• Title/Summary/Keyword: Calbindin

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Optimized Immunohistochemical Analysis of Cerebellar Purkinje Cells Using a Specific Biomarker, Calbindin D28k

  • Kim, Byung-Joo;Lee, So-Yeon;Kim, Hyung-Woo;Park, Eun-Jung;Kim, Jun;Kim, Sang-Jeong;So, In-Suk;Jeon, Ju-Hong
    • The Korean Journal of Physiology and Pharmacology
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    • v.13 no.5
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    • pp.373-378
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    • 2009

  • Cerebellar Purkinje cells (PCs) play a crucial role in motor functions and their progressive degeneration is closely associated with spinocerebellar ataxias. Although immunohistochemical (IHC) analysis can provide a valuable tool for understanding the pathophysiology of PC disorders, the method validation of IHC analysis with cerebellar tissue specimens is unclear. Here we present an optimized and validated IHC method using antibodies to calbindin D28k, a specific PC marker in the cerebellum. To achieve the desired sensitivity, specificity, and reproducibility, we modified IHC analysis procedures for cerebellar tissues. We found that the sensitivity of staining varies depending on the commercial source of primary antibody. In addition, we showed that a biotin-free signal amplification method using a horseradish peroxidase polymer-conjugated secondary antibody increases both the sensitivity and specificity of ICH analysis. Furthermore, we demonstrated that dye filtration using a $0.22\;{\mu}m$ filter eliminates or minimizes nonspecific staining while preserving the analytical sensitivity. These results suggest that our protocol can be adapted for future investigations aiming to understand the pathophysiology of cerebellar PC disorders and to evaluate the efficacy of therapeutic strategies for treating' these diseases.

  • https://doi.org/10.4196/kjpp.2009.13.5.373 인용 PDF KSCI

Spontaneous Electrical Activity in Cerebellar Purkinje Neurons of Postnatal Rats

  • Nam, Sang-Chae
    • The Korean Journal of Physiology and Pharmacology
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    • v.1 no.4
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    • pp.355-366
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    • 1997

  • Although cerebellar Purkinje cells display spontaneous electrical activity in vivo and in slice experiments, the mechanism of the spontaneous activity generation has not been clearly understood. The aim of this study was to investigate whether cerebellar Purkinje cells of postnatal rats generate spontaneous electrical activity without synaptic inputs. Dissociated cerebellar Purkinje cells were used for reducing synaptic inputs in the present study. Cerebellar Purkinje cells with dendrites were dissociated from postnatal rats using enzymatic treatment followed by mechanical trituration. Spontaneous electrical activities were recorded from dissociated cells without any stimulus using whole-cell patch clamp configuration. Two types, spontaneously firing or quiescent, of dissociated Purkinje cells were observed in postnatal rats. Both types of cells were identified as Purkinje cells using immunocytochemical staining technique with anti-calbindin after recording. Spontaneously active cells displayed two patterns of firing, repetitive and burst firings. Two thirds of dissociated Purkinje cells displayed repetitive firing and the rest of them did burst firing under same recording condition. Repetitive firing activities were maintained even after further isolation using either physical or pharmacological techniques. Neither high magnessium solution nor excitatory synaptic blockers, AP-5 and DNQX, block the spontaneous activity. These results demonstrate that spontaneous electrical activity of isolated cerebellar Purkinje cells in postnatal rats is generated by intrinsic membrane properties rather than synaptic inputs.

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TRIIODTHYRONINE (T3) ENHANCES THE STIMULATORY EFFECT OF 1, 25-DIHYDROXYVITAMIN D3 ON CALBINDIN-D28k mRNA EXPRESSION IN THE KIDNEY AND INTESTINE BUT NOT IN CEREBELLUM OF THE CHICK

  • Sechman, A.;Shimada, K.;Saito, N.;Ieda, T.;Ono, T.
    • Asian-Australasian Journal of Animal Sciences
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    • v.9 no.1
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    • pp.37-44
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    • 1996

  • The present study was conducted to investigate the role of thyroid hormones in the regulation of gene expression of calbindin-$D_{28k}$ (CaBP-D28K) in the chicken. By employing slot blot and RIA analyses, levels of CABP-D28K mRNA and CaBP-D28K protein in the intestine, kidney, cerebellum and liver were measured 6 and 12 h after i.m. injection of 1, 25-dihydroxyvitamin $D_3$ [1, 25 $(OH)_2D_3$; 250 ng/chick] and 3, 5, 3'-triiodothyronine ($T_3$; 500 ng/chick) in one-day-old chicks. The abundant messages of CaBP-D28K mRNA were detected in the intestine, kidney and cerebellum while there was little message in the liver. After 1, 25 $(OH)_2D_3$ treatment (6 + 12 hours), levels of CaBP-D28K mRNA increased in the intestine, but there was no change in the mRNA levels in the kidney and cerebellum. Although $T_3$ alone had no effect on CaBP-D28K mRNA levels, simultaneous administration of $T_3$ enhanced the 1, 25 $(OH)_2D_3$ effect of levels of CaBP-D28K mRNA in the intestine both 6 and 12 h post-treatment, and in the kidney 12 h post-treatment. At a protein level, co-treatment with 1, 25 $(OH)_2D_3$ and $T_3$ elicited a significant increase in CaBP-D28K expression in the intestine 12 h post-treatment, as compared to treatment with only 1, 25 $(OH)_2D_3$, whereas no differences were observed in the CaBP-D28K protein levels in the kidney and cerebellum. These results suggest that thyroid hormones may play a synergistic role with 1, 25 $(OH)_2D_3$ for CaBP-D28K gene expression in the intestine and kidney in chicks.

  • https://doi.org/10.5713/ajas.1996.37 인용 PDF