Isobel J. Lever

Noxious stimulation induces Trk receptor and downstream ERK phosphorylation in spinal dorsal horn

Several lines of evidence suggest that the brain-derived neurotrophic factor (BDNF) acts as central pain neuromodulator. We examined the ability of different types of peripheral stimulation to activate the BDNF high-affinity receptor, TrkB, in the spinal cord. We found that noxious chemical, mechanical, or thermal stimuli, but not innocuous stimuli, caused Trk phosphorylation in the spinal cord. These changes were rapid and transient and restricted to somatotopically appropriate spinal segments. We observed, both in vitro and in vivo, that exogenous BDNF induced a rapid activation of ERK, a signaling kinase important in the development of acute pain. Finally, we found that sequestering BDNF in vivo with a TrkB-IgG fusion molecule significantly reduced the activation of ERK evoked by noxious stimulation. These data suggest that BDNF, once released with activity from primary afferent nociceptors, exerts a neuromodulatory role in pain processing through stimulation of postsynaptic TrkB receptors and subsequent activation of ERK.

BDNF modulates sensory neuron synaptic activity by a facilitation of GABA transmission in the dorsal horn.

Topical application of brain-derived neurotrophic factor (BDNF) to the adult rat isolated dorsal horn with dorsal root attached preparation inhibited the electrically evoked release of substance P (SP) from sensory neurons. This effect of BDNF was dose dependent (EC(50) 250 pM) and reversed by the tyrosine kinase inhibitor, K-252a. BDNF-induced inhibition of SP release was blocked by the GABA(B) receptor antagonist CGP 55485 but not by naloxone. Acute application of BDNF significantly increased potassium-stimulated release of GABA in the dorsal horn isolated in vitro and this effect was blocked by K-252a. Intrathecal injection of BDNF into the rat lumbar spinal cord induced a short-lasting increase in hindpaw threshold to noxious thermal stimulation that was blocked by CGP 55485 and was associated with activation of ERK in dorsal horn. These data suggest that exogenous BDNF can indirectly modulate primary sensory neuron synaptic efficacy via facilitation of the release of GABA from dorsal horn interneurons.

The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn

The stimulation of C-fiber sensory neurons is known to induce activation of the ERK MAP kinase signaling pathway in the spinal cord dorsal horn. In this study we have elucidated some of the signaling components of C-fiber transmission responsible for ERK activation. Using an in vitro slice preparation of the mouse spinal cord dorsal horn, we compared the release of substance P (SP) and BDNF with the activation of ERK in postsynaptic neurons. We observed that primary afferent stimulation recruiting C-fibers was required for both SP and BDNF release and ERK activation in post-synaptic dorsal horn neurons. Glutamate transmission via NMDA and mGluR1 but not AMPA receptors was critical to this ERK activation. BDNF signaling via TrkB receptors but not SP signaling via NK(1) were also involved in ERK recruitment. In conclusion, glutamate and BDNF are the important C-fiber signaling components for ERK activation in dorsal horn neurons.

Glial cell line-derived neurotrophic factor increases calcitonin gene-related peptide immunoreactivity in sensory and motoneurons in vivo.

Calcitonin gene‐related peptide (CGRP) is expressed at high levels in roughly 50% of spinal sensory neurons and plays a role in peripheral vasodilation as well as nociceptive signalling in the spinal cord. Spinal motoneurons express low levels of CGRP; motoneuronal CGRP is thought to be involved in end‐plate plasticity and to have trophic effects on target muscle cells. As both sensory and motoneurons express receptors for glial cell line‐derived neurotrophic factor (GDNF) we sought to determine whether CGRP was regulated by GDNF. Rats were treated intrathecally for 1–3 weeks with recombinant human GDNF or nerve growth factor (NGF) (12 µg/day) and dorsal root ganglia and spinal cords were stained for CGRP. The GDNF treatment not only increased CGRP immunoreactivity in both sensory and motoneurons but also resulted in hypertrophy of both populations. By combined in situ hybridization and immunohistochemistry we found that, in the dorsal root ganglia, CGRP was up‐regulated specifically in neurons expressing GDNF but not NGF receptors following GDNF treatment. Despite the increase in CGRP in GDNF‐treated rats, there was no increase in thermal or mechanical pain sensitivity, while NGF‐treated animals showed significant decreases in pain thresholds. In motoneurons, GDNF increased the overall intensity of CGRP immunoreactivity but did not increase the number of immunopositive cells. As GDNF has been shown to promote the regeneration of both sensory and motor axons, and as CGRP appears to be involved in motoneuronal plasticity, we reason that at least some of the regenerative effects of GDNF are mediated through CGRP up‐regulation.

CB1 receptor antagonist SR141716A increases capsaicin‐evoked release of Substance P from the adult mouse spinal cord

Cannabinoids have an antinociceptive action in many pain models. We have investigated a possible modulatory role for Type 1 Cannabinoid receptors (CB1) on the release of excitatory transmitter Substance P from the adult mouse spinal cord after stimulation of nociceptor terminals by capsaicin. Capsaicin (0.1 – 10 μM) was applied to superfused cord sections and evoked a dose dependent release of SP above basal outflow of (23.36±2.96 fmol 8 ml−1). Maximum evoked SP release was obtained with 5 μM Capsaicin (262.4±20.8 fmol 8 ml−1). Higher capsaicin concentrations (50 – 100 μM) evoked less SP release. Superfusion of CB1 antagonist SR141716A (5 μM) increased evoked SP release with capsaicin (0.1 – 10 μM) and reversed the reducing effect of high dose capsaicin (100 μM). Antagonism of CB1 receptors in the spinal cord during capsaicin stimulation, is evidence of tonic CB1 activity inhibiting the release of excitatory transmitters after activation of nociceptive neurones and is also indicative of endocannabinoid production during noxious stimulation.

Intrathecally delivered glial cell line‐derived neurotrophic factor produces electrically evoked release of somatostatin in the dorsal horn of the spinal cord

Glial cell line‐derived neurotrophic factor (GDNF) is a trophic factor with an established role in sensory neuron development. More recently it has also been shown to support adult sensory neuron survival and exert a neuroprotective effect on damaged sensory neurons. Some adult small‐sized dorsal root ganglion (DRG) cells that are GDNF‐sensitive sensory neurons express the inhibitory peptide somatostatin (SOM). Thus, we tested the hypothesis that prolonged GDNF administration would regulate SOM expression in sensory neuron cell bodies in the dorsal root ganglia (DRG) and activity‐induced release of SOM from axon terminals in the dorsal horn. Continuous intrathecal delivery of GDNF for 11–13 days significantly increased the number of small DRG cells that expressed SOM. Furthermore, GDNF treatment evoked SOM release in the isolated dorsal horn following electrical stimulation of the dorsal roots that was otherwise undetectable in control rats. Conversely capsaicin‐induced release of SOM (EC50 50 nm) was not modified by GDNF treatment. These results show that GDNF can regulate central synaptic function in SOM‐containing sensory neurons.

Basal and activity-induced release of substance P from primary afferent fibres in NK1 receptor knockout mice: evidence for negative feedback

The concept that NK1 receptors are located pre-junctionally on substance P (SP)-containing nerves, acting as autoreceptors to inhibit SP release, has been suggested, but remains a controversial issue. To further investigate the existence of this receptor on central and peripheral terminals of primary afferent fibres, NK1 receptor knockout mice and an NK1 receptor antagonist were used in nerve-attached tissue preparations. These were the isolated dorsal horn of the spinal cord with dorsal roots attached, and the hairy skin of the hind paw with attached saphenous nerve. The results reveal that in the dorsal horn preparation, basal release of SP is significantly higher in NK1(-/-) mice than NK1(+/+) mice (P<0.05, n=7 mice/strain). However, a difference in SP release evoked in the dorsal horn by electrical stimulation of the dorsal roots or capsaicin application was not observed. In contrast, antidromic electrical stimulation of the saphenous nerve caused a substantially greater release of SP in the skin of NK1(-/-) mice than in NK1(+/+) mice (P<0.05, n=5 to 6 mice/strain). These results provide evidence for the existence of NK1 autoreceptors on sensory nerves in skin, which may be relevant to the modulation of their peripheral pathophysiological effector functions.

A novel control mechanism based on GDNF modulation of somatostatin release from sensory neurones

Small‐diameter sensory neurones found in the rat dorsal root ganglia (DRG) include cells sensitive to glial cell line‐derived neurotrophic factor (GDNF), which express the inhibitory peptide somatostatin (SOM). Here we addressed the functional relationship between GDNF and sensory neurone‐derived SOM. Topical application of GDNF through the rat isolated dorsal horn of the spinal cord promoted activity‐induced release of SOM from central terminals of sensory neurones. Once released by sensory neurones, SOM is known to act, at least in part, by opposing the action of Substance P (SP) in neurogenic inflammation. Therefore, we evaluated GDNF ability to modulate two well‐documented effects of peripherally and centrally administered SP. Local application of GDNF in the mouse air pouch reduced SP‐induced leukocyte migration. This effect of GDNF was mimicked by the SOM analog octreotide (OCT) and required intact SOM neuronal pools. Intrathecal injection of GDNF activated rat lumbar dorsal horn neurones and inhibited intrathecal SP‐induced thermal hypersensitivity. This effect of GDNF was reversed by the SOM antagonist c‐SOM and mimicked by OCT. In conclusion we propose GDNF regulation of neuronal SOM release as a novel mechanism that, if explored, may lead to new therapeutic strategies based on local release of somatostatin.