Immunohistochemical localization of calbindin‐D28k and calretinin in the brainstem of anuran and urodele amphibians

Abstract

Calbindin-D28k (CB) and calretinin (CR) are calcium binding proteins present in distinct sets of neurons; they act as buffers regulating the concentration of intracellular calcium. CB and CR immunohistochemistry was studied in the brainstem of anuran and urodele amphibians in combination with other markers (choline acetyltransferase, tyrosine hydroxylase, and nitric oxide synthase), which served to clarify the localization and signature of many cell groups. CR labeled the retinorecipient layers of the optic tectum, and CB and CR labeled distinct tectal cell populations. The two proteins were largely complementary in the torus semicircularis and marked auditory and lateral line sensory regions, depending on the species. CB and CR in the mesencephalic and isthmic tegmentum specified the boundaries of basal and medial longitudinal bands. In the cerebellum, CB labeled Purkinje cells in all species, whereas CR was mainly found in fibers and labeled Purkinje cells only in Rana. In the parabrachial region, CB and CR allowed the distinction of the laterodorsal tegmental nucleus, isthmic nucleus, locus coeruleus, and rostral octavolateral nuclei. The distribution of CB- and CR-immunoreactive cells in the reticular formation and central gray was consistent with the current models of brainstem segmentation in amphibians. CR was found in the auditory fibers and nuclei in Rana and in mechanosensory lateral line fibers in Xenopus and urodeles, whereas CB mainly labeled vestibular fibers and nuclei in all species. These results highlight the anatomical complexity of the amphibian brainstem and help in an understanding of its regional organization that is not cytoarchitectonically evident.

Calbindin-D32k Is Localized to a Subpopulation of Neurons in the Nervous System of the Sea Cucumber Holothuria glaberrima (Echinodermata)

“…The evidence for this is the following: First, in other organisms in which these have been studied (mostly mammals), these markers specifically label neural structures [9], [10], [16]. Second, in the holothurian studied here, immunoreactivity to these markers is observed in cells and fibers of the radial nerve cords, podial nerves and peripheral nerves, which are the classically described neural structures in the echinoderms [27], [29], [30], [38]–[40].…”

“…In vertebrates, specifically in mammals, calbindin 1, parvalbumin and calbindin 2 are present in different populations of neurons and interneurons of mice adult spinal cord [10]. In amphibians, differential immunoreactivity to anti-calbindin 1 and anti-calbindin 2 in cells and fibers throughout the brainstem serves to determine the localization and signature of many cell groups [9]. Lastly, calbindin 1 and calbindin 2-like immunoreactivities are present in the retina of the lamprey, the basal vertebrate studied at present [16].…”

Regional expression of Pax7 in the brain of Xenopus laevis during embryonic and larval development

“…The specificity of the antibodies used has been assessed by the commercial companies (Table 2) and, in addition, the immunoblotting conducted in our previous studies with Xenopus laevis showed that all antibodies used labeled a single band, which corresponded well (with minor variations) to the bands labeled in the rat lanes (see Morona and González, 2008, 2009; Morona et al, 2011; Moreno et al, 2012a; Domínguez et al, 2013a). …”

“…Their relative localization was framed attending to the neuromeric organization of the brain. We used the same nomenclature as in our previous studies (Morona and González, 2008, 2009, 2013; Domínguez et al, 2011, 2013a,b; Morona et al, 2011). Single and double-labeled sections were analyzed with an Olympus BX51 microscope equipped for fluorescence with appropriate filter combinations, and selected sections were photographed using a digital camera Olympus DP72.…”

“…The immunohistochemical techniques employed in the present study allow high-resolution analysis of expressing cells (Hitchcock et al, 1996; Wullimann and Rink, 2001; González and Northcutt, 2009; Domínguez et al, 2011, 2013a,b; Ferreiro-Galve et al, 2012; Joven et al, 2013a,b). To identify accurately the cell groups expressing Pax7, we used combined immunohistofluorescence to simultaneously reveal several neuronal markers, which served to highlight the boundaries and landmarks of numerous brain regions, as previously reported (González et al, 1994, 2002; Tuinhof et al, 1994; Barale et al, 1996; López and González, 2002; Morona and González, 2008, 2009, 2013; Domínguez et al, 2013a,b). These markers included the γ-amino butyric acid (GABA), calretinin (CR), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), and the transcription factors Nkx2.1 and Otp.…”