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Regional differences in the temporal expression of non-apoptotic caspase-3-positive bergmann glial cells in the developing rat cerebellum.

Finckbone V, Oomman SK, Strahlendorf HK, Strahlendorf JC - Front Neuroanat (2009)

Bottom Line: The current study seeks to further correlate active/cleaved caspase-3 expression with the developmental phenotype of Bergmann glia by examining regional differences in the temporal pattern of expression of cleaved caspase-3 immunoreactivity in lobules of the cerebellar vermis.Compared to intermediate or late maturing lobules, early maturing lobules had higher levels of active caspase-3 at earlier postnatal times.This period of postnatal development is precisely the time during which Bergmann glia initiate differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center Lubbock, TX, USA.

ABSTRACT
Although caspases have been intimately linked to apoptotic events, some of the pro-apoptotic caspases also may regulate differentiation. We previously demonstrated that active caspase-3 is expressed and has an apparent non-apoptotic function during the development of cerebellar Bergmann glia. The current study seeks to further correlate active/cleaved caspase-3 expression with the developmental phenotype of Bergmann glia by examining regional differences in the temporal pattern of expression of cleaved caspase-3 immunoreactivity in lobules of the cerebellar vermis. In general, we found that the expression pattern of cleaved caspase-3 corresponds to the reported developmental temporal profile of the lobes and that its levels peak at 15 days and declines thereafter. Compared to intermediate or late maturing lobules, early maturing lobules had higher levels of active caspase-3 at earlier postnatal times. This period of postnatal development is precisely the time during which Bergmann glia initiate differentiation.

No MeSH data available.


Panels (A) and (B) are fluorescently labeled images from 15-day-old cerebellum using cleaved caspase-3 and S-100b immunohistochemistry visualized with Alexa 488 and Alexa 546, respectively. It is apparent that caspase-3 labels the nuclei of Bergmann glia. Asterisks identify the Bergmann glia in which approximately 60% contain cleaved caspase-3 immunoreactivity. Panel (C) is the triple labeled fluorescent image of caspase-3 (Alexa 488), S-100b (Alexa 546) and calbindin (Alexa 633) to identify and quantitate the total number of Bergmann glia within each lobule at the different ages, surrounding the Purkinje neurons. Panel (D) is a double labeled image using cleaved caspase-3 and GFAP immunoreactivity (visualized with Alexa 488 (green) and Alexa 546 (red), respectively). In Panel (E) cells are double labeled with S-100b (red fluorescence) and Glast (green fluorescence), two markers of Bergmann glia, respectively, to further verify the presence of caspase-3 in the nuclei of Bergmann glia surrounding Purkinje neurons obtained from 15-day-old cerebellum. Panel (F) is a modified image of an original cleaved caspase-3 labeling visualized with green wavelength using a Alexa 488 fluorescent dye coupled with an auto-fluorescent label of the tissue at the red wavelength (594 nm), showing the location of immunoreactive cells within the Purkinje cell layer in a 21-day-old cerebellum. P – Purkinje cell; B – Bergmann glia; ML – molecular layer.
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Figure 2: Panels (A) and (B) are fluorescently labeled images from 15-day-old cerebellum using cleaved caspase-3 and S-100b immunohistochemistry visualized with Alexa 488 and Alexa 546, respectively. It is apparent that caspase-3 labels the nuclei of Bergmann glia. Asterisks identify the Bergmann glia in which approximately 60% contain cleaved caspase-3 immunoreactivity. Panel (C) is the triple labeled fluorescent image of caspase-3 (Alexa 488), S-100b (Alexa 546) and calbindin (Alexa 633) to identify and quantitate the total number of Bergmann glia within each lobule at the different ages, surrounding the Purkinje neurons. Panel (D) is a double labeled image using cleaved caspase-3 and GFAP immunoreactivity (visualized with Alexa 488 (green) and Alexa 546 (red), respectively). In Panel (E) cells are double labeled with S-100b (red fluorescence) and Glast (green fluorescence), two markers of Bergmann glia, respectively, to further verify the presence of caspase-3 in the nuclei of Bergmann glia surrounding Purkinje neurons obtained from 15-day-old cerebellum. Panel (F) is a modified image of an original cleaved caspase-3 labeling visualized with green wavelength using a Alexa 488 fluorescent dye coupled with an auto-fluorescent label of the tissue at the red wavelength (594 nm), showing the location of immunoreactive cells within the Purkinje cell layer in a 21-day-old cerebellum. P – Purkinje cell; B – Bergmann glia; ML – molecular layer.

Mentions: Figures 2A–C illustrate that cells immunoreactive-positive for S-100b and cleaved caspase-3 are situated as classic Bergmann glia surrounding the calbindin-positive Purkinje neurons. S-100b-positive cells also co-labeled for cleaved caspase-3 immunoreactivity in the nuclei rather than cytosol, suggesting a unique role related to nuclear function. We confirmed the apparent authenticity of the S-100b-labeled cells as Bergmann glia by performing additional immunohistochemical studies using antigens known to localize to Bergmann glia, including Glast, and GFAP (Feng et al., 1994; Kuhar et al., 1993). As seen with S-100b, GFAP colocalized with cleaved caspase-3 in the same cells that co-labeled with Glast and S-100b (Figures 2D,E). Figure 2F is a modified image (original active caspase-3 labeled as green fluorescence merged with enhanced Purkinje neuron autofluorescence labeled as red fluorescence) which clearly demonstrates the close proximity of these cells to Purkinje neurons. In Figure 2F, to visualize Purkinje neurons, autofluorescence derived from endogenous, intrinsic fluorophores such as NADPH and flavin coenzymes present in mitochondria and lysosomes was intensified by increasing the gain of the microscope detector. Our lab previously demonstrated (Oomman et al., 2005, 2006) that the S-100b labeled cells failed to label with postmitotic neuronal markers TUJ1, a beta III tubulin cytosolic marker (Cameron and McKay, 2001; Yan et al., 2001), and NeuN, a neuron-specific neuronal marker (Hoshimaru et al., 1996; Yan et al., 2001), further verifying the active caspase-positive cells are not neurons.


Regional differences in the temporal expression of non-apoptotic caspase-3-positive bergmann glial cells in the developing rat cerebellum.

Finckbone V, Oomman SK, Strahlendorf HK, Strahlendorf JC - Front Neuroanat (2009)

Panels (A) and (B) are fluorescently labeled images from 15-day-old cerebellum using cleaved caspase-3 and S-100b immunohistochemistry visualized with Alexa 488 and Alexa 546, respectively. It is apparent that caspase-3 labels the nuclei of Bergmann glia. Asterisks identify the Bergmann glia in which approximately 60% contain cleaved caspase-3 immunoreactivity. Panel (C) is the triple labeled fluorescent image of caspase-3 (Alexa 488), S-100b (Alexa 546) and calbindin (Alexa 633) to identify and quantitate the total number of Bergmann glia within each lobule at the different ages, surrounding the Purkinje neurons. Panel (D) is a double labeled image using cleaved caspase-3 and GFAP immunoreactivity (visualized with Alexa 488 (green) and Alexa 546 (red), respectively). In Panel (E) cells are double labeled with S-100b (red fluorescence) and Glast (green fluorescence), two markers of Bergmann glia, respectively, to further verify the presence of caspase-3 in the nuclei of Bergmann glia surrounding Purkinje neurons obtained from 15-day-old cerebellum. Panel (F) is a modified image of an original cleaved caspase-3 labeling visualized with green wavelength using a Alexa 488 fluorescent dye coupled with an auto-fluorescent label of the tissue at the red wavelength (594 nm), showing the location of immunoreactive cells within the Purkinje cell layer in a 21-day-old cerebellum. P – Purkinje cell; B – Bergmann glia; ML – molecular layer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691149&req=5

Figure 2: Panels (A) and (B) are fluorescently labeled images from 15-day-old cerebellum using cleaved caspase-3 and S-100b immunohistochemistry visualized with Alexa 488 and Alexa 546, respectively. It is apparent that caspase-3 labels the nuclei of Bergmann glia. Asterisks identify the Bergmann glia in which approximately 60% contain cleaved caspase-3 immunoreactivity. Panel (C) is the triple labeled fluorescent image of caspase-3 (Alexa 488), S-100b (Alexa 546) and calbindin (Alexa 633) to identify and quantitate the total number of Bergmann glia within each lobule at the different ages, surrounding the Purkinje neurons. Panel (D) is a double labeled image using cleaved caspase-3 and GFAP immunoreactivity (visualized with Alexa 488 (green) and Alexa 546 (red), respectively). In Panel (E) cells are double labeled with S-100b (red fluorescence) and Glast (green fluorescence), two markers of Bergmann glia, respectively, to further verify the presence of caspase-3 in the nuclei of Bergmann glia surrounding Purkinje neurons obtained from 15-day-old cerebellum. Panel (F) is a modified image of an original cleaved caspase-3 labeling visualized with green wavelength using a Alexa 488 fluorescent dye coupled with an auto-fluorescent label of the tissue at the red wavelength (594 nm), showing the location of immunoreactive cells within the Purkinje cell layer in a 21-day-old cerebellum. P – Purkinje cell; B – Bergmann glia; ML – molecular layer.
Mentions: Figures 2A–C illustrate that cells immunoreactive-positive for S-100b and cleaved caspase-3 are situated as classic Bergmann glia surrounding the calbindin-positive Purkinje neurons. S-100b-positive cells also co-labeled for cleaved caspase-3 immunoreactivity in the nuclei rather than cytosol, suggesting a unique role related to nuclear function. We confirmed the apparent authenticity of the S-100b-labeled cells as Bergmann glia by performing additional immunohistochemical studies using antigens known to localize to Bergmann glia, including Glast, and GFAP (Feng et al., 1994; Kuhar et al., 1993). As seen with S-100b, GFAP colocalized with cleaved caspase-3 in the same cells that co-labeled with Glast and S-100b (Figures 2D,E). Figure 2F is a modified image (original active caspase-3 labeled as green fluorescence merged with enhanced Purkinje neuron autofluorescence labeled as red fluorescence) which clearly demonstrates the close proximity of these cells to Purkinje neurons. In Figure 2F, to visualize Purkinje neurons, autofluorescence derived from endogenous, intrinsic fluorophores such as NADPH and flavin coenzymes present in mitochondria and lysosomes was intensified by increasing the gain of the microscope detector. Our lab previously demonstrated (Oomman et al., 2005, 2006) that the S-100b labeled cells failed to label with postmitotic neuronal markers TUJ1, a beta III tubulin cytosolic marker (Cameron and McKay, 2001; Yan et al., 2001), and NeuN, a neuron-specific neuronal marker (Hoshimaru et al., 1996; Yan et al., 2001), further verifying the active caspase-positive cells are not neurons.

Bottom Line: The current study seeks to further correlate active/cleaved caspase-3 expression with the developmental phenotype of Bergmann glia by examining regional differences in the temporal pattern of expression of cleaved caspase-3 immunoreactivity in lobules of the cerebellar vermis.Compared to intermediate or late maturing lobules, early maturing lobules had higher levels of active caspase-3 at earlier postnatal times.This period of postnatal development is precisely the time during which Bergmann glia initiate differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center Lubbock, TX, USA.

ABSTRACT
Although caspases have been intimately linked to apoptotic events, some of the pro-apoptotic caspases also may regulate differentiation. We previously demonstrated that active caspase-3 is expressed and has an apparent non-apoptotic function during the development of cerebellar Bergmann glia. The current study seeks to further correlate active/cleaved caspase-3 expression with the developmental phenotype of Bergmann glia by examining regional differences in the temporal pattern of expression of cleaved caspase-3 immunoreactivity in lobules of the cerebellar vermis. In general, we found that the expression pattern of cleaved caspase-3 corresponds to the reported developmental temporal profile of the lobes and that its levels peak at 15 days and declines thereafter. Compared to intermediate or late maturing lobules, early maturing lobules had higher levels of active caspase-3 at earlier postnatal times. This period of postnatal development is precisely the time during which Bergmann glia initiate differentiation.

No MeSH data available.