SEARCH

SEARCH BY CITATION

Keywords:

  • neuronal nitric oxide synthase;
  • brain;
  • mouse;
  • embryogenesis;
  • development;
  • immunofluorescent

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The distribution of neuronal nitric oxide synthase (nNOS) in the process of normal mouse brain growth from embryonic (E) Day 11 to postnatal (P) Day 1 was investigated by means of immunohistochemical and immunofluorescent methods. Our results demonstrated that nNOS positive neurons appeared early in superficial cortex at E11. At E13, nNOS positive neurons were located in lateral hypothalamus and amygdala, and temporarily in medullar and ventral hypothalamic neuroepithelia. From E15 to P0, nNOS positive neurons were distributed in superior and inferior colliculi, positive staining could also be seen in superior and inferior tectal neuroepithelium at E15. From E17 to birth, the medial geniculate nucleus had a high density of nNOS labeling. The distribution of nNOS gradually increased and extended laterally in embryo brain, which in turn implies that NO might be involved in the development of mouse brain. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Nitric oxide (NO) was first described about two decades ago as an important regulator of vasodilation (Moncada, 1992; Han et al., 2010). Subsequently, it has also been implicated in a range of other physiological processes, including a role to modulate neurotransmission and the immune system (Bredt and Snyder, 1994a; Vincent, 2010). NO is produced by nitric oxide synthase (NOS), via the conversion of L-arginine to citrulline. There are three isoforms of NOS and they have a common large heme moiety and binding sites for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) (Nathan and Xie, 1994; Alderton et al., 2001b; Vincent, 2010). The isoforms are named according to the tissues in which they were first identified: endothelial NOS (eNOS), neuronal NOS (nNOS), and the macrophage inducible form (iNOS) (Alderton et al., 2001b).

NO functions as a unique gaseous modulator for neurotransmission, plasticity, and cytotoxicity in adult animals (Bidmon et al., 1997; Nakajima et al., 1998; Scott, 1999; Pisu et al., 2002). NO also appears to play a role in development by regulating synapse formation and patterning (Hindley et al., 1997; Mogi et al., 2007; Scala et al., 2007; Vincent, 2010). Recent studies indicate that NO contributes to triggering a switch from cellular proliferation to differentiation during neurogenesis (Gibbs, 2003; Yuan et al., 2006; Vincent, 2010). Thus, the expression of nNOS in the mouse basolateral amygdalar complex (BLC) from embryonic Day 15.5 to adult has been studied (Olmos et al., 2005). It appears that each nucleus of the complex displays a distinct nNOS expression pattern, which is established during ontogenesis, with minor changes in the adult (Guirado et al., 2003; Guirado et al., 2008; Lu et al., 2010). nNOS and GABA are colocalized in the mouse claustrum, and nNOS expressing neurons in the BLC are both GABAergic and non-GABAergic (Suarez et al., 2005).

nNOS signaling regulates diverse cellular processes during brain development and molecular mechanisms required for higher brain function, has also been associated with prefrontal cortical functioning, including cognition; nNOS knockout mice exhibit impairments in contextual fear conditioning (Zoubovsky et al., 2011).

It is known that the NADPH diaphorase activity of NOS remains active after tissue fixation, so extensive methods have been used to histochemically visualize the location of NOS in the nervous system(Cao and Deng, 1999; Lackova et al., 2006; Schlechtweg et al., 2009). The distribution of nNOS in the adult brain has been well documented, with recent interest focusing on the substantia nigra, striatum and frontal cortex (Abe et al., 2010; Eto et al., 2010).

However, the distribution of nNOS in the embryonic brain is poorly understood (Hu et al., 2011). In this study#, therefore, we attempted to highlight the distribution patterns of nNOS positive neurons in the process of brain growth during embryonic development.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Animals

Thirty-five pregnant ICR mice were obtained from the Laboratory Animal Services Center of the Zhejiang University, and housed under a 12-hr light/dark cycle with freely available food and water. Mice were sacrificed at ages from embryonic Days (E) 11, 13, 15, 17, and 19, postnatal Day 0 (P0, the day of birth), and postnatal Day 1 (P1), N = 5. The brains of fetal mice of both sexes were processed for the analysis of nNOS expression. Animal care and experimental procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and conformed to the requirements of the animal ethics committee of Zhejiang University.

Tissue Preparation

Pregnant mice at E11-19 were injected with 7% chloralhydrate (35 mg/100g body weight) intraperitoneally, and the embryos dissected free and collected. The embryos at E19, the litters at P0 and P1 were perfused transcardially with 0.85% normal saline solution (30 mL) followed by 4% paraformaldehyde in 0.01 M phosphate buffer (PBS)(pH 7.4, 100 mL). The brains were dissected out and postfixed in the same fixative at 4°C for 2–3 hr. Afterward tissues were cryoprotected in 30% sucrose in 0.01 M PBS overnight and were embedded in OCT compound after frozen in isopentane chilled in liquid nitrogen. Coronal sections (30 μm in thickness) were obtained using a cryostat. The embryos collected before E17 were not subjected to fixation via cardiac perfusion. Instead, the brains were simply removed and fixed in the same fixative at 4°C for 10 hr.

Immunohistochemistry and Immunofluorescent Labeling

Brain slices were stained according to the immuno-ABC-technique (Han et al., 2010). Sections were rinsed thrice for 10 min in 0.01 M PBS (pH 7.4), preincubated for 20 min with 0.3% H2O2 in 0.01 M PBS, rinsed by PBS, then preincubated with 10% normal goat serum (NGS) 1 hr, and incubated overnight at 4°C with rabbit anti-nNOS polyclonal antibody (Santa Cruz sc-648, CA) in 0.01M PBS with 1% NGS and 0.3% Triton X-100. After rinsing, sections were incubated for 3 hr at 4°C with secondary goat anti-rabbit immunoglobulin G (Vectastain ABC kit; Vector Laboratories, Burlingame, CA), rinsed in PBS, incubated for 2 hr with tertiary antibody (avidin DH/biotinylated horseradish peroxidase H complex, Vectastain ABC kit). A peroxidase reaction was performed to visualize nNOS immunolabeling by incubating with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB) and 0.01% H2O2 for 5 min at room temperature. After rinsing, sections were mounted on gelatin-coated glass slides, dehydrated in ethanol, cleared in xylol, and coverslipped.

FITC-conjugated goat-anti-rabbit secondary antibody (sc-2012 Santa Cruz, CA) was used instead of biotinylated secondary antibody, then sections were coverslipped in antifade medium (Invitrogen) and examined under NIKON fluorescence microscope (Nikon E600, Japan). To test the specificity of nNOS antibody, a blocking peptide (Santa Cruz, sc-648p) was purchased. We combined the nNOS antibody with a fivefold (by weight) excess of blocking peptide in 0.01 M PBS, and incubated with this antibody/peptide mixture overnight at 4°C, no significant staining was observed in this case.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The distribution and relative intensity of nNOS positive neurons in the developing brain are shown in Table 1. Typically stained positive nNOS neurons are shown in Figs. 1–4.

thumbnail image

Figure 1. A: Immunoflourescent staining, note the nNOS-immunopositive neurons in E11 superficial cortex (arrow pointing to a neuron labeled), two times amplificatory image was inserted. LV, lateral ventricle. B: nNOS labeled neurons in E13 Neocortex (C3). C: nNOS labeled neurons in E13 Ventral hypothalamic neuroepithelium (H1v), 3V: 3nd ventricle. D: nNOS labeled neurons in E13 anterior hypothalamic area. E, F: nNOS labeled neurons in E13 Medullary neuroepithelium (Yl). Scale bar = 100 μm.

Download figure to PowerPoint

Table 1. Distribution and relative intensitya of nNOS positive neurons in the developing mouse brain
 E13E15E17E19P0
  • a

    The intensity of staining was divided into four grades based on the number of positive neurons in one HP (40×): 0–19: +, 20–39: ++, 40–59: +++, > 59: ++++.

Cortex     
Rhinencephalon (R3) ++   
Olfactory tubercle (Tu)   + 
Entorhinal cortex (Ent)    Terminus
Anterior olfactory nucs (AO)   +Terminus
Neocortex (C3)+++++  
Neocortical subventricular zone (C2)  ++  
Cortical subplate (CxS)   + 
Frontal cortex (Fr)    +
Parietal cortex, area 2 (Par2)   ++
Occipital cortex (Oc)   ++ 
Amygdala (A3)+++++++  
Anterior cortical amygdaloid nuc (Aco)   ++ 
Anterior amygdaloid nuc (AAD)   ++ 
Medial amygdaloid nuc (Me)   +++
Basomedial amygdaloid nuc (BM)   ++ 
Basomedial amygdaloid nuc,anterior par (BMA)    ++
Basolateral amygdaloid nuc (BL)   + 
Posterolateral cortex amygdaloid nuc (PLCo)   ++ 
Posteromedial cortex amygdaloid nuc (PMCo)  TerminusTerminusTerminus
Ventrobasal nuc complex (T28) ++   
Caudate putamen (Cpu)   ++++
Striatum (B3)  ++  
Globus pallidus (GP)   ++
Dorsal endopiriform nuc (Den)    ++
Septum (Spt)   + 
Lateral septum (S5)  ++  
External capsule (ec)    Fibers
Diencephalon     
Suprachiasmatic nuc (SCh)    +
Thalamic neuroepithelium (T1) ++  
Ventral hypothalamic neuroepithelium (H1v)++    
Anterior hypothalamic neuroepithelium (H1a) ++   
Anterior hypothalamic area, anterior par(AHA)   + 
Lateroanterior hypothalamic nuc (LA)   + 
Lateral hypothalamic area (LH)+++++++++++
Ventral hypothalamus (H3v) ++   
Anterior thalamus (T3a)  +  
Centrolateral thalamic nuc (CL)   + 
Laterodorsal thalamic nuc (LD)   + 
Mediodorsal thalamic nuc(MD)   ++
Lateral posterior thalamic nuc (LP)   ++++
Paraventricular thalamic nuc (PV)  + ++
Subthalamic nuc (STh)   + 
Medial geniculate nuc (MG)  ++++++++++
Peripeduncular nuc (PP) +++  ++
Paracentral thalamic nuc (PC)   +++
Parafascicular thalamic nuc (PF)   + 
Lateral habenular nuc (LHb)  ++++
Medial habenular nuc (MHb)    +
Medial tuberal nuc (Mtu)   ++ 
Anterodorsal nuc (H24)  +  
Rhomboid nuc (T17)  +  
Ventrolateral nuc complex (T27)  ++  
Subcommissural organ (SCO)    +
Mesencephalon     
Superior colliculus (SC) ++++++++++++
Inferior colliculus (IC) +++++++++
Intercollicular nuc (InCo)   ++ 
Superior tectal neuroepithelium (M1) ++   
inferior tectal neuroepithelium (M31) +++   
Anterior pretectal nuc (M24)  ++++  
Central (periaqueductal) gray (CG)   +++++++
Deep mesencephalic nuc (DpMe)    +
Midbrain reticular formation (M9) ++++++  
Red nucleus, magnocellular par (RMC)   ++++++
Median raphe nuc (MnR)  +++++++
Raphe magnus nuc (RMg)    +
Superior central raphe nuc (X8)  ++++  
Dorsal raphe nuc (DR)   ++++++
Venteal tegmental area (VTA)    ++
Paranigral nuc (PN)    +
Paralemniscal nuc (PL)    +++
Ventral periolivary nuc (VPO)   + 
Pons     
Pontine nuc (Pn)++   ++
Anterior pons (X3a) ++   
Posterior pontine neuroepithelium (Xlp) ++++++  
Central gray of the pons (CGPn)   ++ 
Pontine reticular nuc, caudal part (PnC)   ++++
Nucleus of the trapezoid body (Tz)    ++
Parabrachial nuc (PB)   + 
Pedunculopontine tegmental nuc (PPTg)   ++++++
Medulla     
Prepositus nuc (Y7)  ++  
Medulla (Y3)++    
Medullary neuroepithelium (Yl)++ ++  
Medulla raphe (Y5) ++++++++
Medullary reticular formation (Y6) ++++  
Medulla tegmentum (M13) +++++  
Superior olivary nuc (X24) +   
Inferior olive (Y8)  +++  
Solitary nuc (Y16) ++   
Lateral reticular nuc (LRN)    ++
Gigantocellular reticular nuc (Gi)    +++
Cerebellum     
Inferior cerebellar peduncle (icp)   Terminus 
Cerebellar hemisphere (L3h)  ++ ++

nNOS positive neurons first appeared in the superficial cortex at E11 (Fig. 1A). At E13 they were expressed mainly in lateral hypothalamus neocortex (Fig. 1B) and amygdala, and they temporarily appeared in ventral hypothalamic neuroepithelium (Fig. 1C,D) and medullar neuroepithelium (Fig. 1E,F). The cells had short dendrites and connected with each other. nNOS positive neurons also appeared in the superior and inferior colliculi from E15 to P0. Positive neurons also could be seen in the superior and inferior tectal neuroepithelium at E15 (Fig. 2A,B), and posterior pontine neuroepithelium. Fiber networks comprising dendrites were seen in the midbrain reticular formation (Fig. 2C,D). The medial geniculate nucleus expressed nNOS strongly from E17 to birth. However, nNOS positive neurons appeared transiently in the superior central raphe nucleus, anterior pretectal nucleus (Figs. 2E,F and 3A,B), and inferior olive at E17. The intense staining in the medullary reticular formation was moderate with multiangular cell bodies. The paraventricular thalamic nucleus, lateral habenular nucleus, and cerebella also began to exhibit nNOS positive neurons. At the same time, nNOS positive terminuses could be seen in amygdale (Fig. 3C).

thumbnail image

Figure 2. Fluorescent micrographs of nNOS-immunopositive neurons (arrows) in E15 Superior tectal neuroepithelium (M1) (A), Tegmentum (B), brain reticular formation(M9) (C, D). E: nNOS labeled neurons in E17 Anterior pretectal nuc (M24) (a), Medial geniculate nuc (MG) (b), Lateral habenular nuc (LHb) (c), and Thalamic neuroepithelium (T1) (d). F: nNOS labeled neurons in E17 Anterior pretectal nuc (M24).

Download figure to PowerPoint

thumbnail image

Figure 3. A: The nNOS-immunoreactive neurons in E17 Anterior pretectal nuc(M24) (a), Medial geniculate nuc (MG) (b), Lateral habenular nuc (LHb) (c), and Thalamic neuroepithelium (T1) (d). B: E17 Anterior pretectal nuc(M24). C, E17 Terminus of amygdala. D: E17 Neocortical subventricular zone (C2) and Cajal–Retzius cells. E: E19 Raphe magnus nuc (RMg). F: E19 Raphe magnus nuc (RMg), immuflourencent staining.

Download figure to PowerPoint

The expression of nNOS in neocortical subventrical zone (C2) was stronger than neocortex (C3) at E17, and C2 was densely labeled, and the underlying structure showed a band of immunostained cells in which nearly every cell was stained, so that it was difficult to identify single cells. On the other hand, cells which showed the morphological characteristics of Cajal–Retzius cells (Fig. 3D) externally situated round cells with processes oriented upwardly, could be observed. In contrast to the situation at E13, in which the whole C3 was populated by nNOS-positive cells, the migration of nNOS neurons showed a trend from the superficial cortex to the deep-seated brain. At E19, positive neurons no longer presented in neocortex, but appeared in other regions including the central gray, pedunculopontine tegmental nucleus, and peripeduncular nucleus. The cells in raphe magnus nucleus were stained strongly with long dendrites which connected with lateral cells (Fig. 3E,F). Along with the development of the nuclei, the expressions of nNOS positive neurons in hypothalamus and amygdala were more extensive, but the density of cells had no change. nNOS positive neurons distributed broadly at P0 in areas such as magnocellular part of red nucleus, dorsal raphe nucleus, paralemniscal nucleus, and gigantocellular reticular nucleus. In the central (periaqueductal) gray (CG) the cell bodies were enlarged and arranged from the aqueduct to peripheral zone (Fig. 4A). Besides, positive neurons showed a tendency to extend from medulla raphe to lateral reticular nucleus (LRN) through gigantocellular reticular (Gi) nucleus. There were positive fibers in the internal capsule, and many termini presented in amygdala. The distribution of nNOS-immunofluorescence appeared similar to the distribution of nNOS-immunohistochemistry as described above, but more positive fibers and terminuses could be visualized (Fig. 4B). Before E15, there were cells with rotundate or olivary bodies, short dendrites, and less ramification. At the later stage, the cells showed varying morphological features: some were bipolar while others were multipolar, but they had acquired larger sizes, with more dendrites that were like beads (Fig. 4C–F).

thumbnail image

Figure 4. A: Distribution of nNOS-immunoreactivity in P0 Central (periaqueductal) gray (CG) (a), Median raphe nuc (MnR) (b), Pedunculopontine tegmental nuc (PPTg)(c), and Pontine reticular nuc and caudal part (PnC) (d). B: Note the nNOS-immunopositive neurons in P0 Medulla raphe (Y5). C: Showing nNOS-immunopositive neurons in E19 Caudate putamen (Cpu). D: E19 cell with dendrites-like beads. E, F: P0 cell with dendrites-like beads.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

There has been much interest in the potential modular action of NO in the central nervous system (CNS). Many studies demonstrated that neurons in central nervous system contain NOS (Ng et al., 1999; Yu et al., 2000; Han et al., 2010; Vincent, 2010). NO not only participates in the regulation of neurotransmission in the central nervous system (Kiss, 2000), but also takes part in the migration of neurons (Ernst et al., 1999), suggesting that NO is related to the development of the nervous system.

Most studies focused on the adult CNS, with NOS being used as a marker to delineate pathways (Downen et al., 1999). This study focused on embryogenesis and revealed that nNOS neurons were distributed in a unique manner in the brain. Our results demonstrated that nNOS positive neurons appeared first in the superficial cortex at E11, and the number and the location of nNOS positive neurons increased during the development of the brain. Our results also revealed that the expressions of nNOS in the embryonic and adult brain are distinctly different, and that the distributions and general appearance of cells and fibers expressing nNOS are also unique.

Cortex

In the developing mouse brain, nNOS is expressed abundantly but temporarily. There is discrepancy between our findings and those in the study by Bredt and Snyder (Bredt and Snyder, 1994a), which showed the occurrence of transient NOS expression in the cerebral cortical plate at E15-E19, then decrease after birth in rat brain. They also revealed prominent nNOS staining in the cortical plate at E15, E17, and E19, whereas no positive staining in either the ventricular zone or the cortical subplate. In our study, nNOS neurons were detected first at E11, and afterwards in the cortical subplate, whereas there was no expression of nNOS in the neocortex at E19. A similar pattern for nNOS distribution has been reported using immunocytochemistry within rat cerebral cortex from E13 to P0 (Santacana et al., 1998). It is reasonable that the discrepancy might come from the difference in animal species, antibody specificity to NOS, as well as different immunohistochemical procedures used.

Cajal–Retzius cells play a significant role throughout the development of the cerebral cortex. We found nNOS was located in Cajal–Retzius cells in C3 at E13, in C2 and C3 at E15 and E17, and in the cortical subplate at E19. Interestingly, evidence showed that the intense immunostaining and morphological characteristics of Cajal–Retzius cells persisted during the embryonic period and only began to decrease at E20, when neuronal migration was coming to an end in the cortex (Santacana et al., 1998). According to Marin-Padilla (Marin-Padilla, 1992), every cell generated during the development of the cerebral cortex establishes contact with Cajal–Retzius cells of the marginal zone (MZ). Considering the role of these neurons during development, the massive expression of nNOS in C3, and its coincidence with the duration of the migrational process, we suggested that NO might be involved in directing the growing axons. This is in agreement with the hypothesis that NO plays a role in the development of neurons in the brain because nNOS positive neurons appeared earlier, and distribute more extensively compared with the neurons containing other neurotransmitters (Alderton et al., 2001a).

The migration, axonal outgrowth, and differentiation of neurons and the formation of neuronal connectivity are related to the adhesion of some cell adhesion molecules such as PSA-NCAM (polysialylated neural cell adhesion, found on the surface of regrowing axons) and cadherin, which could affect neuronal function by modulating cascades of the second-messenger systems (Aubert et al., 1995). In the striatum, NOS has been reported to first appear at E16 (Murata and Masuko, 2003), and in this study, we detected nNOS at E15. Indeed, the distribution gradually extended and the neurons became mature as the brain developed; the neurons became larger and had a greater number of dendrites that were like beads. On the other hand, the first appearance of nNOS in the lateral Caudate putamen (CPU) extended to the whole striatum gradually, but the ventral lateral had the most intense staining, suggesting that the neurons migrated from the lateral to medial in the striatum.

In mature animals, somatostatin (STT), neuropeptide Y (NPY), and NOS coexist in the striatum (Vincent et al., 1983). At the embryo stage, the migration of STT and NPY in striatum is from lateral to medial (Semba et al., 1988), and similar to the spread of nNOS containing neurons. On the other hand, STT and NPY are also noted as contributing to brain development (Woodside and Amir, 1996), and the similar migration pattern observed for nNOS, STT, and NPY. This suggests that the development of the embryo brain and nNOS is linked with STT and NPY.

Diencephalon

Our study provided a comprehensive and detailed description of the distribution of nNOS containing neurons in the developing thalamus of the mouse embryo. We highlighted a remarkable selectivity in the thalamic structures that may release NO and we suggested that NO could play a multifaceted role in thalamic neurotransmission and/or neuromodulation during development.

The expression of nNOS in different thalamic domains appeared to follow a discrete developmental schedule starting at E13. The most dramatic change occurred between E15 and E17, with the distribution of nNOS activity becoming progressively more intense and widespread. Thus, the histochemical positivity of the neurons increased and peaked at birth. Interestingly, when the expression of nNOS was investigated by means of NADPH-diaphorase histochemistry, the peak population of neuronal cells stained was found around the beginning of the second postnatal week and then declined (Bertini and Bentivoglio, 1997). Yet naturally occurring cell death is minimal in the rat thalamus, which peaks at P5 (Spreafico et al., 1995), thus perhaps facilitating the transient nNOS expression in thalamic neurons during embryogenesis up until the second postnatal week. We would like to point out that the innervation of the thalamus is a relatively early event in development, so that fibers may reach their targets even before thalamic neurons are set in place (Jones, 1985). In this context, it is worth stressing that the fibers innervating the anterior thalamic nuclei were devoid of nNOS staining.

Positive staining for NADPH-diaphorase in the paraventricular (PVN) and supraoptic nuclei (SON) implicates a possible role of NO in reproduction and general mechanisms of homeostasis (Woodside and Amir, 1996). Furthermore, NO was shown to inhibit the stimulated release of vasopressin (AVP) from rat hypothalamic explants in vitro, providing further evidence that NO is involved in neuroendocrine regulation (Yasin et al., 1993). In this study, nNOS was detected in the PVN at E17, and increase until P0. On the other hand, it was revealed that NOS only appeared in the SON after birth (Giuili et al., 1994). Therefore, the NO/cGMP system may be controlled by the PVN during embryogenesis, and then by both the PVN and SON after birth.

While it seems likely that neurons in the thalamus may release NO within a relatively narrow time window the developmental schedule of nNOS expression in thalamic inputs indicates that NO could participate in the refinement of connections in thalamus. A similar situation could also occur in the hypothalamus. The NOS positive neurons in rat hypothalamus were already present on the first postnatal day, implied that the phenotypic genesis of rat NOS might have occurred during embryonic development (Torres et al., 1993). Our study confirmed the hypothesis in that nNOS cells appeared first in the ventral hypothalamic neuroepithelium (H1v) at E13 and then migrated towards the lateral direction by P0. In general, the neuronal structure of rat hypothalamus has been reported to migrate from medial to lateral directions (Altman and Bayer, 1978).

Brainstem

This study demonstrated that only a few nNOS cells were present in brainstem at E13. The positive cells were mostly small in size, and were distributed predominantly in the pons. By P0, nNOS cells increased greatly in both size and number. They were found in a number of areas, including the superior colliculus, inferior colliculus, central (periaqueductal) gray, red nucleus magnocellular par, median raphe nucleus, dorsal raphe nucleus, lateral reticular nucleus, gigantocellular reticular nucleus, and pedunculopontine tegmental nucleus, and so on.

In the adult rat brain, NO has been hypothesized to have a variety of functions, depending upon its localization (Vincent, 2010). The distribution of intense nNOS staining found in Medial geniculate (MG) and reticular system from E15 to P0, suggested that NO might play a role in the development of auditory and arousal systems during embryogenesis. Therefore, NO might also be involved in the migration of visual fibers and neurons in the embryo as well in the development of pain pathways since nNOS was also detected in central (periaqueductal) gray, central gray of the pons, dorsal raphe nucleus, lateral habenular nucleus, medial habenular nucleus, and so on. NOS expressing cells have been known to coexist with various neurotransmitters, so NO probably contributes to the regulation of many neurotransmitter systems. Indeed, most of the results found in the present study were consistent with those studies with adult rat brain (Bredt et al., 1991; Vincent and Kimura, 1992; Bredt and Snyder, 1994b; Rodrigo et al., 1994).

nNOS Positive Ependymal Cells

In our study, nNOS could be detected in ependymal cells at E13, E15, E17, with mild intense, rotundate or olivary bodies and dendrites connecting with each other and spreading from ependyma to lateral. It has been proved that ependymal cells are neural stem cells, giving rise to rapidly proliferating cell types that generate neurons migrated to the olfactory bulb (Johansson et al., 1999). A discrete region of the anterior part of the subventricular zone (SVZa) generated an intense number of neurons that differentiated into granule cells and peri-glomerular cells of the olfactory bulb (the two major types of interneurons, and the SVZa appears to constitute a specialized source of neuronal progenitor cells). Certainly, neurons arising in the SVZa would migrate a distance of several millimeters to reach the olfactory bulb (Luskin, 1993; Lois and Alvarez-Buylla, 1994). On the other hand, we found nNOS positive neurons also distributed in the superficial cortex at the early stage of development (E13, E15), and the density appeared more intense than the ependyma. It may be valuable to study whether the migration of nNOS positive neurons is only from the ependymal cells or not.

The Role of NO in the Development of Embryo Brain

The unique distribution model of nNOS positive neurons in the mice brain during embryogenesis suggests that NO not only takes part in the maturation of the brain, and refinement of the connections, it may also fundamentally relate to important events during embryogenesis such as differentiation (Ogura et al., 1996; Hu et al., 2011) and programmed cell death (PCD).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

The authors thank Henry Davies for assistance in the final preparation of this manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
AR_22408_sm_SuppFig1.tif4726KSupporting Information Figure 1.
AR_22408_sm_SuppFig2.tif4433KSupporting Information Figure 2.
AR_22408_sm_SuppFig3.tif6842KSupporting Information Figure 3.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.