Neuronal calcium-binding proteins 1/2 localize to dorsal root ganglia and excitatory spinal neurons and are regulated by nerve injury

Abstract

Neuronal calcium (Ca 2+ )-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca 2+ -binding protein superfamily. To date, NECABs have been explored only to a limited extent and, so far, not at all at the spinal level. Here, we describe the distribution, phenotype, and nerve injury-induced regulation of NECAB1/NECAB2 in mouse dorsal root ganglia (DRGs) and spinal cord. In DRGs, NECAB1/2 are expressed in around 70% of mainly small-and medium-sized neurons. Many colocalize with calcitonin gene-related peptide and isolectin B4, and thus represent nociceptors. NECAB1/2 neurons are much more abundant in DRGs than the Ca 2+ -binding proteins (parvalbumin, calbindin, calretinin, and secretagogin) studied to date. In the spinal cord, the NECAB1/2 distribution is mainly complementary. NECAB1 labels interneurons and a plexus of processes in superficial layers of the dorsal horn, commissural neurons in the intermediate area, and motor neurons in the ventral horn. Using CLARITY, a novel, bilaterally connected neuronal system with dendrites that embrace the dorsal columns like palisades is observed. NECAB2 is present in cell bodies and presynaptic boutons across the spinal cord. In the dorsal horn, most NECAB1/2 neurons are glutamatergic. Both NECAB1/2 are transported into dorsal roots and peripheral nerves. Peripheral nerve injury reduces NECAB2, but not NECAB1, expression in DRG neurons. Our study identifies NECAB1/2 as abundant Ca 2+ -binding proteins in painrelated DRG neurons and a variety of spinal systems, providing molecular markers for known and unknown neuron populations of mechanosensory and pain circuits in the spinal cord.commissural interneurons | GAD67 | neuropathic pain | VGLUT2

Orthopedic surgery modulates neuropeptides and BDNF expression at the spinal and hippocampal levels

“…Tibial fracture surgery with intramedullary pinning was performed essentially as described (22,110). Complete transection of the sciatic nerve (axotomy) was performed as previously described (129,130) (SI Materials and Methods).…”

“…For IHC, mice were deeply anesthetized, perfused transcardially with 4% (wt/vol) paraformaldehyde containing picric acid, and processed for staining as previously described (130,132). For ISH, mice were deeply anesthetized and brains were removed immediately and stored in a −80°C freezer after sacrifice (SI Materials and Methods).…”

“…Sections were dried at room temperature for at least 30 min and then incubated with primary antibodies (Table S2) diluted in PBS containing 0.2% (wt/vol) BSA (Sigma) and 0.03% Triton X-100 (Sigma) in a humid chamber at 4°C for 48 h. Immunoreactivities were visualized using the TSA Plus Kit (PerkinElmer) as previously described (130,132) (SI Materials and Methods). Of note, all micrographs showing BDNF staining are based on the Amgen antibody (Table S2).…”

Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing

“…The payoffs of such a method are optical access throughout large volumes of tissues, enabling the study of cell-to-cell spatial relationships and long-range neural connectivity in the context of preserved tissue morphology (Chung and Deisseroth, 2013; Chung et al, 2013; Kim et al, 2013; Zhang et al, 2014). In conjunction with fluorescent tracers, tissue clearing facilitates the identification of interacting cellular structures, including diverging or converging nerves and vasculature at their target sites throughout the body.…”

“…Although a comprehensive discussion of their respective strengths and weaknesses is beyond the scope of this text, a few critical points merit mention. First, in the original proof-of-principle for each of these techniques, the detailed methods and optimized protocols were only presented for clearing brain tissue, and occasionally for the spinal cord (Erturk et al, 2012; Zhang et al, 2014) or whole embryo (Dodt et al, 2007; Hama et al, 2011). 3DISCO represents, to date, the most complete elucidation of a clearing method in peripheral tissues.…”