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 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.Calcium (Ca
2+) plays a crucial role in many and diverse cellular processes, including neurotransmission (
1). Glutamate and neuropeptides are neurotransmitters released from the central terminals of dorsal root ganglion (DRG) neurons in the spinal dorsal horn, where signals for different sensory modalities, including pain, are conveyed to higher centers (
2–
12). Neurotransmitter release is tightly regulated by Ca
2+-dependent SNARE proteins whose activity is regulated by Ca
2+-binding proteins (CaBPs) (
1,
7,
13).Parvalbumin (PV), calbindin D-28K (CB), calretinin (CR), and secretagogin (Scgn) are extensively studied EF-hand CaBPs, and they have also emerged as valuable anatomical markers for morphologically and functionally distinct neuronal subpopulations (
14–
17). The expression of CaBPs in DRG neurons has been thoroughly studied (
18). Moreover, neuronal Ca
2+ sensor 1 and downstream regulatory element-antagonist modulator (DREAM) are also EF-hand Ca
2+-binding proteins in DRGs and the spinal cord (
19,
20). Despite these advances, a CaBP has so far not been characterized in the majority of small- and medium-sized DRG neurons, many of which represent nociceptors.The subfamily of neuronal Ca
2+-binding proteins (NECABs) consists of three members (NECAB1–NECAB3), probably as a result of gene duplication (
21). NECABs are also EF-hand proteins, with one pair of EF-hand motifs in the N terminus and a putative antibiotic biosynthesis monooxygenase domain in the C terminus, which are linked by a NECAB homogeneous region (
22). NECAB1/2 are restricted to the nervous system, whereas NECAB3 is also expressed in the heart and skeletal muscle (
21).NECAB1 was first identified as the target protein of synaptotagmin I C2A-domain by affinity chromatography, with its expression restricted to layer 4 cortical pyramidal neurons, inhibitory interneurons, and hippocampal CA2 pyramidal cells in mouse brain (
21,
23). The gene of the second member was cloned from mouse and initially named
Necab. It encodes a 389-aa (NECAB2) (
24). NECAB2 was identified as a downstream target of Pax6 in mouse retina, which is involved in retinal development (
24,
25), as well as being a binding partner for the adenosine A
2A receptor (
22). Furthermore, an interaction between NECAB2 and metabotropic glutamate receptor 5 (mGluR5) was demonstrated in rat hippocampal pyramidal cells, possibly regulating mGluR5’s coupling to its signaling machinery (
26). Finally, NECAB3, also known as XB51, was isolated as an interacting target for the neuron-specific X11-like protein and is possibly involved in the pathogenesis of Alzheimer’s disease (
27,
28).Very recently, NECAB1/2 were shown to have complementary expression patterns in mouse hippocampus at the mRNA and protein levels, whereas NECAB3 is broadly distributed in the hippocampus (
29). NECAB1-expressing cells were seen throughout the cell-sparse layers of Ammon’s horn and the hilus of the dentate gyrus. In contrast, NECAB2 is enriched in pyramidal cells of the CA2 region. A minority of NECAB1
+ neurons were GABAergic yet did not coexpress PV, CB, or CR (
29).Here, we investigated the expression of NECAB1/2 in mouse DRGs and spinal cord using quantitative PCR (qPCR), immunohistochemistry (also combined with CLARITY) (
30), and Western blotting. We compared the distribution of NECABs with that of the four CaBPs restricted to neurons, PV, CB, CR, or Scgn. NECAB
+ neurons in the spinal dorsal horn were phenotyped using transgenic mice harboring genetic markers for excitatory [vesicular glutamate transporter 2 (VGLUT2)] (
31) or inhibitory [glutamate decarboxylase 67 (GAD67)] (
32) cell identities. Finally, the effect of peripheral nerve injury was analyzed.
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