Proceedings of The Physiological Society
Cardiff University (2009) Proc Physiol Soc 17, PC16
Responses of lamina I NK1 receptor-expressing projection neurons and interneurons to noxious stimulation
K. S. Al Ghamdi1, E. Polgár1, A. J. Todd1
1. Department of Neuroscience and Molecular Pharmacology, University of Glasgow, Glasgow, United Kingdom.
The neurokinin 1 receptor (NK1r), which is the main target for substance P, is expressed by many neurons in lamina I of the spinal dorsal horn, and these show a bimodal size distribution (1). The first part of this study tested the hypothesis that large NK1r-immunoreactive cells in this lamina are projection neurons, while the small cells are interneurons. In the second part, these findings were used to investigate phosphorylation of extracellular signal-regulated kinases (ERK) in projection neurons following noxious stimulation. To test whether large NK1r-immunoreactive lamina I cells are projection neurons, 3 male Wistar rats were anaesthetised (2-4% isoflurane inhalation) and received injections of cholera toxin B subunit and Fluorogold into caudal ventrolateral medulla and lateral parabrachial area, respectively. Injections into these sites are likely to label all contralateral lamina I projection neurons (2). After 3 days the rats were re-anaesthetized with pentobarbitone (1g.kg-1 i.p.) and perfused with fixative. Brain sections were used to reveal spread of tracers, while horizontal sections from L4 segments were immunoreacted for the tracers and NK1r. To investigate responses to noxious stimuli, 3 further rats were anaesthetised with urethane (1.3g.kg-1 i.p.) and the skin of the left hindpaw was pinched at 12 locations. After 5 minutes they were perfused with fixative. Horizontal sections of L3-L5 segments were immunoreacted to reveal NK1r, gephyrin and phospho-ERK (pERK). Sections were scanned with a confocal microscope and analysed with Neurolucida software (MBF). In the retrograde tracing experiments, we analysed 1341 NK1r-positive cells, of which 441 were retrogradely labelled. Cross-sectional soma areas of projection neurons (128-1198 µm2, median 298 µm2) were larger than those of cells that were not retrogradely labelled (61-568 µm2, median 124 µm2). This difference was highly significant (P<0.0001, Mann-Whitney U test). Nearly all (99.4%) of the NK1r cells that were not retrogradely labelled had soma areas <200 µm2. In contrast, only 9.8% of retrogradely labelled NK1r cells had somata <200 µm2 and only 0.4% were <160 µm2. After applying the noxious pinch stimulus, 221 NK1r-positive lamina I cells were analyzed, of which 91 had soma areas >200 µm2 and were presumed to be projection neurons. pERK was detected in significantly more of these cells (70.3%) than of the small cells (20.8%; p<0.0001, Chi square test). We also examined responses of lamina I projection cells that lack NK1r and have a high density of gephyrin-positive synapses (3) and found that most (62/73, 84.9%) were pERK-positive.These results indicate that NK1r-immunoreactive projection neurons and interneurons in lamina I can be distinguished based on soma size and suggest that relatively more of the projection neurons are activated by noxious pinch stimuli.
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