Neurons containing calcitonin gene-related peptide without substance P in nociception

Author: Garreth Kestell

Kestell, Garreth, 2015 Neurons containing calcitonin gene-related peptide without substance P in nociception, Flinders University, School of Medicine

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The neural processing of pain is not entirely understood. One of the largest problems limiting the current understanding of nociception is the widespread use of oversimplified classification schemes. Nociceptors are classified as either peptidergic or nonpeptidergic. Peptidergic nociceptors usually co-express the peptides calcitonin gene-related peptide (CGRP) and substance P (SP; CGRP+SP+). CGRP enhances the nociceptive effects of SP, but has very little effect by itself. Yet, in mice dorsal root ganglia (DRG), some neurons express CGRP without SP (CGRP+SP-) and these neurons are not yet characterised.

The prevalence and distribution of CGRP+SP- neurons and their neurochemical profile was determined using multiple labelling immunohistochemistry. In DRG CGRP+SP- neurons accounted for half of the CGRP-immunoreactive (IR) DRG neurons. Cutaneous CGRP+SP- fibres were numerous within dermal papillae and around hair shafts, and CGRP+SP- boutons were prevalent in lateral laminae I/II and in lamina IV/V of the spinal dorsal horn. CGRP+SP- neurons were distinguished from CGRP+SP+ neurons by their much larger soma size in DRG, and by their expression of the myelinated marker NF200 and lack of expression of the capsaicin sensitive TRPV1 receptor, which is possessed by most peptidergic nociceptors. Overall, there were two subpopulations of CGRP+SP- neurons: medium myelinated Aδ-fibre CGRP+SP-TRPV1+ neurons and medium-large sized myelinated Aδ/Aβ-fibre CGRP+SP-TRPV1- neurons.

To determine the central and peripheral projections of the CGRP+SP- neurons axonal tracing was coupled with multiple-labelling immunohistochemistry. Retrograde tracing from muscle tissue revealed that muscular CGRP+SP- afferents originated from the larger CGRP+SP- somata. Anterograde tracing revealed dual central projections of CGRP+SP- neurons in the laminae I/II and laminae IV/V of the spinal dorsal horn. The finer, most likely Aδ-fibre, CGRP+SP- neurons terminated in lamina I/II, whereas the larger, most likely Aβ-fibre, CGRP+SP- neurons terminated in lamina IV/V. The laminae IV/V CGRP+SP- fibres were not classical VGluT1-IR mechanoceptors.

The central targets of these neurons were identified using the activated extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB) as markers for neuronal activation. Viable spinal cord slices were stimulated both electrically and with capsaicin. Noxiously activated neurons received contacts from CGRP+SP- afferents in lamina I. In contrast, no cells were activated in lamina IV/V. Therefore, it is likely that the CGRP+SP- afferents of lamina IV/V were not nociceptive.

Overall, this research has lead to an improved understanding of the properties of an uncharacterised population of presumed nociceptors. CGRP-IR neurons could be subdivided based on their SP expression. CGRP+SP- neurons could be further subdivided based on their TRPV1 expression: Aδ-fibre CGRP+SP-TRPV1+ neurons and Aδ/Aβ-fibre CGRP+SP-TRPV1- neurons. CGRP+SP-TRPV1+ and CGRP+SP-TRPV1- neurons are unlikely to be involved in acute nociception. The CGRP+SP-TRPV1+ neurons had central terminations in laminae I/II and likely had a modulatory role in nociception that may contribute to the central sensitisation resulting in hyperalgesia associated with inflammatory and chronic pain states. The CGRP+SP-TRPV1- neurons are likely an uncharacterized population of mechanoceptors. While these neurons may not be involved in nociception, they may synapse on wide dynamic range neurons and could contribute to mechanical allodynia.

Keywords: spinal cord, dorsal root ganglia, nociception, pain, peptides, calciton gene-related peptide

Subject: Medicine thesis, Anatomy and Histology thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Ian Gibbins