Anat Cell Biol.  2011 Jun;44(2):106-115. 10.5115/acb.2011.44.2.106.

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

• Cho YJ
• Lee JC
• Kang BG
• An J
• Song HS
• Son O
• Nam DH
• Cha CI
• Joo KM

• KMID: 1447420
• DOI: http://doi.org/10.5115/acb.2011.44.2.106

Abstract

Nitric oxide (NO) modulates the activities of various channels and receptors to participate in the regulation of neuronal intracellular Ca2+ levels. Ca2+ binding protein (CaBP) expression may also be altered by NO. Accordingly, we examined expression changes in calbindin-D28k, calretinin, and parvalbumin in the cerebral cortex and hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice using immunohistochemistry. For the first time, we demonstrate that the expression of CaBPs is specifically altered in the cerebral cortex and hippocampal region of nNOS-/- mice and that their expression changed according to neuronal type. As changes in CaBP expression can influence temporal and spatial intracellular Ca2+ levels, it appears that NO may be involved in various functions, such as modulating neuronal Ca2+ homeostasis, regulating synaptic transmission, and neuroprotection, by influencing the expression of CaBPs. Therefore, these results suggest another mechanism by which NO participates in the regulation of neuronal Ca2+ homeostasis. However, the exact mechanisms of this regulation and its functional significance require further investigation.

Keyword

Calcium binding proteins; Cerebral cortex and hippocampal region; Immunohistochemistry; Neuronal nitric oxide synthase (nNOS); nNOS knock-out(-/-) mice

MeSH Terms

Animals
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Carrier Proteins
Cerebral Cortex
Homeostasis
Immunohistochemistry
Mice
Neurons
Nitric Oxide
Nitric Oxide Synthase
Synaptic Transmission
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Carrier Proteins
Nitric Oxide
Nitric Oxide Synthase

Figure

• 

  Fig. 1 Changes in calbindin D28k (CB) immunoreactivity in the cerebral cortex of neuronal NO synthase knockout(-/-) (nNOS-/-) mice (D-F) compared with control mice (A-C). (B, E) are high power views of layers II and III and (C, F) are high power views of layer V of the cerebral cortex. The distribution pattern and morphology of CB-immunoreactive neurons were similar in the control and nNOS-/- mice (A, D). CB immunoreactivity in nNOS-/- mice was much lower than that of control mice (D-F) and the high power views show that lengths of the CB-immunoreactive neurites in the nNOS-/- mice were shorter and less branched than those of the control mice (black arrowheads in E and F). Scale bars=200 µm (A, D), 50 µm (B, C, E, F).

• 

  Fig. 2 Decreased calbindin D28k (CB) expression in the hippocampal region of neuronal NO synthase knockout(-/-) (nNOS-/-) mice (A, C-E, I, J for the control mice and B, F-H, K, L for the nNOS-/- mice). (C and F, D and G, and E and H) are high power views of the dentate gyrus, CA1 of Ammon's horn, and CA3 of Ammon's horn, respectively. The overall distribution patterns of CB-immunoreactivity and the morphology of CB-immunoreactive neurons were similar in the control and nNOS-/- mice (A-L). CB immunoreactivity was much reduced in the neuronal cell bodies and the neuropil of nNOS-/- mice (B, F-H, K, L). CA1, field CA1 Ammon's horn; CA2, field CA2 Ammon's horn; CA3, field CA3 Ammon's horn; DGlb, dentate gyrus lateral blade; DGmb, dentate gyrus medial blade; mo, molecular layer; sg, granule cell layer; po, polymorph layer; so, stratum oriens; sp, pyramidal layer; sr, stratum radiatum; slu, stratum lucidum. Scale bars=200 µm (A, B), 50 µm (C-H), 100 µm (I, K), 80 µm (J, L).

• 

  Fig. 3 Changes in calretinin (CR) immunoreactivity in the cerebral cortex (D-F) and in the hippocampal region (H, L-N) of neuronal NO synthase knockout(-/-) (nNOS-/-) mice, compared with those of control mice (A-C for the cerebral cortex and G, I-K for the hippocampal region). (I and L, J and K, and M and N) are high power views of the dentate gyrus and the CA1 of Ammon's horn, respectively. In the cerebral cortex and hippocampal region, the overall distribution patterns of CR-immunoreactivity and the morphology of CR-immunoreactive neurons were similar in the control and nNOS-/- mice (A-N). In the cerebral cortex, nNOS-/- mice had fewer CR-immunoreactive neurons and each CR-immunoreactive neuron showed less CR-immunoreactivity than that in control mice (D-F). High power views showed that the lengths of the CR-immunoreactive neurites in the nNOS-/- mice were shorter than those of the control mice (D-F). Calbindin D28k (CB) immunoreactivity in the neuropil of the dentate gyrus was much reduced in the hippocampal region of nNOS-/- mice (H, L) and the number of CR-immunoreactive neurons was less (H) in nNOS-/- mice, although the morphology of these neurons was preserved (L-N). CA1, field CA1 Ammon's horn; CA3, field CA3 Ammon's horn; DGlb, dentate gyrus lateral blade; DGmb, dentate gyrus medial blade; mo, molecular layer; sg, granule cell layer; po, polymorph layer; so, stratum oriens; sp, pyramidal layer; sr, stratum radiatum. Scale bars=150 µm (A, D), 80 µm (B, C, E, F), 200 µm (G, H), 65 µm (I, L), 50 µm (J, K, M, N).

• 

  Fig. 4 Parvalbumin (PV) immunoreactivity in the cerebral cortex (A-D) and hippocampal region (E-L) of control mice (A, B, E, G-I) and neuronal NO synthase knockout(-/-) (nNOS-/-) mice (C, D, F, J-L). (B and C, G and J, H and K, and I and L) are high power views of the cerebral cortex, the polymorph layer of the dentate gyrus, the granule cell layer of the dentate gyrus, and the pyramidal layer of Ammon's horn, respectively. The numbers of PV-immunoreactive neurons, their distribution patterns, and morphology were unchanged in the cerebral cortex and hippocampal region of nNOS-/- mice (F, J-L). CA1, field CA1 Ammon's horn; CA3, field CA3 Ammon's horn; DGlb, dentate gyrus lateral blade; DGmb, dentate gyrus medial blade; sg, granule cell layer; po, polymorph layer; mo, molecular layer;so, stratum oriens; sp, pyramidal layer; sr, stratum radiatum. Scale bars=150 µm (A, D), 60 µm (B, C), 200 µm (E, F), 40 µm (G-L).

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