Nerve terminals contain multiple sites specialized for the release of neurotransmitters. Release usually occurs with low probability, a design thought to confer many advantages. High probability release sites are not uncommon but their advantages are not well understood. Here we test the hypothesis that high probability release sites represent an energy efficient design. We examined release site probabilities and energy efficiency at the terminals of two glutamatergic motor neurons synapsing on the same muscle fiber in Drosophila larvae. Through electrophysiological and ultrastructural measurements we calculated release site probabilities to differ considerably between terminals (0.33 vs. 0.11). We estimated the energy required to release and recycle glutamate from the same measurements. The energy required to remove calcium and sodium ions subsequent to nerve excitation was estimated through microfluorimetric and morphological measurements. We calculated energy efficiency as the number of glutamate molecules released per ATP molecule hydrolyzed, and high probability release site terminals were found to be more efficient (0.13 vs. 0.06). Our analytical model indicates that energy efficiency is optimal (~0.15) at high release site probabilities (~0.76). As limitations in energy supply constrain neural function, high probability release sites might ameliorate such constraints by demanding less energy. Energy efficiency can be viewed as one aspect of nerve terminal function, in balance with others, because high efficiency terminals depress significantly during episodic bursts of activity.

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.

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. 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). 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. 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. Ryanodine (10 μm) did not significantly change the amplitude of Δ[Ca2+]v in response to short trains. Spontaneous increases in Δ[Ca2+]v were observed in individual varicosities, with coupling in the increase of Δ[Ca2+]v between varicosities. 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%. 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.

1 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. 2 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 þ concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected. 3 In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na þ channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, b-pompilidotoxin (100 mM), which slows Na þ channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K þ channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. 4 The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between b-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na þ channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.