A subpopulation of capsaicin‐sensitive porcine dorsal root ganglion neurons is lacking hyperpolarization‐activated cyclic nucleotide‐gated channels

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to stabilizing resting membrane potential, thus controlling neuron excitability. Subclasses of nociceptive neurons differ in their excitability, therefore, these channels could be a distinguishing marker. We investigated isolated dorsal root ganglion neurons from a non-rodent species, the pig, Sus scrofa domesticus. Single labeling revealed capsaicin-induced cobalt-uptake in 54.3% and transient receptor potential V1 (TRPV1) immunoreactivity in 55.1% of all neurons. Ruthenium red and capsazepine suppressed capsaicin-induced cobalt-uptake. HCN-1 and HCN-2 channel isoform immunoreactivity was detected in 82.6% and 88.3%, respectively, and binding of IB4 in 29.4% of all neurons. Double labeling revealed that out of the capsaicin-positive neurons, 42.3% were IB4-positive, 80.0% immunoreactive for the HCN-1, and 77.3% for the HCN-2 channel isoform, respectively. Neurons lacking HCN-1 or HCN-2 channel isoforms were mostly capsaicin-positive and IB4-negative. The soma size of neurons lacking HCN-1 and/or HCN-2 channels was small to medium. Western blot analysis showed protein products of sizes similar to those of HCN-1 and HCN-2 channel isoforms. Functionally, in patch-clamp experiments, some neurons were unresponsive to membrane hyperpolarization, thus, probably lacking HCN channels. In conclusion, in porcine dorsal root ganglion neurons there is a subset of capsaicin-positive, IB4-negative neurons lacking HCN-1 and/or HCN-2 channel isoforms.

Increased expression of HCN2 channel protein in L4 dorsal root ganglion neurons following axotomy of L5- and inflammation of L4-spinal nerves in rats

“…In the rat trigeminal ganglia, HCN1 isoform has been reported to be expressed by all sizes of neurons (Wells et al, 2007) or to be present predominantly in large-sized neurofilament rich (A-type) neurons (Cho et al, 2009a). In porcine DRGs, a subset of capsaicin-positive (IB4-negative) neurons have been shown to lack HCN-1 and/or HCN-2 channel isoforms (Obreja et al, 2008).…”

Topography and Morphometric Characteristics of NF200+ Neurons in the Gray Matter of the Spinal Cord after Capsaicin Deafferentation

“…The introduction of chemical deafferentation methods into experimental morphology allowed particular attention to be paid to the state of nerve structures in conditions of sensory deficiency. Administration of capsaicin at neurotoxic doses is known to lead to the death of capsaicinsensitive neurons containing TRPV1 receptors [4,5,7] and to induce changes in neurofilaments (NF) in sensory ganglion neurons [6,9,12]. NF are the main structural component of the cytoskeleton of neuron axons.…”

Isolation and cultivation of primary keratinocytes from piglet skin for compartmentalized co-culture with dorsal root ganglion neurons

“…sensory neurons, requires a substrate supporting adhesion of both types. For keratinocyte cultures 3T3 feeder layer and collagen are the preferred substrate [6,27]; for sensory neurites, it is p-L-lysine or p-L-ornithin [8,42]. We therefore tested different substrates to evaluate adhesion and the growth of keratinocytes ( Table 2).…”

“…Therefore, we tested for keratinocytes the commercially available collagen coatings of human, rat and bovine origin and the coatings laminin, poly-L-orinithine and poly-L-lysine mostly used for supporting adhesion of DRG neurons [8,42].…”

“…Dorsal root ganglia were excised from the spinal cord and neurons were isolated as described previously [8]. Neurons were seeded into the central compartment at a density of approx.…”