Spontaneous Excitatory Junction Potentials Research Articles

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5 in Drosophila

ABSTRACT We investigated in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5 is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we studied an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displayed ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of dj-1β, Lrrk, DCTN2-p50, DCTN1-p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the macroautophagy/autophagy machinery or overexpression of human HSPA9 broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects. Abbreviations: AEL: after egg laying; AZ: active zone; brp: bruchpilot; Csp: cysteine string protein; dlg: discs large; eEJPs: evoked excitatory junctional potentials; GluR: glutamate receptor; H2O2: hydrogen peroxide; mEJP: miniature excitatory junctional potentials; MT: microtubule; NMJ: neuromuscular junction; PD: Parkinson disease; Pink1: PTEN-induced putative kinase 1; PSD: postsynaptic density; SSR: subsynaptic reticulum; SV: synaptic vesicle; VGlut: vesicular glutamate transporter.

Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

Systemic scorpion envenomation is characterized by massive neurotransmitter release from peripheral nerves mediated primarily by scorpion venoms neurotoxins. Tityus bahiensis is one of the medically most important species in Brazil, but its venom pharmacology, especially regarding to peripheral nervous system, is poorly understood. Here, we evaluated the T. bahiensis venom activity on autonomic (sympathetic) neurotransmission by using a variety of approaches, including vas deferens twitch-tension recordings, electrophysiological measurements (resting membrane potentials, spontaneous excitatory junctional potentials and whole-cell patch-clamp), calcium imaging and histomorphological analysis. Low concentrations of venom (≤ 3 μg/mL) facilitated the electrically stimulated vas deferens contractions without affecting postsynaptic receptors or damaging the smooth muscle cells. Transient TTX-sensitive sustained contractions and resting membrane depolarization were mediated mainly by massive spontaneous ATP release. High venom concentrations (≥ 10 μg/mL) blocked the muscle contractions and induced membrane depolarization. In neuronal cells (ND7-23wt), the venom increased the peak sodium current, modified the current-voltage relationship by left-shifting the Nav-channel activation curve, thereby facilitating the opening of these channels. The venom also caused a time-dependent increase in neuronal calcium influx. These results indicate that the sympathetic hyperstimulation observed in systemic envenomation is presynaptically driven, probably through the interaction of α- and β-toxins with neuronal sodium channels.

Spontaneous synaptic drive in detrusor smooth muscle: computational investigation and implications for urinary bladder function.

The detrusor, a key component of the urinary bladder wall, is a densely innervated syncytial smooth muscle tissue. Random spontaneous release of neurotransmitter at neuromuscular junctions (NMJs) in the detrusor gives rise to spontaneous excitatory junction potentials (SEJPs). These sub-threshold passive signals not only offer insights into the syncytial nature of the tissue, their spatio-temporal integration is critical to the generation of spontaneous neurogenic action potentials which lead to focal contractions during the filling phase of the bladder. Given the structural complexity and the contractile nature of the tissue, electrophysiological investigations on spatio-temporal integration of SEJPs in the detrusor are technically challenging. Here we report a biophysically constrained computational model of a detrusor syncytium overlaid with spatially distributed innervation, using which we explored salient features of the integration of SEJPs in the tissue and the key factors that contribute to this integration. We validated our model against experimental data, ascertaining that observations were congruent with theoretical predictions. With the help of comparative studies, we propose that the amplitude of the spatio-temporally integrated SEJP is most sensitive to the inter-cellular coupling strength in the detrusor, while frequency of observed events depends more strongly on innervation density. An experimentally testable prediction arising from our study is that spontaneous release frequency of neurotransmitter may be implicated in the generation of detrusor overactivity. Set against histological observations, we also conjecture possible changes in the electrical activity of the detrusor during pathology involving patchy denervation. Our model thus provides a physiologically realistic, heuristic framework to investigate the spread and integration of passive potentials in an innervated syncytial tissue under normal conditions and in pathophysiology.

Investigation of the Syncytial Nature of Detrusor Smooth Muscle as a Determinant of Action Potential Shape.

Unlike most excitable cells, certain syncytial smooth muscle cells are known to exhibit spontaneous action potentials of varying shapes and sizes. These differences in shape are observed even in electrophysiological recordings obtained from a single cell. The origin and physiological relevance of this phenomenon are currently unclear. The study presented here aims to test the hypothesis that the syncytial nature of the detrusor smooth muscle tissue contributes to the variations in the action potential profile by influencing the superposition of the passive and active signals. Data extracted from experimental recordings have been compared with those obtained through simulations. The feature correlation studies on action potentials obtained from the experimental recordings suggest the underlying presence of passive signals, called spontaneous excitatory junction potentials (sEJPs). Through simulations, we are able to demonstrate that the syncytial organization of the cells, and the variable superposition of the sEJPs with the “native action potential”, contribute to the diversity in the action potential profiles exhibited. It could also be inferred that the fraction of the propagated action potentials is very low in the detrusor. It is proposed that objective measurements of spontaneous action potential profiles can lead to a better understanding of bladder physiology and pathology.

Cellular Environment in a Bundle Modulates SEJP Characteristics in Detrusor Smooth Muscle.

Smooth muscle that ensheathes the urinary bladder wall is known as the detrusor. The detrusor has a bundled syncytial architecture where groups of electrically coupled cells form discrete bundles. Electrical activity recorded from detrusor cells is varied. Among the electrical signals recorded from the detrusor, spontaneous excitatory junction potentials (SEJPs) are events that are sub-threshold for spike generation. SEJPs are caused by spontaneous random release of neurotransmitter molecules from varicosities of innervating nerves. SEJPs recorded from different cells of the detrusor vary in amplitude and/or kinetics, and the reasons for variability are obscure. Our hypothesis was that variety in SEJP characteristics may be attributed to the biophysical micro-environment of a cell (in a bundle), where it arises. With the help of computational models, we show how cellular environment factor, such as size of a bundle significantly alters the amplitude as well as kinetics of SEJPs. These findings are congruent with experimental observations. Our results also suggest that characteristically different SEJPs may be observed in identical detrusor cells, with the difference arising from their neighbourhood rather than the inherent nature of the cells. Consequently, SEJP characteristics might be indicative of the cellular environment of electrophysiological recording.

Allostatic Compensatory Mechanisms Enable the Mutant KCNT1 Channels to Induce Seizures and Hyperexcitability

Mutations in the sodium-activated potassium channel (KCNT1) gene are linked to epilepsy. Surprisingly, all KCNT1 mutations examined to date increase K current amplitude. These findings present a major neurophysiological paradox: how do gain-of-function KCNT1 mutations expected to silence neurons cause epilepsy? Here, we use Drosophila to show that expressing mutant KCNT1 in GABAergic neurons leads to seizures, consistent with the notion that silencing inhibitory neurons tips the balance towards hyperexcitation. Unexpectedly, mutant KCNT1 expressed in excitatory neurons also causes seizures. Further observations reveal that mutant KCNT1 causes abnormally large and spontaneous excitatory junction potentials (sEJPs), which are shown to be resulting from local depolarization of synaptic terminals due to a reduction in Shaker and Eag channel levels and more active Na and Ca2 channels. Such compensatory changes are likely mediated by the transcriptional factor Islet. Hence, we provide the first in vivo evidence that both disinhibition of inhibitory neurons and allostatic compensatory plasticity in excitatory neurons might account for the paradoxical effects of gain-of-function mutant KCNT1 in epilepsy.

A1 Adenosine Receptor-Mediated Inhibition of Parasympathetic Neuromuscular Transmission in Human and Murine Urinary Bladder.

The potential role of A1 adenosine receptors in modulating neuromuscular transmission in the detrusor muscle of the urinary bladder has been tested in human and murine preparations with the intent to determine the viability of using adenosine receptor agonists as adjuncts to treat overactive bladder. In human detrusor muscle preparations, contractile responses to electrical field stimulation were inhibited by the selective A1 adenosine receptor agonists 2-chloro-N(6)-cyclopentyladenosine, N(6)-cyclopentyladenosine (CPA), and adenosine (rank order of potency: 2-chloro-N(6)-cyclopentyladenosine > CPA > adenosine). Pretreatment with 8-cyclopentyl-3-[3-[[4(fluorosulphonyl)benzoyl]oxy]propyl]-1-propylxanthine, an irreversible A1 antagonist, blocked the effects of CPA, thus confirming the role of A1 receptors in human detrusor preparations. In murine detrusor muscle preparations, contractions evoked by electrical field stimulation were reduced by CPA or adenosine. Amplitudes of the P2X purinoceptor-mediated excitatory junctional potentials (EJPs) recorded with intracellular microelectrodes were reduced in amplitude by CPA and adenosine with no effect on the spontaneous EJP amplitudes, confirming the prejunctional action of these agents. 8-Cyclopentyltheophylline, a selective A1 receptor antagonist, reversed the effects of CPA on EJP amplitudes with no effect of spontaneous EJPs, confirming the role of A1 receptors in mediating these effects.

Mechanisms Involved in Nicotinic Acetylcholine Receptor-Induced Neurotransmitter Release from Sympathetic Nerve Terminals in the Mouse Vas Deferens

Prejunctional nicotinic acetylcholine receptors (nAChRs) amplify postganglionic sympathetic neurotransmission, and there are indications that intraterminal Ca2+ stores might be involved. However, the mechanisms by which nAChR activation stimulates neurotransmitter release at such junctions is unknown. Rapid local delivery (picospritzing) of the nAChR agonist epibatidine was combined with intracellular sharp microelectrode recording to monitor spontaneous and field-stimulation-evoked neurotransmitter release from sympathetic nerve terminals in the mouse isolated vas deferens. Locally applied epibatidine (1 µM) produced ‘epibatidine-induced depolarisations’ (EIDs) that were similar in shape to spontaneous excitatory junction potentials (SEJPs) and were abolished by nonselective nAChR antagonists and the purinergic desensitizing agonist α,β-methylene ATP. The amplitude distribution of EIDs was only slightly shifted towards lower amplitudes by the selective α7 nAChR antagonists α-bungarotoxin and methyllcaconitine, the voltage-gated Na+ channel blocker tetrodotoxin or by blocking voltage-gated Ca2+ channels with Cd2+. Lowering the extracellular Ca2+ concentration reduced the frequency of EIDs by 69%, but more surprisingly, the Ca2+-induced Ca2+ release blocker ryanodine greatly decreased the amplitude (by 41%) and the frequency of EIDs by 36%. Ryanodine had no effect on electrically-evoked neurotransmitter release, paired-pulse facilitation, SEJP frequency, SEJP amplitude or SEJP amplitude distribution. These results show that activation of non-α7 nAChRs on sympathetic postganglionic nerve terminals induces high-amplitude junctional potentials that are argued to represent multipacketed neurotransmitter release synchronized by intraterminal Ca2+-induced Ca2+ release, triggered by Ca2+ influx directly through the nAChR. This nAChR-induced neurotransmitter release can be targeted pharmacologically without affecting spontaneous or electrically-evoked neurotransmitter release.

Sympathetic overdrive in obesity involves purinergic hyperactivity in the resistance vasculature

While a close correlation exists in obese humans between sympathetic, adrenergic hyperactivity and structural and functional organ damage, a role for the co-transmitter, ATP, in vascular function remains unexplored. We therefore studied sympathetic nerve-mediated responses of pressurised small mesenteric arteries from control and obese rats. Diet-induced obesity significantly increased the amplitude of vasoconstriction to transmural nerve stimulation (1-10 Hz; P <0.05). At 1 and 5 Hz, both adrenergic and purinergic responses were significantly augmented, while only the purinergic component was increased at 10 Hz (P <0.05). Nerve stimulation at 1 Hz evoked contractions and underlying excitatory junction potentials (EJPs), which were both significantly increased in amplitude during obesity (P <0.05) and abolished by αβ-methylene ATP (1 μM; desensitises purinergic receptors). The rise time and rate of decay of these EJPs were significant decreased (P <0.05), without change in resting membrane potential. Amplitude and frequency of spontaneous EJPs and the density of perivascular sympathetic nerves were also significantly increased (P <0.05). Inhibition of sensory neurotransmitter release (capsaicin; 10 μM) significantly increased the amplitude of nerve-mediated contraction (P <0.05), with a greater effect in control than obese animals, although the density of sensory nerves was unaffected by obesity. We demonstrate that sympathetic nerve-mediated vasoconstriction is enhanced by diet-induced obesity due to upregulation of purinergic, in addition to adrenergic, neurotransmission. Changes result from increased perivascular sympathetic innervation and release of ATP. We conclude that augmented sympathetic control of vasoconstriction induced by obesity could contribute directly to hypertension and global organ damage. A decrease in sensitivity to sensory vasodilatory neurotransmitters may also affect these processes.

All ahead stop! How intestinal motility adapts to cope with inflammation induced ulceration

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Prejunctional and postjunctional actions of heptanol and 18β-glycyrretinic acid in the rodent vas deferens

Heptanol and 18β-glycyrrhetinic acid (18βGA) block gap junctions, but have other actions on transmitter release that have not been characterised. This study investigates the prejunctional and postjunctional effects of these compounds in guinea pig and mouse vas deferens using intracellular electrophysiological recording and confocal Ca2+ imaging of sympathetic nerve terminals. In mice, heptanol (2 mM) reversibly decreased the amplitude of purinergic excitatory junction potentials (EJPs; 52±5%, P<0.05) while having little effect on spontaneous excitatory junction potentials (sEJPs). Heptanol (2 mM) reversibly abolished the nerve terminal Ca2+ transient in 52% of terminals. 18βGA (10 μM) decreased the mean EJP amplitude, and increased input resistance in both mouse (137±17%, P<0.05) and guinea pig (354±50%, P<0.001) vas deferens indicating gap junction blockade. Further, 18βGA increased the sEJP frequency significantly in guinea pigs (by 71±25%, P<0.05) and in 5 out of 6 tissues in mice (19±3%, P<0.05). Moreover, 18βGA depolarised cells from both mice (11±1%, P<0.01) and guinea pigs (8±1%, P<0.005). Therefore, we conclude that heptanol (2 mM) decreases neurotransmitter release (given the decrease in EJP amplitude) by abolishing the nerve terminal action potential in a proportion of nerve terminals. 18βGA (10 μM) effectively blocks the gap junctions, but the increase in sEJP frequency suggests an additional prejunctional effect, which might involve the induction of spontaneous nerve terminal action potentials.

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.

Measures of purinergic neurotransmission reveal variable effective packet size

The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other smooth muscle syncytia has been attributed to electrically-attenuated depolarisations resulting from the spontaneous release of uniform-sized ATP packets acting on distant electrically-coupled smooth muscle cells (SMCs). In the present investigation, however, surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176±18 MΩ, n=4) similar to those of dissociated cells.

The effect of epibatidine on spontaneous and evoked neurotransmitter release in the mouse and guinea pig isolated vas deferens

Nicotinic agonists increase sympathetic field-stimulus-evoked contraction of the rodent vas deferens, presumably by increasing evoked neurotransmitter release. This presumption was tested in two species. The effect of the nicotinic acetylcholine receptor (nAChR) agonist epibatidine on neurotransmitter release in mouse and guinea pig isolated vas deferens was investigated using contraction studies and conventional intracellular recording techniques. In 12 of 14 mouse vasa deferentia, slow bath application of epibatidine (100 nM) had no significant effect on excitatory junction potential (EJP) amplitude and spontaneous EJP (SEJP) frequency. However, rapid application of epibatidine to the mouse vas deferens caused an increase in SEJP frequency (by 530%), with no effect on EJP amplitude. Despite the absence of an effect on EJPs, electrically-evoked contractions of the mouse vas deferens were significantly increased in the presence of epibatidine (by 50%). A transient contraction was reliably induced by a higher epibatidine concentration (1 microM). This contraction was significantly reduced in the presence of prazosin, tetrodotoxin, or alpha,beta-methyleneATP. Epibatidine did not induce a contraction in the presence of a combination of prazosin, alpha,beta-methyleneATP and cyclopentolate. In guinea pig vasa deferentia, bath-applied epibatidine potentiated EJP amplitude in a biphasic pattern, lasting for at least 30 minutes. The nAChR-mediated augmentation of neurogenic contraction is indeed prejunctional, but in the mouse arises from an increase in spontaneous neurotransmitter release that primes smooth muscle for subsequent contraction, while in the guinea pig there is a direct augmentation of evoked neurotransmitter (ATP) release.

The origin of the skewed amplitude distribution of spontaneous excitatory junction potentials in poorly coupled smooth muscle cells

The skewed amplitude distribution of spontaneous excitatory junction potentials (sEJPs) in the mouse vas deferens and other electrically-coupled smooth muscle syncytia has been attributed to electrically-attenuated depolarizations resulting from the spontaneous release of quantized packets of ATP acting on remote smooth muscle cells (SMCs). However, in the present investigation surface SMCs of the mouse isolated vas deferens were poorly electrically coupled, with input resistances (176±18 MΩ, range: 141–221 MΩ, n=4) similar to those of dissociated cells. Furthermore, the amplitude of evoked EJPs was more variable in surface compared with deeper SMCs (F test, F=17.4, P<0.0001). Using simultaneous electrophysiology and confocal microscopy to investigate these poorly-coupled cells, it is shown that α-latrotoxin-stimulated sEJPs correlate, in timing (median delay ranged from −30 to −57 ms, P<0.05 in all experiments, n=5) and amplitude (Pearson product moment correlation, ρ>0.55 and P<0.001), with purinergic neuroeffector Ca2+ transients (NCTs) in SMCs. The temporal correlation between sEJPs of widely ranging amplitude with NCTs in the impaled SMC demonstrates that all sEJPs could arise from neurotransmitter action on the impaled cell and that the skewed distribution of sEJPs can be explained by the variable effect of packets of ATP on a single SMC. The amplitude correlation of sEJPs and NCTs argues against the attenuation of electrical signal amplitude along the length of a single SMC. The skewed sEJP amplitude distribution arising from neurotransmitter release on single SMCs is consistent with a broad neurotransmitter packet size distribution at sympathetic neuroeffector junctions.

Effects of carbenoxolone on syncytial electrical properties and junction potentials of guinea-pig vas deferens

The effects of the putative gap junction blocker carbenoxolone on smooth muscle syncytial properties and junction potentials were studied in guinea pig vas deferens (GPVD). Treatment with 50 muM carbenoxolone reversibly and significantly increased input resistance (R (in)) (by 682.5 +/- 326.0 %, P < 0.05) and abolished cable potentials within 6-7 mins of incubation, without disturbing resting membrane potential. Carbenoxolone reversibly and significantly increased the amplitude of spontaneous excitatory junction potentials (sEJPs) by 96.9 +/- 35.45% (P < 0.05), shifted their amplitude distribution rightwards, and reduced their frequency of occurrence by 58.17 +/- 17.7% (P < 0.05), without altering their time courses. Similarly, carbenoxolone increased the amplitude of evoked excitatory junction potentials (eEJPs) by 17.7 +/- 5.88% and tau (decay) by 19.43 +/- 8.29% (P < 0.05). Our results indicate that carbenoxolone alters the electrical properties and junctional potentials of the GPVD by a mechanism consistent with a relatively specific block of gap junctions. These results suggest that gap junction mediated cell-to-cell communication may significantly modulate the electrical properties and junctional potentials of the GPVD and consequently the physiological functioning of this tissue.

Post‐ and prejunctional consequences of ecto‐ATPase inhibition: electrical and contractile studies in guinea‐pig vas deferens

At sites of purinergic neurotransmission, synaptic ecto-ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP-mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto-ATPase inhibitor, on ATP-mediated electrical and contractile activity in the sympathetically innervated guinea-pig vas deferens. ARL 67156 at 100 mum significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% (P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 +/- 0.13 to 0.90 +/- 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto-ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation-evoked quantal release of neurotransmitter, was also increased ( approximately 6-fold; P < 0.01), along with the appearance of DEs at previously 'silent' latencies. Purinergic contractions of the vas deferens were potentiated significantly (P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine (P < 0.01) but left unaffected by the A1 antagonist 8-phenyltheophylline (8-PT). Our results indicate (i) that ecto-ATPase activity, in addition to modulating the ATP-mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors.

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 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.

Simulation framework for electrophysiological networks: Effect of syncytial properties on smooth-muscle synaptic potentials

A building block-based software framework was developed to simulate electrophysiological networks. The synaptic potentials generated during neurotransmission were simulated in an existing discrete bidomain model of smooth muscle, using cubic, three-dimensional grids of varying sizes. The model is automatically derived and numerically solved, and the results of the simulation agree with previous results obtained analytically. An enhanced model was also proposed, incorporating an additional (junctional) capacitance in the network. The correctness of the model was verified, and the effect of the extra capacitance on the synaptic potentials was explored. It was found that, with a junctional capacitance C(i) of 1.4 x 10(-10) F incorporated, the peak amplitude of the spontaneous excitatory junction potential V(peak) declined by approximately 13% at node 0 and by approximately 37% at node 3x for a system size of 9(3). Similar results were obtained for different system sizes. V(peak) also declined as the junctional capacitance Ci was increased. In a system of size 11(30, a 200-fold increase in C(i) induced a 55% reduction at node 0. It is suggested that the type of modular simulation framework developed here may find general applicability for simulations of other physiological systems.

Excitatory effects of Buthus C56 toxin on Drosophila larval neuromuscular junction

Buthus C56 toxin from venom of the Indian red scorpion Mesobuthus tamulus was studied for its effects on spontaneous miniature excitatory junctional potentials (MEJP) on Drosophila larval neuromuscular junctions. C56 toxin was isolated on CM-Cellulose with linear gradient of ammonium acetate buffer, pH 6.0. Toxin purity was determined on SDS slab gel electrophoresis. Effective concentration of C56 toxin was based on contraction paralysis units (CPU) in Drosophila 3rd instar larvae by microinjection (0.1 CPU/ml = 2 x 10-6 g/ml). The toxin-induced excitatory junctional potentials were studied for calcium dependency (0.2 mM to 1.2 mM Ca2+) in Drosophila Ringer. Excitatory junctional potential amplitude was increased with increasing calcium concentration; maximum increase in the frequency at 0.4 mM Ca2+/4 mM Mg2+ Drosophila Ringer. It was suggested that while amplitude of excitatory junctional potentials was increased with concentration, maximum frequency increase at 0.4 mMCa2+/4 mM Mg2+ Drosophila Ringer may be due to augmented Ca2+ influx in 0.4 mM Ca2+, when NMDA receptors were maximally activated in C56 toxin-treated Drosophila larval neuromuscular junction.