Neural cell adhesion molecule (NCAM) and polysialic acid–NCAM expression in developing ICR mice

In the present study, fetal (E17) and postnatal mice (P3, P5, and P7) showed a normal external appearance (Figure 1) with normal sensory and motor function. Normal growth development is indicated by measurements as shown and compared in Table 1. Cortical thickness, brain weight, body weight, and body length were compared between P3, P5, and P7 and increased significantly at P < 0.05 when compared with P3. In addition, as shown in Figure 1, normal brain development was indicated by the 6 cortical cell layers in all groups of mice. Progression of the neocortex shown by NCAM immunoperoxidase staining with hematoxylin-counterstained coronal brain sections demonstrated 6 cortical cell layers starting from E17 to P7. In E17, cortical neurons still aggregate and separate into layers, but in postnatal brains P3, P5, and P7 the cortical neurons migrate to specific targets and then they show overlapped layers. NCAM immunoreactivity is also shown in Figure 1; the intensity increased gradually from E17 to P7 and entirely stained over all layers in the cortex of P7 mice.

The localization of NCAM and PSA–NCAM immunoreactivity in coronal sections through the SGZ of hippocampus is shown and indicated the subfields of the dentate gyrus (DG): po layer, stratum granulosum (gr), and stratum moleculare (mo).

In Figure 2A, NCAM immunoreactivity is localized to the surface of neurons and glia and is more intense in postnatal mice than it is in embryonic mice. In Figure 2B, PSA– NCAM is localized in cytoplasm and membrane of neural cells. In Figure 3, PSA–NCAM immunoperoxidase staining with or without hematoxylin counterstaining is demonstrated. PSA–NCAM immunoreactive neural cells are localized in the 3 layers of dentate and in the lacunosum moleculare (lm) and radiatum (ra) layers of the cornu ammonis 1 area (CA1) of the hippocampus, and the higher magnification in the po layer of the dentate showed PSA–NCAM immunoreactivity in the cytoplasm and nucleus of neural cells. In Figure 4, the localization of NCAM/PSA–NCAM immunoreactivity in SGZ was compared between groups, intense PSA–NCAM staining is seen in the SGZ of dentate in P7, but no or less staining found in E17. Results from image analysis seen in Figure 5 indicated a gradual increase in NCAM and PSA–NCAM immunoreactivity from E17 to P7; the immunoreactivity was significantly higher when compared with that at E17. The western blot analysis showed similar results, and the NCAM and PSA–NCAM immunoreactivity normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was significantly greater in P5 and P7 when compared with P3 as shown in Figure 6. The result of E17 was not included in Western blot analysis, because whole body of E17 was collected.

Figure 3

Figure 4

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Figure 6

The present study found all parameters measured indicated normal ICR mice brain development from embryonic stage E17 to postnatal stages P3, P5, and P7 including the increase in cortical thickness related to the increase in brain and body weights. In addition, the 6 cortical cell layers have been shown in all groups of mice with some differences, dense aggregation of neurons in layers found in the embryonic brain at E17, but more homogeneity of cortex was found in postnatal brains at P3, P5, and P7. In agreement with findings reported previously [16], the cytoarchitecture was established during embryonic development through the coordinated processes of neurogenesis and cell migration. During mammalian embryonic development, newly generated neurons leave the proliferative areas to reach their place of final maturation in precise locations within the cortex. The present study established that most of the migration occurred after birth; in postnatal mice, neurons underwent disaggregation and migrated to specific targets or layers, where they would form contacts or synapses.

Neuronal migration is a complex process involving intrinsic and extrinsic factors including transcription factors, adhesion molecules, secreted cues, and extracellular matrix components that control the different phases and aspects of cell movement. These factors can regulate the cytoskeleton in the control of cell migration in brain including radial, tangential, and axonophilic migration [17].

Previous studies have found a variety of NCAM expressions in the brain development of different species. NCAM was found to appear early in development in homologs of all 3 germ layers in amphibian and avian embryos [18]. However, at later stages, NCAM is also dramatically upregulated in the neural plate and neural tube forming in response to neural induction. Moreover, NCAM is ubiquitously distributed in the neural tube and developing CNS [19].

In a study of early mouse embryo, immunostaining of the cerebral cortex showed NCAM immunoreactivity throughout the width of the telencephalon in E9 [20]. Marín-Padilla [21] described that the prenatal development of mammalian cerebral cortex, including that of humans, is characterized by 2 periods: an early embryonic and a late fetal period [22]. In the present study, NCAM immunoreactivity suggesting expression of the protein in the cerebral cortex and SGZ in the dentate of the hippocampus gradually increases from the late fetal E17 stage and progressively through postnatal stages P3, P5, and P7. However, PSA–NCAM immunoreactivity was coupled with NCAM immunoreactivity after birth in postnatal P3, P5, and P7 suggesting a role in the increase of neuronal migration in both the cerebral cortex and SGZ.

Our findings are consistent with an immunohistological study of motor neuron development [23] that showed the absence of NCAM immunoreactivity in the early development as in E10.5 rat cervical spinal cord and immunoreactivity restricted to motor neurons at E11. Another study of NCAM expression in the early mouse embryo demonstrated positive immunostaining throughout the neuroepithelium in the cross-sections of the E8 and E9 neural tube at the level of the future spinal cord [24]. Our present study confirmed that low levels of both NCAM and PSA–NCAM immunoreactivity in SGZ are detected in the early development as seen here in the embryonic brain at E17 with higher levels gradually increasing in postnatal brains at P3, P5, and P7 as demonstrated by western blotting and histology with immunoperoxidase staining.

The focus on newly generated neurons in the SGZ in hippocampus was studied [25] by demonstrating the coronal section through hippocampus to show subfields in CA1 in the hippocampus, and in the DG. Individual layers in the subfields are indicated as follows: strata oriens (or), pyramidale (py), ra, and lm in CA1; po layer, strata gr, and mo in the DG. As shown in Figures 2 and 3, NCAM immunoreactivity was seen on the surface of neuronal cells when cell–cell interactions, aggregation, and migration occur and may influence cell proliferation, differentiation, and the development of synapses. Dityatev et al. [26] reported that PSA–NCAM is highly expressed throughout the embryonic and juvenile mammalian brain, but by contrast we found no or scant immunoreactivity at E17, PSA–NCAM was mostly found after birth in ICR mice. PSA– NCAM is proposed as involved in a range of developmental processes including cell migration, axon/dendrite growth and remodeling, and synaptic reorganization [26]. PSA–NCAM is also considered to mediate neuronal plasticity by facilitating the remodeling of synapses and neuronal structure.

In all vertebrates [27], including mammals [28] and humans [29], the main neurogenic area of the hippocampus is found at the DG, and not at the subventricular zone. The newly produced hippocampal neurons tend to differentiate to granule cells locally, rather than migrate to other areas [30]. Proliferation of the neuronal cells occurs in the SGZ of DG or in the inner area of the granule cell layer. The majority of these newly produced cell bodies translocate to peripheral layers just a few micrometers distant. They then extend their dendrites and axons to the stratum moleculare and the hilar area, respectively [30]. By 3 weeks after neuronal production, their neurites start to proliferate, while the dendritic spines appear from the 3rd week [31]. Substantial PSA–NCAM expression can be demonstrated in these newly proliferated precursors of the granule cells [32]. To investigate the plasticity of the CNS and neuronal production in developing mice, it may be useful to employ NCAM and PSA-NCAM as markers for implicating the pattern of NCAM expression in brain repair.

The present study is limited because E17 fetal brains are too small to be collected. They were collected as whole body for immunoperoxidase staining, but not included in Western blotting of brain tissue. Therefore, PSA–NCAM immunoreactivity at E17 cannot support its low level as shown in staining. Any comparison using immunoperoxidase staining may find it difficult to demonstrate any difference among groups, and the result may be better after immunofluorescent staining. Nevertheless, immunoperoxidase staining can show other nearby cells and related structures.