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Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

ABSTRACT: INTRODUCTION:Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations. METHODS:This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single-cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease-activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5-hydroxytryptamine receptor agonist (5-HT), bovine adrenal medulla peptide (BAM8-22), substance P, allyl isothiocyanate (AITC), and capsaicin. RESULTS:This study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens. CONCLUSION:Ratiometric calcium imaging demonstrated that small-diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

SUBMITTER: Ganchingco JRC

PROVIDER: S-EPMC6908857 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

Ganchingco Joy Rachel C JRC Fukuyama Tomoki T Yoder Jeffrey A JA Bäumer Wolfgang W

Brain and behavior 20190930 12

<h4>Introduction</h4>Rodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations.<h4>Methods</h4>This study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single-cell ratiometric calcium imaging, with the activatio ...[more]

PMID: 31571393

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Project description:Major aspects of neuronal function are regulated by Ca(2+) including neurotransmitter release, excitability, developmental plasticity, and gene expression. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited both greater excitability and evoked release of neuropeptides compared to wildtype mice. Furthermore, augmented voltage-dependent sodium currents but not potassium currents contribute to the enhanced excitability. To determine the mechanisms giving rise to the enhanced release of substance P and calcitonin gene-related peptide in the Nf1+/- sensory neurons, the potential differences in the total voltage-dependent calcium current (ICa) as well as the contributions of individual Ca(2+) channel subtypes were assessed. Whole-cell patch-clamp recordings from small-diameter capsaicin-sensitive sensory neurons demonstrated that the average peak ICa densities were not different between the two genotypes. However, by using selective blockers of channel subtypes, the current density of N-type (Cav2.2) ICa was significantly larger in Nf1+/- neurons compared to wildtype neurons. In contrast, there were no significant differences in L-, P/Q- and R-type currents between the two genotypes. Quantitative real-time polymerase chain reaction measurements made from the isolated but intact dorsal root ganglia indicated that N-type (Cav2.2) and P/Q-type (Cav2.1) Ca(2+) channels exhibited the highest mRNA expression levels although there were no significant differences in the levels of mRNA expression between the genotypes. These results suggest that the augmented N-type (Cav2.2) ICa observed in the Nf1+/- sensory neurons does not result from genomic differences but may reflect post-translational or some other non-genomic modifications. Thus, our results demonstrate that sensory neurons from Nf1+/- mice, exhibit increased N-type ICa and likely account for the increased release of substance P and calcitonin gene-related peptide that occurs in Nf1+/- sensory neurons.

Dataset Information

Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

Publications

Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

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