The nifedipine-sensitive and nifedipine-resistant components of contractions evoked by 100 pulses at 1 Hz in control and SCI arteries with no pretreatment or pretreated with ryanodine (Ryan; 10 µM) or cyclopiazonic acid (CPZ; 1 µM).

<p>Data are presented as mean ± SE or median and interquartile range (in parentheses) and comparisons between control and SCI values were made with Student’s unpaired <i>t</i>-tests or Mann Whitney U-tests respectively (* indicates significant <i>P</i> values).</p><p>The nifedipine-sensitive and nifedipine-resistant components of contractions evoked by 100 pulses at 1 Hz in control and SCI arteries with no pretreatment or pretreated with ryanodine (Ryan; 10 µM) or cyclopiazonic acid (CPZ; 1 µM).</p

Calcium channel currents recorded from tail artery vascular smooth muscle cells (SMCs) are not changed by spinal cord injury (SCI).

<p>(<b>A, B</b>) Ba²⁺ currents elicited by a 250 ms depolarizing step to +10 mV from a holding potential of −80 mV in a representative SMC from a sham-operated (control) rat (<b>A</b>) and a SCI rat (<b>B</b>) before (basal) and during application of Bay K8644 (1 µM). (<b>C</b>) Leak-subtracted current-voltage relations in control and SCI SMCs under basal conditions (control: 8 cells, <i>n</i> = 5; SCI: 9 cells, <i>n</i> = 5). (<b>D</b>) Normalized conductance-voltage relations for currents from control and SCI SMCs. The activation and inactivation curves in each case are the least squares-fit to the Boltzmann equation. (<b>E</b>) Current-voltage relations in control and SCI SMCs in the presence of Bay K8644 (1 µM) (control: 8 cells, n = 5; SCI: 9 cells, n = 5). (<b>F</b>) Mean increase in current amplitude produced stepping from −80 mV to +10 mV in the absence and in the presence of Bay K8644 (control: 10 cells, <i>n</i> = 5; SCI: 9 cells, <i>n</i> = 5). The data are presented as means and SE and statistical comparisons were made with paired <i>t-</i>tests. **<i>P<</i>0.01.</p

Both ryanodine (Ryan; 10 µM) and cyclopiazonic acid (CPZ; 1 µM) increased the amplitude of nerve-evoked contractions in arteries from sham-operated rats (control arteries) but not in those from spinal cord injured rats (SCI arteries).

<p>(<b>A, D</b>) Averaged traces showing contractions to 100 pulses at 1 Hz in control (<i>left traces</i>) and SCI arteries (<i>right traces</i>) before (<i>black line</i>) and during (<i>grey line</i>) application of ryanodine (<b>A</b>) or CPZ (<b>D</b>). (<b>B, C, E, F</b>) Increases in wall tension measured at the 10^th (<b>B, E</b>) and 100^th pulse (<b>C, F</b>) during the trains of stimuli in control (<i>n</i> = 6) and SCI (<i>n</i> = 6) arteries before (<i>white bars</i>) and during (<i>grey bars</i>) application of ryanodine (<b>B, C</b>) or CPZ (<b>E, F</b>). Data are presented as means and SEs and statistical comparisons were made with paired <i>t</i>-tests. **<i>P</i><0.01.</p

Bay K8644 did not change noradrenaline (NA)-induced oxidation currents evoked by nerve stimulation.

<p>(<b>A</b>) Overlaid averaged traces showing NA-induced oxidation currents that followed the stimulus artifacts (SA) before and during application of Bay K8644 (0.1 µM). In addition, (<b>A</b>) shows an averaged trace recorded during the subsequent addition of Cd²⁺ (0.1 mM) to block neurotransmitter release. (<b>B</b>) Mean stimulus period 2 to stimulus period 1 (S₂/S₁) ratios in tissues with Bay K8644 (BayK) added between the stimulus periods (<i>n</i> = 6; <i>grey bar</i>) and time-matched control tissues (<i>n</i> = 6; <i>white bar</i>). This graph also shows that S₂/S₁ ratios increased significantly in tissues treated with the α₂-adrenoceptor antagonist idazoxan (Idaz) between the stimulus periods (0.1 µM, <i>n</i> = 6; <i>hatched bar</i>). The data are presented as means and SE and statistical comparisons with control were made with unpaired <i>t-</i>tests. *<i>P<</i>0.05.</p

The facilitation of nerve-evoked contraction produced by Bay K8644 (0.1 µM) was greater in arteries from sham-operated rats (control arteries) than in arteries from spinal cord injured rats (SCI arteries).

<p>(<b>A</b>) Averaged traces showing contractions to 100 pulses at 1 Hz in control (<i>upper traces</i>) and SCI arteries (<i>lower traces</i>) before (<i>black line</i>) and during (<i>grey line</i>) application of Bay K8644 (0.1 µM). (<b>B</b>) Increases in wall tension measured at the 100^th pulse during the trains stimuli in control (<i>n</i> = 6) and SCI (<i>n</i> = 6) arteries before (<i>white bars</i>) and during (<i>grey bars</i>) application of Bay K8644. Data are presented as means and SEs and statistical comparisons were made with paired <i>t</i>-tests. *<i>P</i><0.05.</p

Schematic representations of the mechanisms regulating the contribution of Ca²⁺ influx via L-type Ca²⁺ channels to nerve-evoked contractions.

<p>(A) In control arteries, much of the Ca²⁺ entering the cell via L-type Ca²⁺ channels is rapidly sequestered into the sarcoplasmic reticulum (SR) limiting its access the contractile mechanism. (B) In SCI arteries, less of the Ca²⁺ entering through L-type Ca²⁺ channels is sequestered into the SR increasing its access to the contractile mechanism. (C) Bay K8644 increases Ca²⁺ entry via L-type Ca²⁺ channels overcoming the Ca²⁺ buffering capacity of the SR.</p

Both ryanodine (Ryan; 10 µM) and cyclopiazonic acid (CPZ; 1 µM) increased the blockade of nerve-evoked contractions produced by nifedipine (Nif; 1 µM) in arteries from sham-operated rats (control arteries).

<p>By contrast, only ryanodine increased the blockade of nerve-evoked contractions produced by nifedipine in arteries from spinal cord injured rats (SCI arteries). (<b>A–C</b>) Averaged overlaid traces showing contractions to 100 pulses at 1 Hz in control (<i>left traces</i>) and SCI arteries (<i>right traces</i>) in absence (<b>A</b>; <i>black line</i>) or in the presence of ryanodine (<b>B</b>; <i>black line</i>) or CPZ (<b>C</b>; <i>black line</i>) and following addition of nifedipine (<i>grey lines</i>). (<b>D, E</b>) The % blockade of contractions produced by nifedipine at the 10^th (<b>D</b>) and 100^th pulse (<b>E</b>) during the trains of stimuli in control (<i>n</i> = 6) and SCI (<i>n</i> = 6) arteries in the absence (<i>white bars</i>) or in the presence of ryanodine (<i>grey bars</i>) or CPZ (<i>black bars</i>). Data are presented as means and SEs and statistical comparisons were made with unpaired <i>t</i>-tests. *<i>P</i><0.05, **<i>P</i><0.01.</p

The neurochemistry and morphology of functionally identified corneal polymodal nociceptors and cold thermoreceptors

<div><p>It is generally believed that the unencapsulated sensory nerve terminals of modality specific C- and Aδ-neurons lack structural specialization. Here we determined the morphology of functionally defined polymodal receptors and cold thermoreceptors in the guinea pig corneal epithelium. Polymodal receptors and cold thermoreceptors were identified by extracellular recording at the surface of the corneal epithelium. After marking the recording sites, corneas were processed to reveal immunoreactivity for the transient receptor potential channels TRPV1 (transient receptor potential cation channel, subfamily V, member 1) or TPRM8 (transient receptor potential cation channel subfamily M member 8). Polymodal receptor nerve terminals (n = 6) were TRPV1-immunoreactive and derived from an axon that ascended from the sub-basal plexus to the squamous cell layer where it branched into fibers that ran parallel to the corneal surface and terminated with small bulbar endings (ramifying endings). Cold thermoreceptor nerve terminals were TRPM8-immunoreactive (n = 6) and originated from an axon that branched as it ascended through the wing cell and squamous cell layers and terminated with large bulbar endings (complex endings). These findings indicate that modality specific corneal sensory neurons with unencapsulated nerve endings have distinct nerve terminal morphologies that are likely to relate to their function.</p></div

The electrical activity recorded from a cold thermoreceptor nerve terminal at the corneal surface.

<p><b>A</b> and <b>B</b>, show the temperature of the solution superfusing the cornea (<b>A</b>) and the frequency of nerve terminal impulse (NTI) discharge (<b>B</b>). During heating and cooling the frequency of NTIs was decreased and increased, respectively. <b>C</b>, overlaid traces recorded during stimulation of the ciliary nerves with a train of 25 pulses at 1 Hz. At this recording site electrical stimulation evoked a single stimulus locked NTI. The insets in <b>C</b> show averages of the electrically evoked and spontaneous NTIs. The smaller amplitude of the spontaneous NTIs indicates they are likely to be initiated very close to site of recording (see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195108#pone.0195108.ref025" target="_blank">25</a>]). In <b>C</b>, the stimulus artifact (SA) is indicated and during the flat line immediately following the SA the signal was out of the analogue-to-digital recording range.</p

The electrical activity recorded from a capsaicin-sensitive nerve terminal at the corneal surface.

<p>A, the frequency of NTI discharge before and during application of capsaicin (0.5 μM). In this receptor, there was a low level of ongoing NTI activity that was markedly increased by capsaicin. B, overlaid traces recorded during stimulation of the ciliary nerves with a train of 25 pulses at 1 Hz. At this recording site electrical stimulation evoked a single stimulus locked NTI. In B, the stimulus artifact (SA) is indicated and during the flat line immediately following the SA the signal was out of the analogue-to-digital recording range.</p