Immunohistochemical Localization of Calbindin-D28k and Calretinin in the Spinal Cord of Lungfishes

Abstract

A common pattern of distribution of neurons and fibers containing the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the spinal cord of terrestrial vertebrates has been recently demonstrated. Lungfishes are considered the closest living relatives of tetrapods, but practically no experimental data exist on the organization of their spinal cord. By means of immunohistochemical techniques, the localization of CB and CR was investigated in the spinal cord of the African (Protopterus dolloi) and Australian (Neoceratodus forsteri) lungfishes. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. A large population of immunoreactive cells was found in the dorsal column of the gray matter in both species, and abundant cells were distributed in the lateral and ventral columns. Ventrolateral motoneurons and multipolar cells were only intensely CB and CR immunoreactive in Neoceratodus. For the most part, separate cell populations contained either CB or CR, but a small subset of dorsally located neurons contained both in the two lungfishes. Colocalization was found in motoneurons and in ventrolaterally located cells only in Neoceratodus. Fiber labeling showed a predominance of CR-containing axons in the lateral and ventral funiculi of presumed supraspinal origin. These results show that lung-fishes and tetrapods have many features in common, suggesting that primitive anatomical, and likely functional, organization of the spinal cord of tetrapods is present in lungfishes.

Regional chemoarchitecture of the brain of lungfishes based on calbindin D‐28K and calretinin immunohistochemistry

“…The lack of cross‐reactivity between the CB and CR antibodies in the present study was clear, because many cells were distinctly labeled for either CB or CR, and only a small portion of neurons was double labeled in the combined experiments. Similar observations were reported previously for the retina and spinal cord of these lungfish species (Morona, Northcutt, & González, ).…”

“…The detailed distribution of CBir and CRir cell populations in the spinal cord of these lungfish species has been detailed in a previous report (Morona et al, ). Our new observations confirmed that study and corroborated the presence of separated cell populations, with some double labeled cells, in the dorsal and ventral horns.…”

“…The specificity of the TH monoclonal antibody was corroborated by Western blot analysis in rat, mouse, ferret, cat, and Aplysia (see specification data sheet; ImmunoStar), diverse amphibians (Morona & González, ), and the turtle Pseudemys scripta (Moreno, Morona, López, & González, ) in which it selectively labels a single band at approximately 62 kDa. The Western blot performed with brain extracts from the two lungfishes (Morona et al, ; López & González, ) detected a single band at the same molecular weight (about 62 kDa) as that of the major product detected in rat and Xenopus brain extract (Figure ).…”

Localization of Calbindin-D28k and Calretinin in the Brain of Dermophis Mexicanus (Amphibia: Gymnophiona) and Its Bearing on the Interpretation of Newly Recognized Neuroanatomical Regions

“…The abundant CBir and CRir spinal cells revealed in Dermophis are distinctly distributed in the dorsal and ventral gray fields largely resembling the situation found in urodeles and lungfish [Morona and González, 2009;Morona et al, 2010]. Significantly, in anurans the distribution of CB and CR in the spinal cord includes a variety of cells with different localizations and morphologies in close resemblance with the distributions reported in amniotes [Morona and González, 2006a].…”

Immunohistochemical distribution of calretinin and calbindin (D‐28k) in the brain of the cladistian Polypterus senegalus

“…Both proteins act as intracellular calcium buffers (Miller, 1995), neuromodulators (Schwaller et al, 2002), or calcium sensors (McCue et al, 2010), among other functions. Antibodies raised against these proteins have been widely used as neuronal markers to characterize neuronal populations or brain regions in tetrapods (mammals: García-Segura et al, 1984;Celio, 1990;S equier et al, 1990;Jacobowitz and Winsky, 1991;Resibois and Rogers, 1992;Andressen et al, 1993;birds: Roth et al, 1981;Rogers et al, 1990;Puelles et al, 1994;reptiles: D avila et al, 1997amphibians: Necchi et al, 1999;Mil an and Puelles, 2000;Gonz alez, 2008, 2009;Morona et al, 2011a) and fishes (lampreys: Pombal and Puelles, 1999;Megías et al, 2003;elasmobranchs: Rodríguez-Moldes et al, 1990a,b;Anad on et al, 2009;chondrosteans: Graña et al, 2012;holosteans: Northcutt, 2009;teleosts: Díaz-Regueira and Anad on, 2000;Castro et al, 2003Castro et al, , 2006aNorthcutt, 2009;lungfish: Gonz alez et al, 2010;Morona et al, 2010).…”