Supplementary Material for: Developmental Changes in Voltage-Gated Calcium Channel α<sub>2</sub>δ-Subunit Expression in the Canine Dorsal Root Ganglion

The voltage-gated calcium channel subunit α<sub>2</sub>δ plays a fundamental role in propagation of excitatory signals associated with release of glutamate and neuropeptides substance P (SP) and calcitonin gene-related protein (CGRP). It can be selectively inhibited by gabapentinoids. Hence, investigation of the α<sub>2</sub>δ subunit may predict the efficacy of gabapentinoid therapy in neuropathic pain. Since sensory processing underlies significant age-related changes, this study was conducted in order to elucidate the role of the α<sub>2</sub>δ subunit in the sensory transmission during canine development. Dorsal root ganglia (DRG) were harvested from four spinal segments of 16 puppies and 10 adult dogs without a history of neurological signs, pain, spinal disease or orthopedic disorders. α<sub>2</sub>δ-Subunit expression and coexpression with SP and CGRP was evaluated immunohistochemically regarding the number of immunopositive ganglion cells, staining intensity and subcellular distribution. All tested ganglia were immunopositive for α<sub>2</sub>δ. Cell counts and expression levels were significantly lower in pups than in adult dogs (p < 0.05). In the cervical segments of both groups, the number and percentage of immunopositive neurons was significantly higher than in lumbar DRG (p < 0.05). Multilabeling studies in all tested animals confirmed the coexpression of α<sub>2</sub>δ and pain peptides SP and CGRP. This anatomical study for the first time documents the involvement of α<sub>2</sub>δ subunits in sensory signal processing in dogs. The proportion of positive neurons and the intracellular expression levels show a net increase from early postnatal life to adulthood. A significant portion of α<sub>2</sub>δ-positive cells in the dogs exhibited C- and Aδ-phenotypes compatible with nociceptive neurons. The coexpression of α<sub>2</sub>δ, SP and CGRP imply that these neurons are involved with peptidergic nociception. The cervicolumbar gradient of α<sub>2</sub>δ expression in adults reflects functional differences in between forelimbs and hind limbs. These data will facilitate translational studies on neuropathic pain states in this species such as common canine nerve entrapment syndromes.