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

Significance Calcium-binding proteins (CaBPs) are key determinants of cellular functions, as well as useful anatomical markers for neural subpopulations. Here, we reveal the distribution and phenotypes of neurons expressing neuronal calcium-binding proteins 1 and 2 (NECAB1/2) in intact mouse dorsal root ganglia (DRGs) and spinal cord and after nerve injury using immunohistochemistry and the CLARITY method. In DRGs, NECAB1/2 are expressed in high numbers (∼70%) of all DRG neurons, including nociceptors. Axonal injury down-regulates NECAB2 in DRGs. In spinal cord, NECAB1/2 show a complementary distribution, mostly in excitatory neurons, and represent unique molecular markers for commissural neurons originally described by Ramón y Cajal. Our characterization of NECABs at the spinal level provides a basis for exploring their role in sensory functions, particularly pain. Neuronal calcium (Ca2+)-binding proteins 1 and 2 (NECAB1/2) are members of the phylogenetically conserved EF-hand Ca2+-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 Ca2+-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 Ca2+-binding proteins in pain-related 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.

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