Anat Cell Biol.  2010 Mar;43(1):64-71. 10.5115/acb.2010.43.1.64.

Immunohistochemical study on the expression of calcium binding proteins (calbindin-D28k, calretinin, and parvalbumin) in the cerebellum of the nNOS knock-out(-/-) mice

• Lee JC
• Chung YH
• Cho YJ
• Kim J
• Kim N
• Cha CI
• Joo KM

• KMID: 2168898
• DOI: http://doi.org/10.5115/acb.2010.43.1.64

Abstract

Nitric Oxide (NO) actively participates in the regulation of neuronal intracellular Ca2+ levels by modulating the activity of various channels and receptors. To test the possibility that modulation of Ca2+ buffer protein expression level by NO participates in this regulatory effect, we examined expression of calbindin-D28k, calretinin, and parvalbumin in the cerebellum of neuronal NO synthase knock-out (nNOS(-/-)) mice using immunohistochemistry. We observed that in the cerebellar cortex of the nNOS(-/-) mice, expression of calbindin-D28k and parvalbumin were significantly increased while expression of calretinin was significantly decreased. These results suggest another mechanism by which NO can participate in the regulation of Ca2+ homeostasis.

Keyword

Calbindin-D28k; calretinin; parvalbumin; cerebellum; neuronal nitric oxide synthase (nNOS) knock-out (-/-) mice

MeSH Terms

Animals
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Cerebellar Cortex
Cerebellum
Homeostasis
Immunohistochemistry
Mice
Neurons
Nitric Oxide
Nitric Oxide Synthase
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Nitric Oxide
Nitric Oxide Synthase

Figure

• 

  Fig. 1 Tests for specificity of primary antibodies (A, B). A sample of sections reacted without primary antiserum (A), and a different sample exposed to primary antiserum preabsorbed for 24 hours with CB (B) do not exhibit any immunoreactivity. No immunoreactivity is observed in both samples exposed to primary antiserums preabsorbed for 24 hours with CR and PV (data not shown). Scale bar = 150 µm.

• 

  Fig. 2 Changes of CB-immunoreactivity in the cerebellum of nNOS(-/-) mice (B, D), compared with that of the control mice (A, C). In control mice, CB-immunoreactivity is observed in Purkinje cells, neuropil of the molecular layer of the cerebellar cortex (A) and neuropil of the deep cerebellar nuclei (C). This expression pattern is preserved in the nNOS(-/-) mice (B, D) but in all cerebellar cortical layers (B) and deep cerebellar nuclei (D), CB-immunoreactivity is increased. In image analysis (E), these differences are statistically significant. Mean density is the sum of the white values of all the pixels in the selection divided by the number of pixels within the selection. Values are the mean±standard deviations. A t-test was performed (*P<0.01). D, deep cerebellar nuclei; FN, fastigial nucleus; G, granular layer; IP, interposed nucleus; M, molecular layer; P, Purkinje cell layer. Scale bar (A, B) = 100 µm; (C, D) = 400 µm.

• 

  Fig. 3 Decreased CR expression in the cerebellum of nNOS(-/-) mice (A, C for the control mice and B, D for the nNOS(-/-) mice). In the cerebellar cortex of both group, granule cells and neuropil of the molecular layer express CR but Purkinje cells express little CR (A, B). Compared with control mice (A), CR-immunoreactivity is significantly decreased in all cerebellar cortical layers of nNOS(-/-) mice (B, E). In contrast to the cerebellar cortex, CR-immunoreactivity of the deep cerebellar nuclei is very low (C, D). In many animals CR expression seems to be decreased in these regions in the nNOS(-/-) mice although their differences are not statistically significant (E). Mean density is the sum of the white values of all the pixels in the selection divided by the number of pixels within the selection. Values are the mean±standard deviations. A t-test was performed (*P<0.01). The graph in E shows results from image analysis. D, deep cerebellar nuclei; FN, fastigial nucleus; G, granular layer; IP, interposed nucleus; M, molecular layer; P, Purkinje cell layer. Scale bar (A, B) = 100 µm; (C, D) = 400 µm.

• 

  Fig. 4 Changes of PV-immunoreactivity in the cerebellum of the nNOS(-/-) mice (B, D) compared with that of control mice (A, C). In the cerebellar cortex of the control mice, the Purkinje cells, neuropil, and cells of the molecular layer express PV (A). Although PV expression patterns of nNOS(-/-) mice are similar to that of control mice, PV-immunoreactivity of Purkinje cells and the molecular layer is significantly increased (A, B, E) and that of the granular layer is significantly decreased (A, B, E) in nNOS(-/-) mice. In the deep cerebellar nuclei of the control and the nNOS(-/-) mice, cell bodies as well as neuropil express PV (C, E). The change of immunoreactivity was minor but cell bodies of the nNOS(-/-) mice seemed to express higher PV-immunoreactivity (C~E). The graph in E shows results from image analysis. Mean density is the sum of the white values of all pixels in the selection divided by the number of pixels within the selection. Values are the mean±standard deviations. A t-test was performed (*P<0.01). D, deep cerebellar nuclei; FN, fastigial nucleus; G, granular layer; IP, interposed nucleus; M, molecular layer; P, Purkinje cell layer. Scale bar (A, B) = 100 µm; (C, D) = 400 µm.

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