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

https://doi.org/10.1016/S1044-7431(03)00200-8Get rights and content

Abstract

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 NK1 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.

Introduction

Small diameter sensory neurons in the dorsal root ganglia project unmyelinated axons to the dorsal horn of the spinal cord, where they make synaptic contact with neurons located in superficial layers. The peripheral branches of these neurons transduce a variety of sensory stimuli, including those considered as noxious. Several transmitter molecules have been identified in the terminals of C-fiber afferents in the dorsal horn. The amino acid transmitter glutamate is found in small synaptic release vesicles and is often stored in the same terminals as the neuropeptide substance P (SP) De Biasi and Rustioni, 1988, Merighi et al., 1991. Brain-derived neurotrophic factor (BDNF) is also found in association with the peptidergic vesicle population in these terminals Michael et al., 1997, Luo et al., 2001. In slice preparations of the rat dorsal horn, the electrical stimulation of attached dorsal roots and topical application of capsaicin are established ways of activating primary afferent fibers Kangrga and Randic, 1991, Urban and Dray, 1991. Such stimulation is sufficient to promote the release of transmitters measurable in superfusate outflow from the dorsal horn Kangrga and Randic, 1991, Malcangio and Bowery, 1993 and to initiate changes in the membrane potential of postsynaptic dorsal horn neurons Yang et al., 1998, Randic et al., 1993.

The release of glutamate can be measured in the extracellular superfusing fluid Kangrga and Randic, 1991, Lever et al., 2001, after recruitment of low threshold A-fibers or high-threshold C-fiber afferents and A-fibers, by stimulating dorsal roots at either low or high intensity. Glutamate is also released in the dorsal horn after capsaicin superfusion (Ueda et al., 1993) selectively activating C-fiber terminals expressing VR1 (TRPV1) receptors (Michael et al., 1999). SP is only released from dorsal horn preparations after C-fiber recruitment using capsaicin or high-intensity electrical stimulation Del Bianco et al., 1991, Malcangio and Bowery, 1993. In addition to glutamate and SP, high-frequency stimulation of C-fibers in bursts also induces BDNF release (Lever et al., 2001). Released glutamate can bind to ionotropic and metabotropic receptors expressed on dorsal horn neurons (see Carpenter and Dickenson, 2001). The fast excitatory response recorded from dorsal horn neurons after C-fiber stimulation is mainly mediated by ionotropic AMPA/kainate receptors (Yoshimura and Jessel, 1990). However, under sustained stimulus conditions, antagonists to NMDA and Group 1 metabotropic receptors profoundly affect the responses of dorsal horn neurons to C-fiber input Woolf and Thompson, 1991, Young et al., 1997, Willis, 2002. The Neurokinin 1 (NK1) receptor for SP and the TrkB receptor for BDNF are also found on post-synaptic neurons in the superficial dorsal horn Todd et al., 2002, McLeod et al., 1999, Zhou et al., 1993, Groth and Aaonsen, 2002. NK1 signaling also contributes to the facilitation of dorsal horn neuron responses Urban et al., 1994, Liu and Sandkuhler, 1997, Willis, 2002. Likewise, TrkB receptors are activated in the spinal cord after nociceptor stimulation (Pezet et al., 2002b). Although TrkB activity has not been localized to particular neurons, signaling through this receptor has been demonstrated to modulate the excitability of neurons that are responsive to nociceptive input in the dorsal horn Kerr et al., 1999, Mannion et al., 1999, Heppenstall and Lewin, 2001, Groth and Aaonsen, 2002.

Two mitogen activated protein kinases (MAP kinases), p42 and p44, referred to as extracellular signaling kinases (ERK 1 and 2), are also activated (phosphorylated) in dorsal horn cells after specific stimulation of C-fibers in vitro (Ji et al., 1999). The same neurons activate ERK MAP kinases following noxious but not non-noxious stimulation of primary afferents in vivo Ji et al., 1999, Ji et al., 2002, Karim et al., 2001, Galan et al., 2002. ERK1/2 function as intracellular signaling proteins that transduce extracellular signals from the plasma membrane to the cell interior (Sweatt et al., 2001). Their activation in the CNS is associated with the generation of activity-dependent changes to synaptic efficacy, including those related to the production of LTP in the hippocampus English and Sweatt, 1996, Impey et al., 1999, Dudek and Fields, 2001 and the potentiation of C-fiber evoked dorsal horn neuron responses Ji et al., 1999, Ji et al., 2002, Karim et al., 2001, Galan et al., 2002. The mechanism of ERK activation, studied in cultured neurons, occurs in response to rises in cytosolic Ca2+ levels ([Ca2+]i). This typically occurs after influxes of Ca2+, which are concurrent with membrane depolarization, so that the presence of phosphorylated ERK protein (pERK) can be used as a marker of neuronal activation Rosen et al., 1994, Agell et al., 2002. ERK1/2 can also be specifically activated by neurotrophins and neurotransmitters, including BDNF and glutamate Ying et al., 2002, Barnea and Roberts, 2002, Bading and Greenberg, 1991, Fiore et al., 1993.

In this study, we applied a novel approach to the investigation of primary afferent signaling function at synapses in the superficial dorsal horn of the spinal cord. Using an in vitro preparation of the adult mouse spinal cord, with attached dorsal roots, we have been able to monitor the activity of both pre- and postsynaptic elements at these synapses during afferent stimulation. Transmission from C-fiber afferents is assessed by measuring the activity-generated release of signaling molecules SP or BDNF in cord superfusates after capsaicin superfusion and electrical stimulation of dorsal roots. The response of post-synaptic neurons in dorsal horn tissue can also be monitored from the same preparation, using immunological detection of an activity-regulated signaling molecule (pERK). Using specific receptor antagonists, we show that individual signaling components of C-fiber transmission (glutamate, SP, and BDNF) make a variable contribution to the activation of the ERK MAP kinase pathway in dorsal horn neurons.

Section snippets

Isolated mouse spinal cord preparation

Adult mice of both sexes (25–30 g) were the CD1 strain obtained from Charles River (UK). The mouse spinal cord was exposed by laminectomy from the thoracic to sacral region and the dura mater removed. A 16-mm-long section of the lumbar enlargement region, with L4 and L5 dorsal roots attached, was excised and longitudinally hemisected using a Vibratome (Campden Instruments, UK). The resulting dorsal horn slices (∼400 μm thick) were mounted into the central chamber of a three-compartment organ

Results/discussion

In this study, the release of signaling molecules SP and BDNF was used to assess the activity of C-fiber central terminals in a mouse spinal cord preparation. The resulting activation of post-synaptically located dorsal horn neurons was compared using immunolabeling of phosphorylated ERK protein. The use of a superfused tissue preparation allowed receptor antagonists to be applied to the dorsal horn tissue. This enabled us to assess the relative contribution of different signaling factors

Acknowledgements

This work was supported by a Wellcome Trust Career Development Fellowship to M.M. We thank Sanofi-Sythelabo, Montpellier, France, for the gift of SR140333.

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