Calbindin and calretinin immunoreactivities identify different types of neurons in the adult lamprey spinal cord

Abstract

The central pattern generator for locomotion in vertebrates is composed of different spinal neuronal populations that generate locomotor movement. In the lamprey spinal cord, several classes of interneurons have been identified based on morphologic and physiological criteria and integrated in the spinal cord circuits implicated in the generation of locomotion. However, the lack of histochemical markers for most of the interneurons makes it difficult to study whole populations along the spinal cord. We have investigated the immunoreactivity with antibodies raised against calbindin and calretinin. Several types of neurons could be classified: (1). strongly immunoreactive neurons located dorsomedially, (2). moderately immunoreactive neurons located laterally, (3). small weakly immunoreactive neurons, d). ventromedial neurons, (4). liquor contacting cells, and (5). motoneurons. The ventromedial group of calbindin-immunoreactive neurons also is immunoreactive for serotonin and, therefore, represents the ventromedial group of dopamine/serotonin spinal neurons. Some of the lateral calbindin-immunoreactive neurons may be CC-type cells (cells with caudal-crossed axons), because they are retrogradely labeled by tracer injections into the contralateral spinal cord. Other well-characterized cell types, such as sensory dorsal cells, lateral interneurons, descending propriospinal edge cells, and spinobulbar giant interneurons are negative for both calbindin and calretinin. Therefore, calbindin and calretinin are useful markers for the study of cell populations that may be integrated in locomotor circuits.

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

“…The CBir and CRir cell populations are more numerous in Xenopus [Morona et al, 2005[Morona et al, , 2006b]. Apart from lampreys [Megías et al, 2003], comparable cells have not been reported in the spinal cord of any other group of fishes, and in terms of location, morphology, and calcium-binding protein content, the cells detected in the ventral columns of lungfishes are very similar to those of tetrapods.…”

“…The actual proteins CB and CR have not been isolated in lungfishes, but the Western blot analysis we performed and previously published data suggest that these proteins are conserved in evolution [Wassermann and Taylor, 1966;Parmentier et al, 1987;Denizot et al, 1988] and that the use of antibodies against mammalian or avian proteins is therefore a valuable tool for identifying CBir and CRir structures in the nervous system of a wide variety of vertebrates [Martínez-Guijarro and Freund, 1992;Guirado et al, 1999;Pritz and Siadati, 1999;Cuenca et al, 2000;Dávila et al, 2000;Hackney et al, 2003;Megías et al, 2003;Bennis et al, 2005;González, 2008, 2009]. Furthermore, the present results show that the antibodies used in our analysis of the spinal cord of P. dolloi and N. forsteri yield specific immunostaining and that the simultaneous double-labeling experiments corroborate the lack of cross-reactivity in the case of lungfishes.…”

“…The immunoreactive cells do not belong to any specific cell type, and their location includes dorsal and ventral spinal regions of the gray matter. It should be noted that previous studies of diverse species have suggested a comparable distribution of these calcium-binding proteins in the spinal cord of tetrapods [Morona et al, 2005[Morona et al, , 2006a[Morona et al, , b, 2007, unlike the pattern seen in fishes [Díaz-Regueira and Anadón, 2000;Rodríguez-Moldes et al, 1990;Denizot et al, 1988;Megías et al, 2003]. Because of the phylogenetic importance of lungfishes as the closest living relatives of tetrapods, the wide distribution of labeled cells found in this study (which is readily comparable to that in tetrapods) represents an interesting finding.…”

“…Notably, no cells have been reported in the dorsal part of the spinal cord in other fishes [Denizot et al, 1988;Rodríguez-Moldes et al, 1990;Díaz-Regueira and Anadón, 2000]. Only in lampreys has a cell population in the dorsal spinal cord been reported to be CBir/CRir, but it revealed a different pattern of dendritic arborization [Megías et al, 2003].…”

Quantitative Investigation of Calcium Signals for Locomotor Pattern Generation in the Lamprey Spinal Cord

“…At least in the short term, the endogenous buffering allows motoneurons to rapidly recover from Ca 2ϩ transients at a low energy cost (Palecek et al 1999). Recent evidence shows that lamprey spinal cord putative CPG neurons and motoneurons are indeed rich in Ca 2ϩ buffering molecules like calbindin and calretinin (Megias et al 2003).…”