James A. Brock

Tetrodotoxin-resistant impulses in single nociceptor nerve terminals in guinea-pig cornea

1 Extracellular recording techniques have been used to study nerve impulses in single sensory nerve terminals in guinea‐pig cornea isolated in vitro. 2 Nerve impulses occurred spontaneously and were evoked by electrical stimulation of the ciliary nerves. 3 The nerve impulses were identified as originating in polymodal receptors, mechano‐receptors or ‘cold’ receptors. All three types are believed to be nociceptors. 4 Tetrodotoxin (TTX, 1 μm) blocked nerve impulses evoked by electrical stimulation of the ciliary nerves. However, ongoing and/or naturally evoked nerve impulses persisted in the presence of TTX in all three types of receptors. Lignocaine (lidocaine; 1 mm) blocked all electrical activity. 5 TTX‐resistant sodium channels therefore play a major role in generating the action potentials that signal pain to the brain.

Converting cold into pain

Cold temperature can evoke a wide spectrum of perceptual sensations that range from freshness to unpleasant cold or overt pain. In mammals, the detection of cold temperature is accomplished by the activation of different subsets of sensory terminals innervating the skin and mucosae. Direct recordings of corneal nerve endings, combined with studies of thermoreceptive neurons in culture, have allowed the characterization of ionic mechanisms involved in cold temperature sensing. In recent years, major progress has also taken place in the identification and operation of thermally gated ion channels, especially of the transient receptor potential (TRP) family. However, it is still uncertain how individual sensory endings can be activated with different thermal thresholds. In this review, we have considered the known properties of cold-sensitive receptors and their transduction mechanisms and related them to the sensations they evoke. We analyzed the evidence linking specific ion channels to the activation of particular sets of afferent fibers. In our view, cold thermotransduction is complex and involves the concerted operation of several ion channels. Excitatory effects of cationic channels (e.g., TRPs) balance their activity with several excitability brakes (e.g., potassium channels), leading to tunable levels of sensory thresholds and activity. Alteration in this fine balance may result in altered cold sensitivity, a frequent symptom in patients with peripheral nerve injury.

Differences between nerve terminal impulses of polymodal nociceptors and cold sensory receptors of the guinea‐pig cornea

Extracellular recording techniques were used to study nerve terminal impulses (NTIs) recorded from single polymodal nociceptors and cold‐sensitive receptors in guinea‐pig cornea isolated in vitro. The amplitude and time course of NTIs recorded from polymodal nociceptors was different from those of cold‐sensitive receptors. Bath application of tetrodotoxin (1 μm) changed the time course of spontaneous NTIs recorded from both polymodal and cold‐sensitive receptors. Bath application of lignocaine (lidocaine; 1–5 mm) abolished all electrical activity. Local application of lignocaine (2.5 and 20 mm) through the recording electrode changed the time course of the NTIs recorded from polymodal nociceptors but not that of NTIs recorded from cold‐sensitive nerve endings. It is concluded that action potentials propagate actively in the sensory nerve endings of polymodal nociceptors. In contrast, cold‐sensitive receptor nerve endings appear to be passively invaded from a point more proximal in the axon where the action potential can fail or be initiated.

Effects of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246, on Y2 receptors at peripheral neuroeffector junctions

This study investigated the effects of BIIE0246, a novel neuropeptide Y (NPY) Y2 receptor antagonist, on the inhibition of cholinergic neuroeffector transmission in rat heart and guinea‐pig trachea and purinergic neuroeffector transmission in guinea‐pig vas deferens produced by the NPY Y2 receptor agonist, N‐acetyl [Leu28,31] NPY 24‐36. In pentobarbitone anaesthetized rats, supramaximal stimulation every 30 s, of the vagus nerve innervating the heart, increased pulse interval by approximately 100 ms. This response was attenuated by intravenous administration of N‐acetyl [Leu28,31] NPY 24‐36 (10 nmol kg−1). Transmural stimulation of segments of guinea‐pig trachea at 1 min intervals with 5 s trains of stimuli at 0.5, 5, 10, 20 and 40 Hz evoked contractions which were reduced in force by N‐acetyl [Leu28,31] NPY 24‐36 (2 μM). In guinea‐pig vasa deferentia, the amplitude of excitatory junction potentials evoked by trains of 20 stimuli at 1 Hz was reduced in the presence of N‐acetyl [Leu28,31] NPY 24‐36 (1 μM). In all preparations BIIE0246 attenuated the inhibitory effect of N‐acetyl [Leu28,31] NPY 24‐36 but had no effect when applied alone. The findings support the view that the nerve terminals of postganglionic parasympathetic and sympathetic neurones possess neuropeptide Y Y2 receptors which, when activated, reduce neurotransmitter release.

Effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline release and purinergic neuroeffector transmission in rat tail artery.

The effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline (NA) and adenosine 5′‐triphosphate (ATP) release from postganglionic sympathetic nerves have been investigated in rat tail arteries in vitro. Intracellularly recorded excitatory junction potentials (e.j.ps) were used as a measure of ATP release and continuous amperometry was used to measure NA release. Varying the extracellular Ca2+ concentration similarly affected the amplitudes of e.j.ps and NA‐induced oxidation currents evoked by trains of ten stimuli at 1 Hz. The N‐type Ca2+ blocker, ω‐conotoxin GVIA (ω‐CTX GVIA, 0.1 μM) reduced the amplitudes of both e.j.ps (evoked by trains of ten stimuli at 1 Hz) and NA‐induced oxidation currents (evoked by trains of ten stimuli at 1 Hz and 50 stimuli at 10 Hz) by about 90%. The ω‐CTX GVIA resistant e.j.ps and NA‐induced oxidation currents evoked by trains of 50 stimuli at 10 Hz were abolished by the non‐selective Ca2+ channel blocker, Cd2+ (0.1 mM), and were reduced by ω‐conotoxin MVIIC (0.5 μM) and ω‐agatoxin IVA (40 nM). Nifedipine (10 μM) had no inhibitory effect on ω‐CTX GVIA resistant e.j.ps and NA‐induced oxidation currents. Thus both varying Ca2+ concentration and applying Ca2+ channel blockers results in similar effects on NA and ATP release from postganglionic sympathetic nerves. These findings are consistent with the hypothesis that NA and ATP are co‐released together from the sympathetic nerve terminals.

ENHANCED EXCITATORY JUNCTION POTENTIALS IN MESENTERIC ARTERIES FROM SPONTANEOUSLY HYPERTENSIVE RATS

Excitatory junction potentials (EJPs) were examined using intracellular recording techniques in mesenteric arteries isolated from 12- to 15-week-old spontaneously hypertensive (SHR), Wistar Kyoto (WKY) and Sprague Dawley (SD) rats. The amplitudes of EJPs evoked by single supramaximal stimuli were larger in arteries from SHRs (12.9±0.7 mV,n=16) than in arteries from either WKYs (5.2±0.5 mV,n=24) or SDs (8.6±0.8 mV,n=15). The time constant of decay of EJPs did not differ significantly, suggesting that the passive electrical properties of the vascular smooth muscle are similar in the three rat strains. Spontaneous EJPs recorded in tissues from SHRs and WKYs had similar amplitude frequency distributions, suggesting that the quantal size is also similar between strains. In some arteries from SHRs, EJPs evoked by single stimuli triggered muscle action potentials (MAPs). Visible constriction only occurred following a MAP. In tissues from all three strains, summation of EJPs triggered MAPs. As EJPs are generated by the sympathetic co-transmitter adenosine 5′-triphosphate (ATP), the findings of the present study indicate that purinergic transmission is enhanced in mesenteric arteries from SHRs, probably as a result of an increase in quantal release. A consequence is that when nerves are activated SHR arteries more readily undergo constriction that is dependent on voltage-activated Ca2+ influx.

Co-ordination of contractile activity in guinea-pig mesenteric lymphatics.

1. Intraluminally perfused lymphatic vessels from the mesentery of the guinea‐pig were examined in vitro to investigate their contractile activity and the co‐ordination of this activity between adjacent lymphangions. 2. Lymphangions constricted at fairly regular intervals and exhibited ‘refractory’ periods of up to 3 s during which constrictions did not occur. 3. The contractile activity of adjacent lymphangions was highly co‐ordinated. 4. The smooth muscle was found to be continuous between the adjacent lymphangions for the majority of valve regions examined morphologically (52 of 63 preparations). 5. Mechanical and electrical coupling between adjacent lymphangions was indicated, as some lymphangions underwent transient dilatations just prior to constriction, whereas direct electrophysiological measurements showed that the smooth muscle of most adjacent lymphangions was electrically coupled across the valve (15 out of 20 pairs of lymphangions). 6. It is concluded that perfused lymphangions of guinea‐pig mesenteric lymphatic vessels rhythmically constrict, with the contractile activity of adjacent lymphangions highly co‐ordinated. The findings also indicate that transmission of both mechanical and electrical signals between the adjacent lymphangions contribute to the co‐ordination of their contractile activity.

Tail arteries from chronically spinalized rats have potentiated responses to nerve stimulation in vitro.

Patients with severe spinal cord lesions that damage descending autonomic pathways generally have low resting arterial pressure but bladder or colon distension or unheeded injuries may elicit a life‐threatening hypertensive episode. Such episodes (known as autonomic dysreflexia) are thought to result from the loss of descending baroreflex inhibition and/or plasticity within the spinal cord. However, it is not clear whether changes in the periphery contribute to the exaggerated reflex vasoconstriction. The effects of spinal transection at T7–8 on nerve‐ and agonist‐evoked contractions of the rat tail artery were investigated in vitro. Isometric contractions of arterial segments were recorded and responses of arteries from spinalized animals (‘spinalized arteries’) and age‐matched and sham‐operated controls were compared. Two and eight weeks after transection, nerve stimulation at 0.1–10 Hz produced contractions of greater force and duration in spinalized arteries. At both stages, the α‐adrenoceptor antagonists prazosin (10 nm) and idazoxan (0.1 μm) produced less blockade of nerve‐evoked contraction in spinalized arteries. Two weeks after transection, spinalized arteries were supersensitive to the α1‐adrenoceptor agonist phenylephrine, and the α2‐adrenoceptor agonist, clonidine, but 8 weeks after transection, spinalized arteries were supersensitive only to clonidine. Contractions of spinalized arteries elicited by 60 mm K+ were larger and decayed more slowly at both stages. These findings demonstrate that spinal transection markedly increases nerve‐evoked contractions and this can, in part, be accounted for by increased reactivity of the vascular smooth muscle to vasoconstrictor agents. This hyper‐reactivity may contribute to the genesis of autonomic dysreflexia in patients.

Effects of Heating and Cooling on Nerve Terminal Impulses Recorded from Cold-sensitive Receptors in the Guinea-pig Cornea

An in vitro preparation of the guinea-pig cornea was used to study the effects of changing temperature on nerve terminal impulses recorded extracellularly from cold-sensitive receptors. At a stable holding temperature (31–32.5°C), cold receptors had an ongoing periodic discharge of nerve terminal impulses. This activity decreased or ceased with heating and increased with cooling. Reducing the rate of temperature change reduced the respective effects of heating and cooling on nerve terminal impulse frequency. In addition to changes in the frequency of activity, nerve terminal impulse shape also changed with heating and cooling. At the same ambient temperature, nerve terminal impulses were larger in amplitude and faster in time course during heating than those recorded during cooling. The magnitude of these effects of heating and cooling on nerve terminal impulse shape was reduced if the rate of temperature change was slowed. At 29, 31.5, and 35°C, a train of 50 electrical stimuli delivered to the ciliary nerves at 10–40 Hz produced a progressive increase in the amplitude of successive nerve terminal impulses evoked during the train. Therefore, it is unlikely that the reduction in nerve terminal impulse amplitude observed during cooling is due to the activity-dependent changes in the nerve terminal produced by the concomitant increase in impulse frequency. Instead, the differences in nerve terminal impulse shape observed at the same ambient temperature during heating and cooling may reflect changes in the membrane potential of the nerve terminal associated with thermal transduction.

INHIBITION OF TRANSMITTER RELEASE FROM SYMPATHETIC NERVE ENDINGS BY ω‐CONOTOXIN

1. The effects of the calcium channel blocker, ω‐conotoxin, on sympathetic neuroeffector function in the guinea‐pig vas deferens have been investigated using a combination of mechanical and electrophysiological recording techniques.