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

Abstract

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 Ca(2+) 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, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's 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 beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.

Figure 1

Oxaliplatin induces multiple EPPs in response to a single stimulation and increases MEPP frequency. Example traces of (a) control, (b) early effect (c) late effect of 500 μM oxaliplatin on mouse phrenic nerve/hemidiaphragm preparation. (ai) Evoked potentials at a low temporal resolution, (aii) single EPPs signal averaged from 30 consecutive potentials, (aiii) single spontaneous MEPP record, signal averaged from 30 detected events. (bi) Low temporal resolution EPPs 39 min after application of oxaliplatin, on some occasions a single stimulus evoked multiple EPPs. (bii) Example of a multiple EPP from a single stimulus, typical of an early oxaliplatin effect. (biii) Signal averaged MEPP at 34 min oxaliplatin exposure. (ci) Repeated trains of EPPs following single stimuli at 59 min oxaliplatin exposure, typical of a late effect. (cii) An example of a sustained train of excitation following a single stimulus, recorded 75 min after oxaliplatin exposure. (ciii) Multiple overlapping MEPP activity observed between stimuli after 75 min oxaliplatin exposure. (d) Changes in MEPP frequency during exposure to H₂O vehicle (star, n=4 preparations), or 500 μM oxaliplatin (circle, n=4). For each treatment group and each preparation, three to four preincubation recordings were made and the mean±s.e.m. are displayed, labelled preincubation on x-axis. Time on x-axis indicates time after addition of vehicle or oxaliplatin. Oxaliplatin MEPP frequency data were directly counted from continuous records. Vehicle MEPP frequency data was either calculated from interval between detected events (n_p=3) or counted from continuous records (n_p=1).

Figure 2

Oxaliplatin has no effect when muscle is not stimulated. (a) During oxaliplatin treatment the muscle was not stimulated and MEPP frequency was directly assessed from continuous records (n=4). Three to four endplates were studied pretreatment and the mean±s.e.m. plotted as preincubation. An example of MEPP activity after 80 min is shown in (bi), at this time the muscle was subjected to a single stimulus, while in the maintained presence of oxaliplatin, and the resultant massive increase in MEPP activity is shown in (bii) 8 s after the single stimulus. (c) Neuronal Na-channels were blocked by TTX (1 μM), MEPP frequency was directly assessed from continuous records (n=4). Three to four endplates were studied pretreatment and the mean±s.e.m. plotted as preincubation, the effect of TTX alone was assessed and the mean±s.e.m. plotted as TTX. Between each assessment of MEPP frequency the phrenic nerve was stimulated for 1 min at 1 Hz.

Figure 3

Pharmacological investigation of oxaliplatin effect. Example traces of signal averaged EPPs and MEPPs (up to 30 records) are shown for each treatment (solid lines), for reference a control trace is aligned for each example (broken line). (a) Apamin (10 μM) after 61 min incubation. (b) 4-AP (0.3 mM) after 6 min incubation. (c) Carbamazepine (0.3 mM) after 14 min incubation. (d) Carbamazepine (0.3 mM) and oxaliplatin (0.5 mM) after 69 min oxaliplatin incubation.

Figure 4

β-pompilidotoxin mimics some of the actions of oxaliplatin. (ai) Example trace of β-pompilidotoxin induced multiple EPPs following stimulation. (aii) Example trace of an EPP associated with elevated MEPP activity in presence of β-pompilidotoxin. (b) Example of two variations of β-pompilidotoxin-induced EPP abnormalities within one continuous trace recorded from a single endplate, 33 min after toxin application. (c) MEPP frequency during β-pompilidotoxin incubation (n=4), three to four endplates were studied pretreatment in each preparation and the mean±s.e.m. plotted as preincubation (error bars are contained within the symbol). Triangles are MEPP frequencies calculated from the interval between detected events, circles are directly measured in continuous records, each symbol represents an individual endplate.

Figure 5

Oxaliplatin reduces the amplitude of EJPs and increases sEJP frequency in the vas deferens. (a) Shows a representative trace of the membrane potential recorded in the mouse vas deferens. Field stimuli were applied every 3 s (arrows) and result in a stimulus artefact (downward deflection) and excitatory junction potential (depolarisation). sEJPs, which report the spontaneous release of ATP, are occasionally detected. After 40 min in oxaliplatin (0.5 mM); (b) the amplitude of EJPs was lower and bursts of sEJPs were recorded. The first few seconds of this recording have been magnified in (c), and the frequency of sEJPs over time is shown in (d), with the data collected into 5 min bins. Pretreatment with either TTX (e) or carbamazepine (f) prevents oxaliplatin's effect on sEJP frequency. In parts (d), (e) and (f) data (collected into 5 min bins) from representative experiments are plotted.

Figure 6

Confocal nerve terminal Ca²⁺ imaging in the presence of oxaliplatin. (a) Shows consecutive confocal images of part of a nerve terminal in the mouse vas deferens filled with the Ca²⁺ indicator Oregon-BAPA-1. A nerve terminal varicosity is marked with an arrow on the first frame. Images were acquired every 0.214 s and field stimuli (sufficient to induce a single nerve terminal action potential) applied at the #. This results in an increase in the fluorescent intensity, indicating an increase in Ca²⁺ concentration within every nerve terminal varicosity ([Ca²⁺]_v). Upon cessation of the stimulus train, the [Ca²⁺]_v returns towards its resting concentration. (b) Shows the same site 40 min after the application of oxaliplatin. The change in the fluorescent signal from the varicosity marked with the arrow is quantified in (c). This figure shows the relative change in fluorescence (ΔF/F), averaged over eight recordings taken 3 min apart, in absence and then presence of oxaliplatin. The error bars show the s.e.m. Oxaliplatin had no significant effect on the stimulation-evoked change in [Ca²⁺]_v.