Abstract
Spinal afferent neurons play a major role in detecting noxious and innocuous stimuli from visceral organs, such as the gastrointestinal tract. However, all our understanding about spinal afferents has been obtained from recordings of spinal afferent axons, or cell bodies that lie outside the gut wall, or peripheral organ they innervate. No recordings have been made directly from spinal afferent nerve endings, which is where sensory transduction occurs. We developed a preparation whereby recordings could be made from rectal afferent nerve endings in the colon, to characterize mechanisms underlying sensory transduction. Dorsal root ganglia (L6-S2) were removed from mice, whilst retaining neural continuity with the colon. Fluo-4-AM was used to record from rectal afferent nerve endings in myenteric ganglia and circular muscle at 36°C. In slack (unstretched) preparations of colon, no calcium transients were recorded from spinal afferent endings. However, in response to a maintained increase in circumferential diameter, a maintained discharge of calcium transients occurred simultaneously in multiple discrete varicosities along single axons of rectal afferents in myenteric ganglia and circular muscle. Stretch-activated calcium transients were resistant to hexamethonium and nifedipine, but were abolished by tetrodotoxin, CPA, BAPTA-AM, cobalt, gadolinium, or replacement of extracellular Na+ with NMDG. In summary, we present a novel preparation in which stretch-activated firing of spinal afferent nerve endings can be recorded, using calcium imaging. We show that circumferential stretch of the colon activates a maintained discharge of calcium transients simultaneously in varicosities along single rectal afferent endings in myenteric ganglia and circular muscle. Non-selective cation channels, TTX-sensitive Na+ channels and both extracellular calcium influx and intracellular Ca2+ stores are required for stretch-activated calcium transients in rectal afferent endings.
Highlights
Few studies have recorded from sensory nerve terminals in vertebrates (Mendelson and Lowenstein, 1965; Firestein et al, 1990; Brock et al, 1998, 2001; Carr and Brock, 2002; Carr et al, 2009)
Whilst much has been learnt from extracellular recordings of spinal afferents that lie outside the gut wall, this technique does not reveal direct information about the mechanisms underlying sensory transduction, since this process only occurs within the afferent nerve endings located within the peripheral organ
EFFECTS OF CIRCUMFERENTIAL STRETCH ON NERVE ENDINGS OF RECTAL AFFERENTS IN MYENTERIC GANGLIA AND CIRCULAR MUSCLE We used the calcium indicator, Fluo-4 to image the nerve endings of spinal afferents that have been shown previously to innervate myenteric ganglia and circular muscle of isolated whole mouse colon
Summary
Few studies have recorded from sensory nerve terminals in vertebrates (Mendelson and Lowenstein, 1965; Firestein et al, 1990; Brock et al, 1998, 2001; Carr and Brock, 2002; Carr et al, 2009). Whilst much has been learnt from extracellular recordings of spinal afferents that lie outside the gut wall, this technique does not reveal direct information about the mechanisms underlying sensory transduction, since this process only occurs within the afferent nerve endings located within the peripheral organ This we demonstrated by studies which showed that physiological stimuli applied to the sensory nerve endings of spinal afferents leads to the generation of action potentials (Zagorodnyuk et al, 2003; Spencer et al, 2008a; Song et al, 2009), but not when the same stimuli are applied to the axons of spinal afferents that lie away from the transduction sites (Zagorodnyuk et al, 2003; Spencer et al, 2008a). In light of our limited understanding of the mechanisms underlying sensory transduction in spinal afferents, it was our intention to develop a preparation whereby recordings could be made directly from spinal afferent endings, to characterize mechanisms underlying their activation by circumferential stretch
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