Presumptive Renshaw cells contain decreased calbindin during recovery from sciatic nerve lesions.

Abstract

A subpopulation of calbindin-immunoreactive neurons in lamnina VII of the spinal cord has been identified by its location as Renshaw cells, the anatomical substrate for recurrent inhibition. The expression of calbindin (28 kDa) in these calbindin-containing rat ventral horn interneurons was studied with immunocytochemistry after sciatic nerve injuries.One week after axotomy calbindin immunoreactivity was strongly reduced on the lesioned side between levels L4 and L6, while calbindin-containing neurons and fibers were still numerous contralateraily and cranially to the lesioned levels. Low-affinity nerve growth factor receptor (LNGFR) immunoreactivity was used as marker for peripherally lesioned a-motoneurons, since previous studies showed that motoneurons express LNGFR during regeneration after sciatic nerve injuries (22, 24). MATERIALS AND METHODSSciatic Nerve Lesions. For this study, six female SpragueDawley rats weighing 200-220 g were anesthetized intraperitoneally with a mixture of ketamine, acepromazine, and xylazine, and their right sciatic nerve was crushed with forceps at the mid-thigh level three times for 5 sec and left in place. Six other animals received a sciatic nerve cut with removal ofa 2-cm segment to prevent reconnection ofthe two stumps. Six sham-operated rats were used as controls. Three further rats received an intramuscular injection of 5 ng of botulinum toxin A (Sigma) in 5 ,u of saline in the right tibialis anterior muscle. Three control rats received an injection of saline. Four normal unperturbed rats were also used.Immunohistochemistry. Rats were deeply anesthetized and perfused intracardially with ice-cold heparinized 0.1 M phosphate buffer (pH 7.4) followed by 250 ml of ice-cold 4% paraformaldehyde in phosphate buffer.Spinal cords were treated as described (24). Briefly, the spinal cord was removed after laminectomy, anchored to a dental wax sheet to prevent curvature or distortions, and postfixed in 4% paraformaldehyde in phosphate buffer for 16 hr at 4°C. The spinal cords were then rinsed in phosphate buffer for 24 hr and cryoprotected in 10% and 30% sucrose in phosphate buffer for 24 hr each. Prior to sectioning, the spinal cords were divided under the dissecting microscope into three segments by cuts across the dorsal roots at C8 and T10; in the present study, only the caudal segment was analyzed. The dorsal root entry zone on the right side of Li was marked with a small cut for orientation. The spinal cord segments were then sectioned on a freezing microtome, and individual sections (40 ,um) were collected and stored at 4°C in Millonig's buffer until immunocytochemistry.Sections were incubated free floating at room temperature in 0.1 M Tris (pH 7.4) containing 1.4% NaCl and 10%o (vol/vol) heat-inactivated horse serum (Tris-serum) for 30 min. The sections were then treated in Tris-serum with a mouse anti-calbindin monoclonal antibody (25) used at a dilution of 1:6000 overnight or with the mouse anti-LNGFR monoclonal antibody (26) 192-IgG used at 5 ug/ml for 30 hr. LNGFR...

Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells by Motor Axon Excitatory Inputs

“…BoNT-A effects began to recover ϳ6 -8 d after injection (P11-P13). These short time courses of toxin-induced paralysis contrast with the long-lasting paralysis (15-20 d for TeNT and 1-3 months for BoNT-A) induced in adult animals (Sanna et al, 1993;Moreno-Ló pez et al, 1997;Gonzalez-Forero et al, 2003).…”

“…2, available at www.jneurosci.org as supplemental material) (Sanna et al, 1993). The number of calbindin-IR neurons in the ventral 200 m of laminas VII and IX (Renshaw cells) was, however, similar in both spinal cord sides in all of these animals.…”

“…Renshaw cells were identified by their intense calbindin immunoreactivity (Antal et al, 1990;Ardvidsson et al, 1992;Sanna et al, 1993;Carr et al, 1998;Fallah and Clowry, 1999;Geiman et al, 2000;McDonough et al, 2001). Immunoreactive sites were visualized with donkey anti-rabbit, anti-mouse, or anti-goat IgGs coupled to cyanine 3 (Cy3) or FITC (Jackson ImmunoResearch, West Grove, PA).…”

Postnatal phenotype and localization of spinal cord V1 derived interneurons

“…Calbindin is a good marker for Renshaw cells (RCs) in the spinal cord ventral horn (Ardvisson et al, 1992;Sanna et al, 1993;Carr et al, 1998;Alvarez et al, 1999;Geiman et al, 2000). In addition a large number of other adult ventral interneurons express parvalbumin or calretinin (Antal et al, 1990;Ren and Ruda, 1994).…”

Androgen Mitigates Axotomy-Induced Decreases in Calbindin Expression in Motor Neurons

“…Changes in calbindin expression have been observed in the C NS after injury. For instance, kindling reduces calbindin content in granule cells of the dentate gyrus (Köhr and Mody, 1991); Renshaw interneurons decrease calbindin immunoreactivity after axotomy of the presynaptic motor neurons (Sanna et al, 1993), and the number of calbindinimmunopositive cells increases after axotomy of sympathetic ganglion cells (Sánchez-Vives et al, 1994). In patients with Huntington's and Alzheimer's diseases, calbindin expression is downregulated in the forebrain and striatum (Iacopino and Christakos, 1990a).…”