Secretagogin is expressed in sensory CGRP neurons and in spinal cord of mouse and complements other calcium-binding proteins, with a note on rat and human

Abstract

Background: Secretagogin (Scgn), a member of the EF-hand calcium-binding protein (CaBP) superfamily, has recently been found in subsets of developing and adult neurons. Here, we have analyzed the expression of Scgn in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), and in spinal cord of mouse at the mRNA and protein levels, and in comparison to the well-known CaBPs, calbindin D-28k, parvalbumin and calretinin. Rat DRGs, TGs and spinal cord, as well as human DRGs and spinal cord were used to reveal phylogenetic variations.

Results: We found Scgn mRNA expressed in mouse and human DRGs and in mouse ventral spinal cord. Our immunohistochemical data showed a complementary distribution of Scgn and the three CaBPs in mouse DRG neurons and spinal cord. Scgn was expressed in ~7% of all mouse DRG neuron profiles, mainly small ones and almost exclusively co-localized with calcitonin gene-related peptide (CGRP). This co-localization was also seen in human, but not in rat DRGs. Scgn could be detected in the mouse sciatic nerve and accumulated proximal to its constriction. In mouse spinal cord, Scgn-positive neuronal cell bodies and fibers were found in gray matter, especially in the dorsal horn, with particularly high concentrations of fibers in the superficial laminae, as well as in cell bodies in inner lamina II and in some other laminae. A dense Scgn-positive fiber network and some small cell bodies were also found in the superficial dorsal horn of humans. In the ventral horn, a small number of neurons were Scgn-positive in mouse but not rat, confirming mRNA distribution. Both in mouse and rat, a subset of TG neurons contained Scgn. Dorsal rhizotomy strongly reduced Scgn fiber staining in the dorsal horn. Peripheral axotomy did not clearly affect Scgn expression in DRGs, dorsal horn or ventral horn neurons in mouse.

Conclusions: Scgn is a CaBP expressed in a subpopulation of nociceptive DRG neurons and their processes in the dorsal horn of mouse, human and rat, the former two co-expressing CGRP, as well as in dorsal horn neurons in all three species. Functional implications of these findings include the cellular refinement of sensory information, in particular during the processing of pain.

Figure 1

Scgn expression in DRGs. (a) Quantitative (real-time) PCR detection of Scgn mRNA in mouse olfactory bulb (OB), ventral and dorsal horns (VH/DH) of L4-5 spinal cord segments, and L4-5 DRGs. Scgn mRNA is found expressed at significantly lower levels in VH and DRGs than in the OB. Scgn mRNA is under the detection threshold in DH. Gapdh is used as internal control. (b-c) Scgn mRNA expression in human DRG (hDRG). Semitransparent purple and green shades outline the surface area of Scgn mRNA-positive and negative cells, respectively. Note that tissues are counterstained by cresyl violet after emulsion-radiography of the mRNA hybridization signal. Scgn-LI in control L5 mDRGs (d-m”). Immunofluorescence micrographs of sections incubated with antiserum to Scgn (d-m), CGRP (f’), IB4 (g’), TRPV1 (h’), GRP (i’), NF200 (j’), CB (k’), PV (l’) or CR (m’). Color micrographs show merged micrographs after double-staining (f-f”, g-g”, h-h”, i-i”, j-j”, k-k”, l-l”, m-m” show, respectively, the same section). (d) Several Scgn-IR cells are seen. Boxed area shows Scgn staining also in the nucleus. Arrowheads indicate Scgn-positive fibers. (e) Double labeling shows the same staining pattern present after incubation with two different Scgn antibodies (arrowheads). (f-m”) Arrowheads indicate coexistence of Scgn (yellow) with CGRP (f-f”), TRPV1 (h-h”), NF200 (j-j”) and CR (m-m”), most pronounced for Scgn plus CGRP (f-f”). Scgn-LI cannot be seen in IB4-positive (g-g”), GRP (i-I”), CB-(k-k”) or PV-IR (l-l”) neurons. Scale bars indicate 10 μm (b,c), 50 μm (d,e-m”).

Figure 2

Scgn-LI in control L5 rDRGs. (a-e) Double-immunofluorescence micrographs of sections incubated with antiserum to Scgn (a-e; green) plus CGRP (a; red), IB4 (b; red), CB (c; red), PV (d; red) or CR (e; red). Arrows indicate coexistence of Scgn with CB (c; yellow). Scale bar indicates 50 μm (a-e).

Figure 3

Scgn-LI in hDRG and human spinal cord. (a-d) Immunofluorescence micrographs of section incubated with antiserum against Scgn (a,b,d) or/and CGRP (c,d). (b-d) show same section processed for double-immunofluorescence. Arrowheads indicate Scgn-IR neurons (a), and neurons co-expressing (solid arrowheads in d; yellow) Scgn- (b, d) and CGRP-LI (c, d). Note that all Scgn-IR neurons are CGRP-IR. (e-g) Immunofluorescence micrographs of section incubated with antiserum against Scgn (e, g) or/and IB4 (f, g). e-g show same section processed for double-immunofluorescence. Arrowheads indicate Scgn-IR fibers (e, g). Note that no Scgn-IR neuron is IB4-positive. (h-p) Immunofluorescence micrographs of a section incubated with antiserum against Scgn (h,j,k,m,n and p) or/and CGRP (i,j,l,m,o and p). h-j show the double-labeling of Scgn and CGRP in human spinal cord at low magnification. Scgn-IR fibers are concentrated in the lateral superficial layer and some of them show co-localization with CGRP as indicated by arrowheads (inset in j). k-m show Scgn-IR interneurons surrounded by some positive nerve endings in inner layer of lamina II (arrowheads indicate Scgn-IR interneurons). n-p show the co-localization of Scgn-LI and CGRP-LI in axons in the dorsal roots (arrowheads, yellow). Scale bars indicate 200 μm (a, h-j), 100 μm (b-g), 20 μm (k-m, n-p and inset in j).

Figure 4

Scgn-LI in mTG and rTG. (a-d) Immunofluorescence micrographs showing mTG (a,b) and rTG (c,d) after double-staining with antiserum against Scgn (a-d; green) and CGRP (a,c; red) or IB4 (b,d; red). Arrows indicate coexistence of Scgn (yellow) with CGRP (a,c) or IB4 (d). Note coexistence of Scgn with CGRP in m- and rTGs, and of Scgn with IB4 in rTG but not in mTG. Scale bars indicate 50 μm (a=b; c=d).

Figure 5

Scgn-LI in control mouse lumbar spinal cord. (a-t) Immunofluorescence micrographs of sections incubated with antiserum against Scgn (a-g, i-t), CGRP (h, i), TRPV1 (j-j”), GRP (k, k’), PKC gamma (l, l’), CB (m, m’, r-t), PV (n, o), or/and CR (p, q). Scgn-IR fibers are seen in the superficial dorsal horn (arrowheads in a, b) and also processes in lamina III (a, b). Scgn-IR cell bodies are present in lamina II (small-sized in c), a few in III (d) or medial (multi-polar in e) and lateral (small-sized in f) parts of lamina IV. (g-i) Ventral horn neurons express Scgn-LI (g, i) and are mostly CGRP-IR (arrowhead in i). Scgn-IR (j”, k’, I’; green) neurons do not express TRPV1 (j,j”), GRP (k, k’) or PKC gamma (l, l’) in dorsal horn. (m-q) Filled Arrowheads indicate coexistence (yellow) of Scgn-LI (m’, o, q, green) and CB (m, red), PV (o; red) or CR (q; red) in the dorsal horn. (r-t) Triple-labeling of superficial dorsal horn showing (r and r’ same section) a local neuron (light blue; filled arrowhead) coexpressing Scgn (r’; green) and CB (r’; read), but not PV (r’; dark blue). s and t show neurons (filled arrowhead; light blue) containing Scgn (green) and CB (red), but not CR (dark blue). Empty arrowheads indicate a dorsal horn neuron only express Scgn-LI (j'',k',l',o,q,t) or a Scgn-IR fiber (j'; green) does not overlap with TRPV1-IR fiber (j'; red). Scale bars indicate 200μm (a), 50 μm (b=m=n=p; c=d=e=f; g=h=i; j=k=l; j’=j”; k’=l’; m’=o=q; r=s; r’=t).

Figure 6

Scgn-LI in mouse spinal cord and sciatic nerve after rhizotomy or nerve ligation. (a-f) Immunofluorescence micrographs of sections incubated with antiserum against Scgn- (a,c,d,f) and CGRP- (b,c,e,f) LIs in lumbar spinal cord 10 days after unilateral, dorsal rhizotomy (d-f). (a-f) On the contralateral side both Scgn- and CGRP-LIs are strong in the superficial dorsal horn, the former displaying two bands (arrows in a, c), the latter filling out layers I and outer II (arrows in b,c). The merged micrograph (c) shows that the deeper band of Scgn-LI, consisting mainly of cell bodies, essentially remains in inner lamina II, without much overlap with the CGRP-LI (d-f). There is a strong ipsilateral decrease of both Scgn-LI (cf. d, f with a, c; arrows) and CGRP-LI (cf. e, f with b, c), compared to a contralateral side (a-c). (g-i) Scgn-LI is weekly (arrows in g, i), and CGRP more extensively (arrow in h, i) expressed in the control sciatic nerve, and both accumulate, on the proximal side of a-10-hour ligation (arrows in j, k). Double-staining shown in merged color micrographs indicates possible coexistence (arrows in i, l). Scale bars indicate 100 μm (a-f; g-l).

Figure 7

Scgn -LI in control rat spinal dorsal horn. (a-f) Immunofluorescence micrographs after double-staining with antiserum against Scgn (a,c,d,f), CGRP (b,c) or IB4 (e,f). (a-f) Scgn-IR neurons (arrows) are seen in the dorsal horn, both in superficial and deeper layers (a,d). They partly overlap with CGRP-LI (b) and IB4 staining (e), as seen in merged color micrographs (c,f). (g-o) Double-immunofluorescence micrographs of sections incubated with Scgn antiserum (g,i,j,l,m,o; green) plus CB (h, i; red), PV (k,l; red) or CR antiserum (n,o; red). Coexistence is often seen for CB in cell bodies (arrows in g-i) and processes (arrowheads in g-i), less so for PV (arrow/arrowheads in j-l) and, here, none for CR (m-o). Arrows indicate coexistence (yellow) of Scgn-LI (i, l) and CB (i) and PV (l) in the dorsal horn neurons. Arrowheads indicate coexistence of Scgn-IR fibers (i,l) and CB (i) or PV (l). Curved arrows indicate a Scgn-positive neuron that does not express CB (g-i). Arrows indicate a CR-positive but Scgn negative neuron (m-o). Bar in f indicates 100 μm (a-f) and 50 μm (g-o).

Figure 8

Expression of Scgn protein after sciatic nerve axotomy as shown with western blot. (a, b) L4-5 DRGs (a) and L 4–5 spinal cord segments (b) after unilateral nerve transection using antiserum against Scgn and β-Actin. No obvious changes in Scgn expression are seen in ipsi- and contralateral DRGs (a) or spinal cord (b), respectively. Quantification of three western blots shows similar levels of Scgn between ipsi- and contralateral DRGs or spinal cord.