Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC47
BDNF-Induced Ca2+ Oscillations in Rat Organotypic Spinal Cord Slices and their Possible Relevance to Neuropathic Pain
J. E. Biggs1, V. B. Lu2, S. R. Alles1, K. Ballanyi1, P. A. Smith1
1. Centre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada. 2. Laboratory of Molecular Physiology, NIAAA/NIH, Rockville, Maryland, United States.
Peripheral nerve injury promotes the release of brain derived neurotrophic factor (BDNF) from spinal microglia and primary afferent terminals. This induces changes in the properties of neurons in the dorsal horn that lead to the ‘central sensitization’ that underlies neuropathic pain. In accordance with procedures approved by the University of Alberta Animal Care and Use Committee, we used defined medium organotypic cultures of rat spinal cord slices to examine changes in the properties of dorsal horn neurons produced by 6d exposure to 100-200ng/ml BDNF (Lu et al., 2009). This mimics the prolonged increase in dorsal horn BDNF levels that accompanies peripheral nerve injury. All BDNF treated cultures exhibited synchronous oscillations in cytosolic Ca2+ concentration as monitored by fluo-4 confocal Ca2+ imaging. These occurred at 0.14±0.01Hz (n=7) and were synchronous with extracellularly recorded field potentials. Oscillations in BDNF-treated slices were eliminated by removal of extracellular Ca2+, or by superfusion of 200µM Cd2+, 0.1µM TTX or 10µM NBQX. This indicates the involvement of voltage gated Ca2+ and Na+ channels, the release of glutamate and the participation of AMPA receptors in the oscillations. The NMDA receptor blocker AP-5 (50µM) reduced the amplitude (P<0.001, unpaired T-test) but not the frequency of oscillations and the Ca2+ permeable AMPA receptor blocker, IEM1460 (50μM) reduced both amplitude and frequency (P<0.001 for both unpaired T-test. Oscillations were augmented by bicuculline 10µM + strychnine 1µM and eliminated by 1mM GABA. Activation of astrocytes with 10µM phenylephrine (Ruangkittisakul et al., 2009) promoted elevation of Ca2+ in populations of cells distinct from those displaying oscillations. The possible contribution of gap junctions was inferred from an increase in width of each oscillation in the presence of carbenoxolone (100μM). These findings suggest that BDNF oscillations are primarily neuronal in origin and involve glutamatergic transmission. We also found that Ca2+ signals produced by acute superfusion of 10µM AMPA or 50µM NMDA were augmented in BDNF treated cultures. This suggests that the BDNF-induced oscillations may result, at least in part, from increased neuronal sensitivity to glutamate. This type of long range oscillatory activity may enable interactions between different sensory modalities within the dorsal horn and may thereby contribute to the etiology of nerve-injury induced neuropathic pain.
Where applicable, experiments conform with Society ethical requirements