Capsaicin-responsive corneal afferents do not contain TRPV1 at their central terminals in trigeminal nucleus caudalis in rats

Abstract

We examined the substrates for ocular nociception in adult male Sprague-Dawley rats. Capsaicin application to the ocular surface in awake rats evoked nocifensive responses and suppressed spontaneous grooming responses. Thus, peripheral capsaicin was able to activate the central pathways encoding ocular nociception. Our capsaicin stimulus evoked c-Fos expression in a select population of neurons within rostral trigeminal nucleus caudalis in anesthetized rats. These activated neurons also received direct contacts from corneal afferent fibers traced with cholera toxin B from the corneal surface. However, the central terminals of the corneal afferents that contacted capsaicin-activated trigeminal neurons did not contain TRPV1. To determine if TRPV1 expression had been altered by capsaicin stimulation, we examined TRPV1 content of corneal afferents in animals that did not receive capsaicin stimulation. These studies confirmed that while TRPV1 was present in 30% of CTb-labeled corneal afferent neurons within the trigeminal ganglion, TRPV1 was only detected in 2% of the central terminals of these corneal afferents within the trigeminal nucleus caudalis. Other TRP channels were also present in low proportions of central corneal afferent terminals in unstimulated animals (TRPM8, 2%; TRPA1, 10%). These findings indicate that a pathway from the cornea to rostral trigeminal nucleus caudalis is involved in corneal nociceptive transmission, but that central TRP channel expression is unrelated to the type of stimulus transduced by the peripheral nociceptive endings.

Select noxious stimuli induce changes on corneal nerve morphology

“…We have combined behavioral assessments of corneal nociception with quantitative imaging tools and qualitative observations to assess the effects of noxious chemical stimulation on corneal nerves. Eye wipe responses to noxious stimuli were used to establish doses of chemical stimuli and are in agreement with previous studies from this laboratory (Hegarty et al, 2014; Aicher et al, 2015) and others (Farazifard et al, 2005). We used β-tubulin, a well-characterized nerve marker to study the overall changes in corneal nerve density in the central area of the cornea (Dvorscak and Marfurt, 2008; Aicher et al, 2015; Hirata et al, 2015), while CGRP was also used to address whether subgroups of nociceptive nerves were specifically affected by select noxious ocular stimuli (Belmonte et al, 2004; Hegarty et al, 2010).…”

“…Rats were overdosed with pentobarbital sodium (150 mg/kg) and perfused transcardially as previously described (Hegarty et al, 2014; Hermes et al, 2016) through the ascending aorta with 10 ml heparinized saline (1000 units/ml) followed by 600 ml 4% paraformaldehyde (in 0.1 M phosphate buffer (PB), pH 7.4). Eyes were enucleated immediately after perfusion and placed in PB.…”

“…Varicosities were operationally defined as punctate enlargements along or at the end of a corneal nerve that were present in two consecutive optical slices (Hegarty et al, 2010; Aicher et al, 2013; Aicher et al, 2014; Hegarty et al, 2014). Two regions were captured per cornea and varicosities were arbitrarily sampled throughout each scanned image.…”

“…Polymodal nociceptors are presumed to contain more than one type of these molecular transducers (Chao et al, 2016). Activity from corneal nerves is transmitted via the ophthalmic branch of the trigeminal nerve to the trigeminal brainstem and supraspinal substrates of nociception, resulting in the perception of ocular pain (Marfurt and Del Toro, 1987; Meng and Bereiter, 1996; Belmonte et al, 2004; Hegarty et al, 2010; Aicher et al, 2013; Aicher et al, 2014; Hegarty et al, 2014). …”

Localization of TRPV1 and P2X3 in unmyelinated and myelinated vagal afferents in the rat

“…The antibodies directed against the tract tracers IB4 (Vector, Cat: AS-2104) and CTb (List labs, Cat: 703), have been previously utilized by our lab, as well as others (Aicher et al , 2013; Aicher and Reis, 1997; Borges and Sidman, 1982; Hermes et al , 2014; Llewellyn-Smith et al , 1990; Stucky and Lewin, 1999). The antibodies directed against the channels P2X3 (Neuromics, Cat: RA10109) and TRPV1 (Chemicon AB5566) have also been utilized in a number of prior studies (Amadesi et al , 2009; Guo et al , 1999; Hegarty et al , 2014; Saeed and Ribeiro-da-Silva, 2012). Per the manufacturer's specifications, the rabbit polyclonal P2X3 antibody recognizes residues 383 to 397 (VEKQSTDSGAYSIGH) of the carboxy terminus of rat P2X3.…”