Antigen retrieval pre-treatment causes a different expression pattern of Cav3.2 in rat and mouse spinal dorsal horn

Abstract

Cav3 channels consist of three isoforms, Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α1I), which produce low-threshold spikes that trigger burst firings in nociceptive neurons of the spinal dorsal horn (SDH) and dorsal root ganglion (DRG). Although Cav3.2 plays a crucial role in pathological pain, its distribution in SDH still remains controversial. One study showed that Cav3.2 is ubiquitously expressed in neurons, but another study implied that Cav3.2 is expressed restricted to astrocytes. To unravel these discrepancies, we used methods of immunohistochemistry either with or without antigen retrieval (AR) pre-treatment to detect Cav3 in SDH and DRG from both rats and mice. Moreover, Cav3.2 mRNA was detected in mice SDH using in situ hybridization. We found that the expression pattern of Cav3.2 but not Cav3.1 and Cav3.3 in SDH were largely different with or without AR pre-treatment, which showed a neuron-like and an astrocyte-like appearance, respectively. Double staining further demonstrated that Cav3.2 was mainly co-stained with the neuronal marker NeuN in the presence of AR but was with glial fibrillary acidic protein (GFAP, marker for astrocytes) in the absence of AR pre-treatment. Importantly, Cav3.2 mRNA was mainly co-localized with Cav3.2 but not GFAP. Together, our findings indicate that AR pre-treatment or not impacts the expression pattern of Cav3.2, which may make a significant contribution to the future study of Cav3.2 in SDH.

Figure 1.

Expression of Ca_v3.1-3.3 isoforms (Alomone) in SDH and DRG of SD rats with or without AR pre-treatment. Representative confocal images of Ca_v3.1 (A), Ca_v3.2 (B), and Ca_v3.3 (C) in SDH without AR pre-treatment (No AR). Representative confocal images of Ca_v3.1 (D), Ca_v3.2 (F), and Ca_v3.3 (F) in SDH with AR pre-treatment (AR). Representative images of Ca_v3.1 (G), Ca_v3.2 (H), and Ca_v3.3 (I) in DRG without AR pre-treatment (No AR). Representative images of Ca_v3.1 (J), Ca_v3.2 (K), and Ca_v3.3 (L) in DRG with AR pre-treatment (AR).

Figure 2.

Expression of Ca_v3.1-3.3 isoforms (Alomone) in mice SDH sections pre-treated with AR or not. Representative images of Ca_v3.1 (A), Ca_v3.2 (B), and Ca_v3.3 (C) in SDH without AR pre-treatment (no AR). Representative images of Ca_v3.1 (D), Ca_v3.2 (E), and Ca_v3.3 (F) in SDH with AR pre-treatment (AR).

Figure 3.

Expression of anti- Ca_v3.2 from two companies in rat SDH with or without AR pre-treatment. A) Representative image of Ca_v3.2 (Alomone, green) in SDH without AR pre-treatment (No AR). B) Representative image of Ca_v3.2 (Santa Cruz, red) in SDH without AR pretreatment (No AR). C) Merged images of (A) and (B). D) Representative image of Ca_v3.2 (Alomone, green) in SDH with AR pre-treatment (AR). E) Representative image of Ca_v3.2 (Santa Cruz, red) in SDH with AR pre-treatment (AR). F) Merged images of (D) and (E).

Figure 4.

Expression pattern of Ca_v3.2 (Alomone) in rat SDH with or without AR pre-treatment. Representative images of Ca_v3.2 (A, green), GFAP (B, red), NeuN (C, magenta), merged images of Ca_v3.2 and GFAP (D), and merged images of Ca_v3.2 and NeuN (E) in SDH without AR pre-treatment (No AR). Representative images of Ca_v3.2 (F, green), GFAP (G, red), NeuN (H, magenta), merged images of Ca_v3.2 and GFAP (I), and merged images of Ca_v3.2 and NeuN (J) in SDH with AR pre-treatment (AR). K) Quantitative colocalization results of Ca_v3.2-IR with NeuN- and GFAP-IR. Representative images of Ca_v3.2 (Alomone) in SDH from wild-type mice (L) and Ca_v3.2 knock-out (KO) mice (M). **P<0.01, ***P<0.001.

Figure 5.

Expression pattern of Ca_v3.2 mRNA in mouse SDH. A) Microscopic images of a DIG-labeled Ca_v3.2 probe for in situ hybridization in mice SDH. Triple-labeling of in situ hybridization sections for Ca_v3.2 mRNA (B, purple) and the following IHC staining of Ca_v3.2 (C, green, Alomone), GFAP (D, red), DAPI (E, magenta), merged images of Ca_v3.2 and GFAP (F), and merged images of Ca_v3.2 and DAPI (G). Note that Ca_v3.2 mRNA is expressed in Ca_v3.2-positive cells (arrows) but not GFAP-positive astrocytes.