Keith L. Brain

Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage‐gated sodium channels

Oxaliplatin, an effective cytotoxic treatment in combination with 5‐fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side‐effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca2+ concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected. In both tissues, an oxaliplatin‐induced increase in spontaneous activity was prevented by the voltage‐gated Na+ channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, β‐pompilidotoxin (100 μM), which slows Na+ channel inactivation, induced multiple EPPs similar to oxaliplatins effect. By contrast, blockers of K+ channels (4‐aminopyridine and apamin) did not replicate oxaliplatin‐induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between β‐pompilidotoxin and oxaliplatin suggests that alteration of voltage‐gated Na+ channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.

Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens

A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA‐1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed ‘neuroeffector Ca2+ transients’ (NCTs). NCTs were abolished by α,β‐methylene ATP (1 μM), but not by nifedipine (1 μM) or prazosin (100 nm), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L‐type Ca2+ channels or α1‐adrenoceptor‐mediated pathways. Action potential‐evoked ATP release was highly intermittent (mean probability 0.019 ± 0.002; range 0.001‐0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty‐eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the α2‐adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 ± 21 % during trains of stimuli at 1 Hz. Frequency‐dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse‐to‐impulse basis.

Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition.

1 The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA‐1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (Δ[Ca2+]v) were determined following single impulses, and short (5‐impulse) and long (200‐impulse) trains at 5 Hz. 2 All varicosities showed a significant Δ[Ca2+]v in response to every single impulse. The elevated Δ[Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 ± 0.05 s) and a moderate phase (3.6 ± 0.4 s). 3 Line scanning confocal microscopy revealed that the Δ[Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities. 4 Blockade of the voltage‐sensitive calcium channels with Cd2+ (in calcium‐free solution) completely blocked the Δ[Ca2+]v on stimulation. The addition of either nifedipine (10 μm), ω‐conotoxin GVIA (100 nM) or ω‐agatoxin TK (100 nm) showed that 47 ± 6% of the evoked response was mediated by N‐type calcium channels. 5 Ryanodine (10 μm) did not significantly change the amplitude of Δ[Ca2+]v in response to short trains. 6 Spontaneous increases in Δ[Ca2+]v were observed in individual varicosities, with coupling in the increase of Δ[Ca2+]v between varicosities. 7 The presynaptic α2‐receptor antagonist yohimbine (10 μm) increased the amplitude of Δ[Ca2+]v in response to five impulses (5 Hz) by 54 ± 14%, while the α2‐receptor agonist clonidine (1 μm) decreased the Δ[Ca2+]v by 55 ± 4%. 8 These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual Δ[Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.

Influence of spherical aberration on axial imaging of confocal reflection microscopy

The influence of spherical aberration on axial imaging of confocal reflection microscopy is investigated. In particular, the effects of lens aperture size and of the first three orders of spherical aberration are inspected. It is shown both theoretically and experimentally that the aberrated axial response can be improved by slightly reducing the lens aperture size. The experimental results concerning the effect of the tube length on the axial response and the aberration compensation are also given.

Spontaneous purinergic neurotransmission in the mouse urinary bladder

Spontaneous purinergic neurotransmission was characterized in the mouse urinary bladder, a model for the pathological or ageing human bladder. Intracellular electrophysiological recording from smooth muscle cells of the detrusor muscle revealed spontaneous depolarizations, distinguishable from spontaneous action potentials (sAPs) by their amplitude (< 40 mV) and insensitivity to the L‐type Ca2+ channel blocker nifedipine (1 μm) (100 ± 29%). Spontaneous depolarizations were abolished by the P2X1 receptor antagonist NF449 (10 μm) (frequency 8.5 ± 8.5% of controls), insensitive to the muscarinic acetylcholine receptor antagonist atropine (1 μm) (103.4 ± 3.0%), and became more frequent in latrotoxin (LTX; 1 nm) (438 ± 95%), suggesting that they are spontaneous excitatory junction potentials (sEJPs). Such sEJPs were correlated, in amplitude and timing, with focal Ca2+ transients in smooth muscle cells (measured using confocal microscopy), suggesting a common origin: ATP binding to P2X1 receptors. sAPs were abolished by NF449, insensitive to atropine (126 ± 39%) and increased in frequency by LTX (930 ± 450%) suggesting a neurogenic, purinergic origin, in common with sEJPs. By comparing the kinetics of sAPs and sEJPs, we demonstrated that sAPs occur when sufficient cation influx through P2X1 receptors triggers L‐type Ca2+ channels; the first peak of the differentiated rising phase of depolarizations – attributed to the influx of cations through the P2X1 receptor – is of larger amplitude for sAPs (2248 mV s−1) than sEJPs (439 mV s−1). Surprisingly, sAPs in the mouse urinary bladder, unlike those from other species, are triggered by stochastic ATP release from parasympathetic nerve terminals rather than being myogenic.

Characterization of action potential-evoked calcium transients in mouse postganglionic sympathetic axon bundles

1 1. Action potential‐evoked Ca2+ transients in postganglionic sympathetic axon bundles in mouse vas deferens have been characterized using confocal microscopy and Ca2+ imaging. 2 Axonal Ca2+ transients were tetrodotoxin sensitive. The amplitude depended on both the frequency of stimulation and the number of stimuli in a train. 3 Removal of extracellular Ca2+ abolished the Ca2+ transient. Cd2+ (100 μm) inhibited the Ca2+ transient by 78 ± 10 %. The N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) reduced the amplitude by −35 ± 4 %, whereas nifedipine (10 μm; L‐type) and ω‐conotoxin MVIIC (0.1 μm; P/Q type) were ineffective. 4 Caffeine (10 mm), ryanodine (10 μm), cyclopiazonic acid (30 μm) or CCCP (10 μm) had no detectable effects. 5 Blockade of large and small conductance Ca2+‐dependent K+ channels with iberiotoxin (0.1 μm) and apamin (1 μm), respectively, or Ca2+‐dependent Cl− channels by niflumic acid (100 μm) did not alter Ca2+ transients. 6 In contrast, the non‐specific K+ channel blockers tetraethylammonium (10 mm) and 4‐aminopyridine (10 mm) markedly increased the amplitude of the Ca2+ transient. Blockade of delayed rectifiers and A‐like K+ channels, by tityustoxin‐K (α) (0.1 μm) and pandinustoxin‐K (α) (10 nm), respectively, also increased the Ca2+ transient amplitude. 7 Thus, Ca2+ transients are evoked by Na+‐dependent action potentials in axons. These transients originate mainly from Ca2+ entry through voltage‐dependent Ca2+ channels (80 % Cd2+ sensitive of which 40 % was attributable to N‐type). Twenty per cent of the Ca2+ transient was not due to Ca2+ entry through voltage‐gated Ca2+ channels. Intracellular stores and mitochondria were not involved in the generation of the transient. Ca2+ transients are modulated by A‐like K+ channels and delayed rectifiers (possibly KV1.2) but not by Ca2+‐activated ion channels.

Real-time stochastic detection of multiple neurotransmitters with a protein nanopore

The detection of several different neurotransmitters with the same sensor in real-time would be a powerful asset to the field of neurochemistry. We have developed a detector for a broad range of neurotransmitters including amino acids, catecholamines, and nucleotides, which relies on the reversible binding of the analytes to a copper(II) complex within an engineered protein nanopore.

The sources and sequestration of Ca2+ contributing to neuroeffector Ca2+ transients in the mouse vas deferens

The detection of focal Ca2+ transients (called neuroeffector Ca2+ transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca2+ in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA‐1 AM and viewed with a confocal microscope. Ryanodine (10 µm) decreased the amplitude of NCTs by 45 ± 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 ± 10 ms to 800 ± 100 ms). Caffeine (3 mm) induced spontaneous focal smooth muscle Ca2+ transients (sparks). Neither of the T‐type Ca2+ channel blockers NiCl2 (50 µm) or mibefradil dihydrochloride (10 µm) affected the amplitude of excitatory junction potentials (2 ± 5 % and −3 ± 10 %) or NCTs (−20 ± 36 % and 3 ± 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the α1‐adrenoceptor antagonist prazosin (100 nm), showing that NCTs can initiate local contractions. Slow (5.8 ± 0.4 µm s−1), spontaneous smooth muscle Ca2+ waves were occasionally observed. Thus, Ca2+ stores initially amplify and then sequester the Ca2+ that enters through P2X receptors and there is no amplification by local voltage‐gated Ca2+ channels.

Calcium in the nerve terminals of chick ciliary ganglia during facilitation, augmentation and potentiation.

1. The calyciform nerve terminals of chick ciliary ganglia were loaded with the calcium indicators calcium green 1 or fura‐2. These were used to determine the change in calcium concentration in the terminal, [Ca2+]t, following short (10 impulses) and long (600 impulses) trains of high‐frequency (30 Hz) stimulation. 2. Following a single impulse or a short train, the elevated [Ca2+]t declined along two exponentials with time constants similar to slow (F2) facilitation (0.52 s) and augmentation (4.0 s). After a long train elevated [Ca2+]t declined eventually along a single exponential with the time constant of post‐tetanic potentiation (162 s). [Ca2+]t was not elevated through long‐term potentiation. 3. Addition of Ba2+ (0.75 mM) to the extracellular solution slowed only the decline of [Ca2+]t associated with augmentation. The addition of the nitric oxide donor sodium nitroprusside did not affect [Ca2+]t following short or long trains. 4. Removal of extracellular calcium (buffered with EGTA) and the blockade of calcium channels with Cd2+ completely prevented the changes in [Ca2+]t. 5. The soma of ciliary ganglion cells were loaded with calcium green and the postganglionic nerves stimulated with a single impulse or a short train of impulses. Following stimuli, the elevated [Ca2+]t declined along a single exponential with a time constant similar to F2 facilitation with no augmentation component evident. 6. The results are discussed in terms of the hypothesis that each impulse in a train gives an equal increment of residual Ca2+ to a compartment for secretion and that Ca2+ is removed from the compartment by three first‐order kinetics processes associated with F2 facilitation, augmentation and post‐tetanic potentiation.

Calcium in sympathetic boutons of rat superior cervical ganglion during facilitation, augmentation and potentiation.

The sympathetic preganglionic nerve terminals of the rat superior cervical ganglion were loaded with the calcium indicator oregon green 488 BAPTA-1 to measure the change in calcium concentration in the terminal boutons, (delta[Ca2+]b) following short (1 or 5 impulses) and long (200 impulses) trains at 30 Hz. The delta[Ca2+]b after a single action potential or a short train declined in two phases: a fast phase with a time constant of 530+/-30 ms and a moderate phase with a time constant of 4.0+/-0.2 s. The delta[Ca2+]b following a long train eventually declined with a time constant of 127+/-34 s (slow phase). The addition of either omega-agatoxin TK (100 nM), omega-conotoxin GVIA (100 nM) or nifedipine (20 microM) to block P-type, N-type or L-type calcium channels respectively showed that the rise in delta[Ca2+ ]b in boutons was predominantly mediated by an influx of calcium through P-type (53+/-7%) and N-type (46+/-4%) calcium channels. Experiments with caffeine, ryanodine and thapsigargin indicate that intracellular caffeine-sensitive calcium stores have a small but statistically significant effect on the fast and moderate phases. The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 2 microM) significantly decreased the amplitude of the slow phase of delta[Ca2+]b relaxation, and sped its time course, suggesting that mitochondria normally dump calcium during this phase. Adenosine reduced the amplitude of delta[Ca2+]b in response to single action potentials by 30+/-6%, suggesting that adenosine-mediated autoinhibition in these boutons reduces Ca2+ influx. Spontaneous increases in delta[Ca2+]b demonstrated Ca2+ coupling between adjacent boutons. The delta[Ca2+]b kinetics are compared with F2 facilitation, augmentation and post-tetanic potentiation.