Frontal section through diencephalen. Rat embryo at E13,
H and E staining |
The marginal zone is densely labeled and the underlying cortical plate showed
a band of stained cells in which nearly every cell was stained, so that it was
difficult to identify single cells.
In the late stage, the most notable feature was a decrease in the histochemical
reaction of the marginal zone. Horizontally coursing NADPH-d histochemical activity
fibers could also be observed in the marginal zone. Also NADPH-d reactivity
was observed in endothelial cells lining the larger blood vessels, but it was
not present in endothelial cells of sinusoidal capillaries or the small vessels
(Fig. 5).
|
Fig. 2: |
Frontal section through the optic rat embryo at E14 optic
vesicle Viewed at higher magnification. At this stage, the diencephalic
vesicle continues to grow and its thickness increases. The marginal zone
has been also thicker and because of growth of the ventricular zone, the
intermediate zone is more prominent. Cryostat section |
|
Fig. 3: |
Frontal section showing the diencephalic vesicle of a rat
embryo at E14 at high magnification. The picture shows two layers
of marginal zone and ventricular zone. There are NOS-positive round cells
in the marginal zone. The nerve fibers are also stained. At the ventricular
zone, only nerve fibers are weakly stained. Cryostat section |
|
Fig. 4: |
Photomicrograph showing the marginal zone rat embryo at E18Staining
is observed in a group of highly active cells. There are two kinds of NOS-positive
cells: big chief cells in the middle part of the marginal zone with long
processes having the characteristics of Cajal-Retzius cells and small round
cells in the lateral portion. Cryostat section |
|
Fig. 5: |
NADPH-d histochemistry in the rat cells embryo at E19.
Here are also shown the cells without reaction. Cryostat section |
DISCUSSION
The first embryonic neurons to express nNOS in the diencephalic vesicle were those situated in the marginal zone. According to the studies of Bayer and Altman (1990) using autoradiography, tritiated thymidine, the Cajal-Retzius cells that are characteristic of this layer are generated at E13 and E14. Cajal-Retzius cells play a significant role throughout the development of the cerebral cortex. According to Marva-Padilla, every cell generated during the development of cerebral cortex established contact with Cajal-Retzius cells of the marginal zone. As maturation proceeds, only pyramidal neurons retain and expand their original connections with the marginal zone, while other neuronal types lose them. Ogawa et al. (1995) have shown that Cajal-Retzius cells express the protein reelin.
Only a few authors have studied nNOS or NADPH-d expression in Cajal-Retzius cells during embryonic development (Bayer and Altman, 1985; Bredt and Synder, 1994; Yan et al., 1996). These cells are seldom seen in mature animals. In the adult rat, layer I cells are not stained for nNOS (Brown, 2001), but others have recently shown nNOS-immunopositive neurons of Cajal-Retzius morphology can be found in layer I of aged rats (Uttenthal in press). These results suggest that the lack of nNOS expression by these cells of layer I is a prolonged, but transient feature of adult life (Florenzano and Guglielmitti, 2000).
In the present study, the intense histochemistry reaction and morphological characteristics of these cells persisted during the embryonic period and only began to decrease at E20, when neuronal migration is coming to an end. We observed that cells populating the marginal zone were abundant from E20 to the first postnatal stages.
Autoradiographic studies have shown that these cells are generated between E12 and E14; we have observed this phenomenon at E14 in the external part of the marginal zone. We suggest that NO may be involved in directing the ingrowing axons and in the migrational process.
During the development of the diencephalic vesicle, numerous afferent fibers reach the cortex from subcortical structures (Bayer and Altman, 1985; Bayer and Altman, 1991). These do not express choline acetyl transferase until the second postnatal week, where as NGF (Nerve Growth Factor) is expressed in the embryonic basal forebrain as early as E13 (Koh and Loy 1989; Weitzberg and Lundberg, 1998; Santacana et al., 1998).
Another important feature at E17 is the presence of migrating cells expressing nNOS in the intermediate zone. The expression of nNOS or NADPH-d and the role of NO cell migration are controversial issues. Some authors (Schilling et al., 1994) suggest that, in the cerebellum, cells express nNOS only when migration is completed. Studies on slice cultures of rat cerebellum (Tanakas et al., 1994; Riccio et al., 2006) have shown that granule cell migration is inhibited by N-nitro-L-arginin (L-NNA), indicating that NO was involved in cell migration and in the differentiation of granule cells.
In this study, the presence of migrating cells expressing nNOS in the intermediate zone of the diencephalic vesicle suggests that NO is involved in migration process.
From E18, a new embryonic layer is clearly observed in Nissl-stained sections, the supra ventricular zone. At the main source of neurogenesis, the ventricular zone, shrinks, many cells are reduced in the supra ventricular zone. This layer does not completely disappear after birth but continues to generate cells. It has been thought that the germinal cells in the supra ventricular zone produce only astrocytes and oligodentrocytes (Moncada et al., 1991). At E18, nNOS-reactive fibers were observed in the intermediate zone (Hanel and Hensey, 2006).
The present study de scribes the expression of nNOS in the diencephalic vesicle during embryonic development. Neuronal NOS expression may be considered to be correlated with the production of NO and may thus provide indirect evidence for a role of NO in development. During the embryonic stages, the role of NO is probably different from that in the adult. This is suggested by much greater expression of nNOS found in the cerebral cortex during the embryonic stages than in later life (Wang et al., 1994).
First of all, in the diencephalic vesicle, the neuronal elements expressing nNOS are much more abundant in embryonic stages than in neonates or adults (Bredt and Suyder, 1994; Giuili et al., 1994; Northington et al., 1996; Drever et al., 2004). In the adult nervous system, the role of NO in physiological conditions is the productoin a neurotransmitter or neuromodulator (Dawson and Dawson, 1996; Koliatsos, 2004). However, the large amount of nNOS in the embryonic diencephalic vesicle suggests an important role at that stage.
The results presented suggest that NO expression whenever there is an activity related to maturational processes.
ACKNOWLEDGMENTS
We would like to express our heartful thanks to Mr. Negahdar for his valuable guidance. Furthermore, we are grateful to Ms Hoseini for type processing and Mr. Gheibi and Mr. Zohrehvand for providing us with special facilities.
endothelial cells and macrophages by a family of three isoenzymes termed
nitric oxide synthases or NOS (Lopez-Costa
et al., 1997). nNOS
is present in neurons. It is constitutively expressed and its activity
is regulated by Ca
2+. Another constitutive and Ca
2+-dependent
type of NOS is present in endothelial cells (eNOS) of the vasculature.
Nitric oxide synthase can produce nitric oxide with the aid of NADPH
in response to changes in intracellular free calcium by deimidating arginine
to citrulline. The selective coexistence of citrulline-like immunoreactivity
in NADPH-diaphorase positive neurons is thus consistent with NADPH diaphorase
being a nitric oxide synthase (Geurges, 1999). Thus the extensive literature
on the histochemistry of NADPH diaphorase should be reexamined in light
of the fact that this simple histochemical technique allows the cellular
localization of nitric oxide synthase in the nervous system (Dawson et
al., 1991; Bruce, 1991).
In 1961, Thomas and Pearse first described the presence of large, isolated
neurons containing outstanding high NADPH-diaphorase activity which were
scattered throughout the cerebral cortex and basal ganglia (Schober et
al., 1994).
The aim of the present study was to clarify if the rat optic vesicle
cells using the NADPH-d as an indicator to the NOS-histochemistry (Rangarajan
et al., 1999). Effect of NOS expression on optic vesicle development
is very important on assessment abnormalities.
MATERIALS AND METHODS
In this study, 11 young adult female Spargue-Dawley rats were used.
The rats were mated and the gestational age was estimated according to
the day of vaginal plaque formation, to mark the first day of life. Fetuses
aged 8-18 days of gestation were obtained, decapitated and washed in solution
consisting of 0.1 M Phosphate Buffer Saline (PBS) in pH = 7.4. The heads
were transformed into fixative for 24 h; The fixative contained fresh
solution of 4% paraformaldehyde in 0.1 M PBS, pH = 7.4, at 4C. Subsequently
the heads were kept in solution consisting of 15% sucrose in PBS for 1
h and then in 30% sucrose at 4C for 2 h. They were then frozen and transverse
and longitudinal sections to 10 μM were cut on a cryostat. Sections
were collected on gelatin-coated slides. They were later rinsed in 0.1
M PBS (pH = 8.0) for 10 min. NADPH-diaphorase histochemistry was performed
by incubating the sections in a solution consisting of 30 μM
malic acid, 1 μM manganese hydrochloride, 0.2 μM nitro blue
tetrazolium chloride, 1 μM NADPH, 0.5% Triton X-100 in buffer of
Tris-HCl in pH 7.4, at 37C, for 90 min in a dark place. Sections were
mounted in entellan and photographs were taken.
RESULTS
At E13, the diencephalic vesicle is composed of only
two layers: the ventricular zone, where all neurons are generated and
the marginal zone, where the first-generated neurons are setting (Fig.
1).
NADPH-d histochemistry was not observed in the diencephalic vesicle at
this stage in either transverse or longitudinal sections.
At E14, the diencephalic vesicle is greatly enlarged and increased
in thickness, mainly due to growth of the ventricular zone (Fig.
2). The marginal zone is also thicker. An incipient intermediate zone
between the ventricular zone and marginal zone is observed. The whole
marginal zone of the diencephalic vesicle was populated by NOS-positive
cells. There were two chief types of NADPH-d histochemistry cells (Fig.
3), large cells in the middle part of marginal zone with long tangentially
oriented processes, which showed the morphological characteristics of
Cajal-Retzius cells and externally situated round cells with processes
oriented upward.
At E16, the diencephalic vesicle has four layers: the ventricular
zone, which occupies roughly one-third of the thickness, the intermediate
zone, with easily visible fibers coursing along it, marginal zone and
the cortical plate and more prominent in the lateral than in the dorsal
aspect of the diencephalic vesicle.
At E18, NADPH-d reactivity is observed at low magnification
showed five clearly defined layers (Fig. 4).
 |
Fig. 1: |
Frontal section through diencephalen. Rat embryo at
E13, H and E staining |
The marginal zone is densely labeled and the underlying cortical plate
showed a band of stained cells in which nearly every cell was stained,
so that it was difficult to identify single cells.
In the late stage, the most notable feature was a decrease in the histochemical
reaction of the marginal zone. Horizontally coursing NADPH-d histochemical
activity fibers could also be observed in the marginal zone. Also NADPH-d
reactivity was observed in
Fig. 2: |
Frontal section through the optic rat embryo at E14 optic
vesicle Viewed at higher magnification. At this stage, the diencephalic
vesicle continues to grow and its thickness increases. The marginal
zone has been also thicker and because of growth of the ventricular
zone, the intermediate zone is more prominent. Cryostat section |
Fig. 3: |
Frontal section showing the diencephalic vesicle of
a rat embryo at E14 at high magnification. The picture
shows two layers of marginal zone and ventricular zone. There are
NOS-positive round cells in the marginal zone. The nerve fibers are
also stained. At the ventricular zone, only nerve fibers are weakly
stained. Cryostat section |
Fig. 4: |
Photomicrograph showing the marginal zone rat embryo
at E18Staining is observed in a group of highly active
cells. There are two kinds of NOS-positive cells: big chief cells
in the middle part of the marginal zone with long processes having
the characteristics of Cajal-Retzius cells and small round cells in
the lateral portion. Cryostat section |
 |
Fig. 5: |
NADPH-d histochemistry in the rat cells embryo at E19.
Here are also shown the cells without reaction. Cryostat section |
endothelial cells lining the larger blood vessels, but it was not present
in endothelial cells of sinusoidal capillaries or the small vessels (Fig.
5).
DISCUSSION
The first embryonic neurons to express nNOS in the diencephalic
vesicle were those situated in the marginal zone. According to the studies
of Bayer and Altman (1990) using autoradiography, tritiated
thymidine, the Cajal-Retzius cells that are characteristic of this layer
are generated at E13 and E14. Cajal-Retzius cells
play a significant role throughout the development of the cerebral cortex.
According to Marva-Padilla, every cell generated during the development
of cerebral cortex established contact with Cajal-Retzius cells of the
marginal zone. As maturation proceeds, only pyramidal neurons retain and
expand their original connections with the marginal zone, while other
neuronal types lose them. Ogawa et al. (1995) have shown
that Cajal-Retzius cells express the protein reelin.
Only a few authors have studied nNOS or NADPH-d expression in Cajal-Retzius
cells during embryonic development (Bayer and Altman, 1985; Bredt and
Synder, 1994; Yan et al., 1996). These cells are seldom seen in
mature animals. In the adult rat, layer I cells are not stained for nNOS
(Brown, 2001), but others have recently shown nNOS-immunopositive neurons
of Cajal-Retzius morphology can be found in layer I of aged rats (Uttenthal
in press). These results suggest that the lack of nNOS expression by these
cells of layer I is a prolonged, but transient feature of adult life (Florenzano
and Guglielmitti, 2000).
In the present study, the intense histochemistry reaction and morphological
characteristics of these cells persisted during the embryonic period and
only began to decrease at E20, when neuronal migration is coming
to an end. We observed that cells populating the marginal zone were abundant
from E20 to the first postnatal stages.
Autoradiographic studies have shown that these cells are generated between
E12 and E14; we have observed this phenomenon at
E14 in the external part of the marginal zone. We suggest that
NO may be involved in directing the ingrowing axons and in the migrational
process.
During the development of the diencephalic vesicle, numerous afferent
fibers reach the cortex from subcortical structures (Bayer and Altman,
1985; Bayer and Altman, 1991). These do not express choline acetyl transferase
until the second postnatal week, where as NGF (Nerve Growth Factor) is
expressed in the embryonic basal forebrain as early as E13 (Koh
and Loy 1989; Weitzberg and Lundberg, 1998; Santacana et al., 1998).
Another important feature at E17 is the presence of migrating
cells expressing nNOS in the intermediate zone. The expression of nNOS
or NADPH-d and the role of NO cell migration are controversial issues.
Some authors (Schilling et al., 1994) suggest that,
in the cerebellum, cells express nNOS only when migration is completed.
Studies on slice cultures of rat cerebellum (Tanakas et al., 1994;
Riccio et al., 2006) have shown that granule cell migration
is inhibited by N-nitro-L-arginin (L-NNA), indicating that NO was involved
in cell migration and in the differentiation of granule cells.
In this study, the presence of migrating cells expressing nNOS in the
intermediate zone of the diencephalic vesicle suggests that NO is involved
in migration process.
From E18, a new embryonic layer is clearly observed in Nissl-stained
sections, the supra ventricular zone. At the main source of neurogenesis,
the ventricular zone, shrinks, many cells are reduced in the supra ventricular
zone. This layer does not completely disappear after birth but continues
to generate cells. It has been thought that the germinal cells in the
supra ventricular zone produce only astrocytes and oligodentrocytes (Moncada
et al., 1991). At E18, nNOS-reactive fibers were observed
in the intermediate zone (Hanel and Hensey, 2006).
The present study de scribes the expression of nNOS in the diencephalic
vesicle during embryonic development. Neuronal NOS expression may be considered
to be correlated with the production of NO and may thus provide indirect
evidence for a role of NO in development. During the embryonic stages,
the role of NO is probably different from that in the adult. This is suggested
by much greater expression of nNOS found in the cerebral cortex during
the embryonic stages than in later life (Wang et al., 1994).
First of all, in the diencephalic vesicle, the neuronal elements expressing
nNOS are much more abundant in embryonic stages than in neonates or adults
(Bredt and Suyder, 1994; Giuili et al., 1994; Northington et
al., 1996; Drever et al., 2004). In the adult nervous system,
the role of NO in physiological conditions is the productoin a neurotransmitter
or neuromodulator (Dawson and Dawson, 1996; Koliatsos, 2004). However,
the large amount of nNOS in the embryonic diencephalic vesicle suggests
an important role at that stage.
The results presented suggest that NO expression whenever there is an
activity related to maturational processes.
ACKNOWLEDGMENTS
We would like to express our heartful thanks to Mr. Negahdar for
his valuable guidance. Furthermore, we are grateful to Ms Hoseini for
type processing and Mr. Gheibi and Mr. Zohrehvand for providing us with
special facilities.