We review many of the recent findings concerning mechanisms and pathways for pain and its modulation, emphasizing sensitization and the modulation of nociceptors and of dorsal horn nociceptive neurons. We describe the organization of several ascending nociceptive pathways, including the spinothalami

Neuroanatomy of the pain system and of the pathways that modulate pain

Synaptic structure and development: the neuromuscular junction.

Neurotrophin secretion: current facts and future prospects

Botulinum toxin type A (BoNT-A) produced by the bacterium Clostridium botulinum is a potent inhibitor of acetylcholine release in the neuromuscular junction and has been used to treat many disorders related to excessive muscle contraction. However, BoNT-A has recently been used in pain therapy to treat myofascial pain, low back pain and various types of headaches, including migraine. The purpose of this study is to investigate the antinociceptive effect of BoNT-A and its underlying mechanism in the rat formalin inflammatory pain model. BoNT-A (3.5, 7, 15 and 30 U/kg) or vehicle was administered to the plantar surface of the right hindpaw of male Sprague–Dawley rats. BoNT-A dose-dependently (P<0.05) inhibited formalin-induced nociceptive behavior during phase 2 but not during phase 1 when administered 5 h to 12 days before formalin challenge. The onset of the antinociceptive effect started at 5 h after pre-treatment and this effect lasted for at least 12 days. BoNT-A (7 U/kg) also reduced edema. Consistent with the lack of effect in the formalin phase 1, BoNT-A, at 15 U/kg, had no effect on acute thermal nociception; no local muscle weakness was observed at this dose. Pre-treatment of rats with BoNT-A (3.5, 7 or 15 U/kg) all significantly reduced formalin-evoked glutamate (Glu) release. These results demonstrate that local peripheral injection of BoNT-A significantly reduces formalin-induced nociceptive behaviors with the absence of obvious muscle weakness. Such an antinociceptive effect of BoNT-A is associated with the inhibition of formalin-induced release of Glu (and/or neuropeptides) from primary afferent terminals.

Subcutaneous administration of botulinum toxin A reduces formalin-induced pain.

Neuropeptides in perspective: The last ten years

By light microscopic immunocytochemistry it has been previously shown that approximately equal to 70% of the neurons in rat dorsal root ganglia are labeled with an antiserum for glutamate conjugated to hemocyanin; the smaller among these neurons are also positive for substance P. By using a postembedding ImmunoGold method and electron microscopy, it is shown here that synaptic terminals in the superficial laminae of the spinal cord of rats selectively stain for the same glutamate antiserum. Immunolabeling is in small dome-shaped and in large scalloped synaptic terminals. Scalloped terminals are of two types. One type consists of dark terminals with many agranular vesicles of different size and a few large granular vesicles; these are probably endings of unmyelinated and small myelinated primary afferent fibers. The other type consists of light terminals with small agranular vesicles homogeneous in size with neurofilaments and many mitochondria; these are probably endings of larger myelinated primary afferent fibers. By means of double-labeling electron microscopic immunocytochemistry with colloidal gold particles of two different sizes, it is also shown here that substance P is present in only the dark type of glutamate-labeled scalloped terminals. The primary afferent origin of the terminals labeled by the antisera for glutamate and for substance P is demonstrated by a triple-labeling strategy: immunocytochemistry for both antisera on sections from rats in which dorsal rhizotomy or dorsal root ganglion injection of horseradish peroxidase conjugated to wheat germ agglutinin was performed. It is proposed that glutamate is the neurotransmitter in primary afferents mediating input from different peripheral receptor classes, including nociceptors. Effects of glutamate and substance P on spinal dorsal horn neurons may result from co-release of these two mediators from the same dorsal root afferent terminal.

https://www.pnas.org/content/pnas/85/20/7820.full.pdf

Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord.

Substance P-containing projections in the dorsal columns of rats and cats

Immunoreactivity (IR) to calcitonin gene-related peptide (CGRP) has recently been found in chick motoneurons. Bath application of this peptide causes an increase in surface acetylcholine receptor (AChR) density and cAMP level in cultured chick muscle cells. These results suggest a role for this peptide in the formation of synaptic specializations. In this study, we examined the development of CGRP IR in larval Xenopus myotomal muscle in relation to synaptogenesis. Using an antiserum against CGRP, a monoclonal antibody against the p65 synaptic vesicle antigen and a fluorescence conjugate of alpha-bungarotoxin, we followed the development of synaptic specializations with fluorescence microscopy in whole-mount specimens. We found that the postsynaptic specialization in the form of AChR clusters was first detected in stage 22 (24 hour) embryos. The presynaptic specializations, including the synaptic vesicle clusters as evidenced by p65 antibody staining and CGRP IR, were first detected at stage 32 (40 hours). The appearance of these two presynaptic specializations followed the same time course. Subsequently, all three structures, the AChR clusters, CGRP IR, and synaptic vesicle clusters, were colocalized at the neuromuscular junction (NMJ). This shows that CGRP is unlikely to be involved in signaling the development of the postsynaptic apparatus. This premise is further examined in cultures of Xenopus myotomal muscle cells. CGRP at concentrations up to 1 microM did not affect the number of AChR clusters, nor did it interfere with the formation of clusters induced by polycation-coated beads. In contrast, an extract from the basement membrane of Torpedo electric organ promoted the formation of AChR clusters and interfered with the clustering activity of the beads. These results suggest that CGRP, an integral component of the presynaptic specialization, is not involved in signaling synaptogenesis at the NMJ.

Development of calcitonin gene-related peptide (CGRP) immunoreactivity in relationship to the formation of neuromuscular junctions in Xenopus myotomal muscle.

S. De Biasi

Papers

Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord.

Substance P-containing projections in the dorsal columns of rats and cats

Development of calcitonin gene-related peptide (CGRP) immunoreactivity in relationship to the formation of neuromuscular junctions in Xenopus myotomal muscle.