Calbindin-Containing Neurons in the Ventral Horn of the Gray Matter of the Spinal Cord in Mice

Abstract

Experiments were performed on four C57black/6 mice to study neurons containing calbindin protein (CB) with a molecular weight of 28 kD and neurofi laments (NF) with a molecular weight of 200 kD in the ventral horn of the gray matter of spinal cord (SC) segments T II , L IV , L V , and L VI . Immunopositive neurons were detected by labeling with antibodies against CB and double labeling with antibodies against CB and NF. The whole cell population was labeled with NeuroTrace Red Fluorescent Nissl stain. The results of these studies showed that CB-immunopositive (CB + ) neurons detected in the ventromedial area of the ventral horn at all levels of the SC were Renshaw cells. CB + interneurons located in the medial part of the ventral horn were present only in the lumbar segments of the SC. CB + motoneurons detected in the medial part of the ventral horn were present in one SC segment -L IV -and also contained NF protein.Keywords: spinal cord, neurons, 28 kD calbindin, 200 kD neurofi laments, mouse.Among the factors regulating the cellular functions and plasticity, one important one is the maintenance of a particular Ca 2+ concentration, and intracellular Ca-binding proteins play a significant role in this process [21,22,24]. Furthermore, axonal transport is a Ca-dependent process, and an excess of calcium ions within the cell degrades the displacement of neurofilament (NF) proteins, which move from the cell body to the axon terminal by slow transport [9,14,15].Motoneurons and interneurons in the ventral horn of the spinal cord (SC), including Renshaw cells, are known to display immunoreactivity for a wide spectrum of Ca-binding proteins: calbindin (CB), calmodulin, parvalbumin, and calretinin [6,11,16]. The neuromodulatory function of Ca-binding proteins is associated with their distribution in cholinergic and NO-ergic neurons in the ventral horn and the neuroprotective properties of these proteins [17,18].Understanding the functional relationships between motoneurons and interneurons in the ventral horn of the SC requires knowledge of whether Ca-binding proteins and NF are colocated.The aim of the present work was to study neurons in the ventral horn of the gray matter of the SC demonstrating immunopositivity for 28-kD CB and 200-kD NF. Materials and MethodsStudies were performed on four C57black/6 mice weighing 20 ± 5 g in compliance with the "Regulations for Studies Using Experimental Animals." SC was fixed in 4% paraformaldehyde in patient-buffered saline (PBS; 0.01 M, pH 7.4) (BioloT, Russia) for 2 h at 4°C, after which specimens were washed three times 30 min in PBS and placed in 30% sucrose for 24 h at 4°C. Segments T II , L IV , L V , and L VI were harvested from SC and frozen in cryogel (Tissue-Tek O.C.T. compound, Sakura Finetek, Holland). Transverse sections of thickness 16 μm were cut from these specimens on a cryostat (Shandon E, Thermo Scientific, UK). The immunohistochemical, morphometric, and topographical characteristics of interneurons were studied in the ventral horn of the gray matter of the SC an...