Immunohistochemical and hodological characterization of calbindin‐D28k‐containing neurons in the spinal cord of the turtle, Pseudemys scripta elegans

Abstract

Neurons and fibers containing the calcium-binding protein calbindin-D28k (CB) were studied by immunohistochemical techniques in the spinal cord of adult and juvenile turtles, Pseudemys scripta elegans. Abundant cell bodies and fibers immunoreactive for CB were widely and distinctly distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. In the dorsal horn, most CB-containing cells were located in close relation to the synaptic fields formed by primary afferents, which were not labeled for CB. Double immunohistofluorescence demonstrated distinct cell populations in the dorsal horn labeled only for CB or nitric oxide synthase, whereas in the dorsal part of the ventral horn colocalization of nitric oxide synthase was found in about 6% of the CB-immunoreactive cells in this region. Choline acetyltransferase immunohistochemistry revealed that only about 2% of the neurons in the dorsal part of the ventral horn colocalized CB, whereas motoneurons were not CB-immunoreactive. The involvement of CB-containing neurons in ascending spinal projections to the thalamus, tegmentum, and reticular formation was demonstrated combining the retrograde transport of dextran amines and immunohistochemistry. Similar experiments demonstrated supraspinal projections from CB-containing cells mainly located in the reticular formation but also in the thalamus and the vestibular nucleus. The revealed organization of the neurons and fibers containing CB in the spinal cord of the turtle shares distribution and developmental features, colocalization with other neuronal markers, and connectivity with other tetrapods and, in particular with mammals.

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

“…Because of their presence in the superficial dorsal horn, among different primary sensory afferents, it has been proposed that CBir and CRir cells serve regulatory roles in the processing of cutaneous and visceral sensory information [Philippe and Droz, 1988;Rogers et al, 1990;Ren et al, 1993;Ren and Ruda, 1994;Papka et al, 1999;Morona et al, 2006aMorona et al, , 2007. However, tracing studies to reveal the organization of the primary afferents to the spinal cord in lungfishes are needed to corroborate this suggestion.…”

“…Experimental data have indicated, however, that these fibers are of intraspinal origin [Ren and Ruda, 1994;Morona et al, 2007]. In lungfishes, no fiber labeling was found in the dorsal roots in any of the segments analyzed, and the fiber labeling found in the dorsolateral aspect of the cord most probably represents dendritic branching of the cells in the dorsal column.…”

“…A major feature of their organization in all amniotes is the predominance of immunoreactive cells in the superficial dorsal horn [Lunam, 1989;Yamamoto et al, 1989;Antal et al, 1990;Yoshida et al, 1990;Résibois and Rogers, 1992;Antal and Polgár, 1993;Ren et al, 1993;Ren and Ruda, 1994;Morona et al, 2006aMorona et al, , 2007, a situation that is readily comparable to the high numbers of immunoreactive cells in the dorsal field of the gray matter in the spinal cord of amphibians [Morona et al, 2005[Morona et al, , 2006b]. In the context of these observations, the numerous immunoreactive cell populations found in lungfishes most likely represent the counterpart of those in tetrapods.…”

“…Although both CBir and CRir cells in the superficial dorsal horn in amniotes form the most numerous in the spinal cord, they are reported to be topographically segregated, most likely reflecting distinct hodological properties [Lunam, 1989;Yamamoto et al, 1989;Antal et al, 1990;Yoshida et al, 1990;Résibois and Rogers, 1992;Antal and Pólgar, 1993;Ren et al, 1993;Ren and Ruda, 1994;Morona et al, 2006aMorona et al, , 2007. Such segregation was not seen in lungfishes, because CBir and CRir cells largely intermingle in the dorsal column.…”

“…In amniotes, CBir and CRir cells are scarce in the deep layers that receive the bulk of the muscle afferents in the dorsal horn [Lunam, 1989;Antal et al, 1990;Antal and Polgár, 1993;Ren and Ruda, 1993;Morona et al, 2006aMorona et al, , 2007. Because of their location, morphology, and weak immunoreactivity, these cells were compared to labeled cells in the lateral field of amphibians [Morona et al, 2005[Morona et al, , 2006b].…”