Resistant Action Potentials Research Articles

Bladder Overactivity and Hyperexcitability of Bladder Afferent Neurons after Intrathecal Delivery of Nerve Growth Factor in Rats

Nerve growth factor (NGF) has been proposed as an important mediator inducing bladder overactivity under pathological conditions such as spinal cord injury, bladder outlet obstruction, or cystitis. We therefore examined the effects of chronic NGF treatment on bladder activity and the properties of bladder afferent neurons. In adult female rats, NGF (2.5 microg/microl) was infused continuously into the intrathecal space at the L6-S1 level of spinal cord for 1 or 2 weeks using osmotic pumps (0.5 microl/h). Bladder afferent neurons were labeled with axonal transport of Fast Blue injected into the bladder wall. After intrathecal injection of NGF, cystometrograms under an awake condition showed bladder overactivity revealed by time-dependent reductions in intercontraction intervals and voided volume. ELISA analyses showed significant increases in NGF levels in L6-S1 dorsal root ganglia of NGF-treated rats. In patch-clamp recordings, dissociated bladder afferent neurons exhibiting tetrodotoxin (TTX)-resistant action potentials from NGF-treated animals were larger in diameter and had significantly lower thresholds for spike activation compared with sham rats. In addition, the number of TTX-resistant action potentials during 600 ms depolarizing pulses was significantly increased time dependently after 1 or 2 weeks of NGF application. The density of slowly inactivating A-type K+ currents was decreased by 52% in bladder afferent neurons with TTX-resistant spikes after 2 week NGF treatment. These results indicate that increased NGF levels in bladder afferent pathways and NGF-induced reduction in A-type K+ current density could contribute to the emergence of bladder overactivity as well as somal hypertrophy and hyperexcitability of bladder afferent neurons.

Ca2+ involvement in the action potential generation of myenteric neurones in the rat oesophagus.

Intracellular recordings were used to study the physiological behaviour of rat oesophageal myenteric neurones, which are embedded in striated muscle. Injection of depolarizing pulses evoked action potentials with a clear 'shoulder' in all neurones. This shoulder disappeared under low Ca2+/high Mg2+ conditions. Tetrodotoxin (TTX; 1 micromol L-1) did not impede spike firing, whereas under combined TTX and low Ca2+/high Mg2+ conditions the action potentials were completely abolished, indicating that TTX- resistant action potentials are mediated by a Ca2+ current. Further experiments with omega-conotoxin GVIA (100 nmol L-1) revealed that these Ca2+ currents enter the cell via N-type voltage-activated Ca2+ channels (see also accompanying paper). Tetraethylammonium (10 mmol L-1) caused broadening of the action potentials, which probably resulted from prolonged Ca2+ influx due to blockade of the delayed rectifier K+ channel. Although Ca2+ appears to be involved in the spike generation of all rat oesophageal myenteric neurones, only a minority (14%) shows a slow afterhyperpolarization. Thus, no strict correlation exists between the presence of a shoulder and a slow afterhyperpolarization. Furthermore, morphological identification of 25 of the impaled neurones revealed that there was no strict correlation between morphology and electrophysiological behaviour. Consequently, rat oesophageal myenteric neurones appear to differ in several aspects from myenteric neurones in smooth muscle regions of the gastrointestinal tract.

Nitric oxide modulates Ca(2+) channels in dorsal root ganglion neurons innervating rat urinary bladder.

The effect of a nitric oxide (NO) donor on high-voltage-activated Ca(2+) channel currents (I(Ca)) was examined using the whole cell patch-clamp technique in L(6)-S(1) dorsal root ganglion (DRG) neurons innervating the urinary bladder. The neurons were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall. Approximately 70% of bladder afferent neurons exhibited tetrodotoxin (TTX)-resistant action potentials (APs), and 93% of these neurons were sensitive to capsaicin, while the remaining neurons had TTX-sensitive spikes and were insensitive to capsaicin. The peak current density of nimodipine-sensitive L-type Ca(2+) channels activated by depolarizing pulses (0 mV) from a holding potential of -60 mV was greater in bladder afferent neurons with TTX-resistant APs (39.2 pA/pF) than in bladder afferent neurons with TTX-sensitive APs (28.9 pA/pF), while the current density of omega-conotoxin GVIA-sensitive N-type Ca(2+) channels was similar (43-45 pA/pF) in both types of neurons. In both types of neurons, the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) (500 microM), reversibly reduced (23.4-26.6%) the amplitude of I(Ca) elicited by depolarizing pulses to 0 mV from a holding potential of -60 mV. SNAP-induced inhibition of I(Ca) was reduced by 90% in the presence of omega-conotoxin GVIA but was unaffected in the presence of nimodipine, indicating that NO-induced inhibition of I(Ca) is mainly confined to N-type Ca(2+) channels. Exposure of the neurons for 30 min to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM), an inhibitor of NO-stimulated guanylyl cyclase, prevented the SNAP-induced reduction in I(Ca). Extracellular application of 8-bromo-cGMP (1 mM) mimicked the effects of NO donors by reducing the peak amplitude of I(Ca) (28.6% of reduction). Action potential configuration and firing frequency during depolarizing current pulses were not altered by the application of SNAP (500 microM) in bladder afferent neurons with TTX-resistant and -sensitive APs. These results indicate that NO acting via a cGMP signaling pathway can modulate N-type Ca(2+) channels in DRG neurons innervating the urinary bladder.

Changes in tetrodotoxin-resistant action potentials after passive transfer of myasthenia gravis patient sera

Muscle electrical activity has been studied in mice after intraperitoneal injection of sera from myasthenia gravis (MG) patients. Myasthenic serum did not modify the electrical properties of innervated muscle fibres. The resting membrane potential and the action potential parameters remained unchanged. However, tetrodotoxin (TTX)-resistant action potentials of denervated muscles were reduced by myasthenic serum, possibly in association with receptor endocytosis induced by the immunoglobulin. However, a direct effect of MG serum on TTX-resistant sodium channels cannot be ruled out.

Effects of ?-endorphin on the development of denervation changes in rat muscle fiber membrane

Recordings were made of post-denervation changes in resting potential and input resistance in muscle fiber membrane, as well as anode break, tetrodotoxin resistant action potentials, and asynaptic sensitivity to acetylcholine during experiments on cultured diaphragm muscle fiber isolated from rats. Addition of β-endorphin to the culture medium prevented increase in the input resistance of muscle fibers and reduced development of asynaptic transmitter sensitivity in the membrane, but failed to change the ability of the denervated muscle membrane to generate anode break and tetrodotoxin-resistant action potentials. The effects of β-endorphin were not abolished by naloxone, which itself had endorphin-like powers as measured by the indices used in this research. It is therefore suggested that β-endorphin or like substances could be claimed as the neurotrophic factors responsible for controlling passive electrical properties of the muscle fiber membrane and contribute to regulating its acetylcholine sensitivity.

The development of tetrodotoxin-resistant action potentials in long-term organ culture of rat muscle.

The development and long-term maintenance of tetrodotoxin (TTX)-resistant action potentials in culture was examined. The amplitude and maximum rate of rise of the action potential in normal Ringer solution fell during culture. At the same time the amplitude and maximum rate of rise of the TTX-resistant action potential increased, demonstrating that TTX resistance can develop fully in culture. Actinomycin D inhibited the onset of denervation changes in culture and was also able to delay the fall in the maximum rate of rise of the action potential in normal Ringer solution. When applied to muscle after 48 h of culture, it was relatively ineffectual, showing that de novo protein synthesis during the first 48 h of culture result in denervation changes which are not reversed by subsequent exposure to actinomycin D. The onset of TTX resistance occurred more rapidly in dissociated muscle fibres, presumably as a result of the early removal of the nerve stump.

Ionic mechanisms underlying the firing properties of rat neonatal motoneurons studied in vitro

Ionic mechanisms underlying the firing properties of spinal motoneurons of neonatal rats (postnatal days 3–10) have been investigated using a hemisected, in vitro spinal cord preparation. These results demonstrate the presence of a high-threshold voltage-dependent calcium response and partial sodium-dependent spikes. The calcium current is evident during the falling phase of the action potential and is the major component of the after-depolarizing potential. The subsequent increase in intracellular calcium concentration activates a calcium-dependent potassium conductance (g K-ca), the major component of the after-hyperpolarizing potential. The g ca, by activating g K-ca, is the primary determinant of firing rate in neonatal motoneurons. For, when g ca, was blocked by Cd 2+, the interspike interval decreased, the maximum firing rate and the slope of the firing frequency-injected current relation increased. The calcium current is particularly robust during the first few postnatal days; during this period, tetrodotoxin resistant action potentials can be elicited by direct stimulation under control conditions. In animals older than 5 days such calcium spikes could be elicited only after decreasing g k with intracellular Cs + or extracellular tetraethylammonium. This was the case even when 1 mM of the bath CaCl 2 was replaced with BaCl 2. The rising phases of calcium spikes recorded from neurons in both age groups demonstrate several components suggesting the calcium spikes comprise several discrete events, which probably originate across the dendritic membrane. When g k was decreased by bath application of tetraethylammonium + and Cs +, neonatal motoneurons generated prolonged Ca-dependent spikes lasting for up to 6 s. Repolarization of Ca spikes occurred in two stages, the first was rapid (− 2.11 ±0.8 V/s, n = 6) but incomplete. The second, was slower (−0.01 ± 0.003 V/s, n = 5) and returned the membrane potential to the resting level after about 1–2 s. It is suggested that accumulation of extracellular potassium may contribute to the slow phase of repolarization. Motoneurons from the younger age group (3–5 days old) demonstrate all-or-none partial spikes rising from the after-depolarization of directly elicited sodium-dependent action potentials. Similar partial spikes were elicited from neurons from older animals during intracellular Cs + loading. The partial spikes had faster rates of rise than the tetrodotoxin-resistant spikes and were not seen after tetrodotoxin treatment, suggesting that they are sodium-dependent. These findings suggest that, in addition to the action potentials arising at the initial segment, neonatal motoneurons generate both sodium-dependent and calcium-dependent electroresponsive events at anatomically discrete sites, probably across the dendritic membrane. Further, in these immature cells an increase in intracellular calcium concentration activates a g k-ca which prolongs the after-hyperpolarization and thus gives the cells a low maximum firing rate compared to mature motoneurons, insuring a firing level well-matched to the properties of the immature sensory and motor systems.

Electrophysiologic denervation changes of human muscle fibers in motoneuron diseases.

Muscle denervation and reinnervation by sprouting of the surviving motoneurons characterizes motoneuron diseases (MND). In mammalian muscles, experimental denervation induces the appearance of extrajunctional acetylcholine (ACh) receptors and tetrodotoxin (TTX) resistant action potentials (AP). These denervation changes have been investigated in muscle biopsies from 10 MND patients and in 2 traumatically denervated normal human muscles. Extrajunctional ACh sensitivity was present in 113 of the 140 (73%) fibers from MND muscles studied. In 50 of 84 (70%) ACh-sensitive fibers, no TTX-resistant AP were present. The remaining fibers (30%) showed small regenerative responses. In contrast, all the traumatically denervated muscle fibers showed extrajunctional ACh sensitivity and TTX-resistant AP. Histochemical analysis of the biopsies showed no direct correlation between the frequency of ACh-sensitive fibers and that of the atrophic or normal-appearing fibers. The absence of TTX-resistant AP in ACh-sensitive fibers and its lack of correlation with the histochemical criteria of denervation suggest the presence of a state of innervation in MMD, where the motoneuron is not able to maintain its fully trophic influence on the muscle fiber membrane.

Denervated frog skeletal muscle. Some electrical and mechanical properties.

The effects of denervation on several mechanical and electrical parameters of frog sartorius muscle have been investigated. In denervated muscles, there is no change in the resting potential and a relatively small change in the action potential. The first alteration in the action potential is a reduction of about 30% in the maximum rate of repolarization in muscles that have been denervated for 40 days or longer. Later, the overshoot and maximum rate of depolarization also decline. No tetrodotoxin resistant action potentials could be detected. Fibrillatory potentials were observed infrequently and in most cases in depolarized fibers. Twitch tension is significantly reduced by denervation while the tetanus tension is practically unaffected by denervation. The experiments suggest that the decline in twitch tension produced by denervation reflect a defect in some step of the excitation contraction coupling sequence. On the other hand, post-tetanic potentiation of the twitch is much larger in denervated than in control muscles. This potentiation in denervated muscles is paralleled by an increased action potential duration which returns to its pretetanic duration with a time course indistinguishable from that of the twitch potentiation.

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na + channel specific action of ATX II. Na + current inactivation is slowed with the time constant τ h increasing towards positive membrane potentials. The steady state inactivation curve h oo was shifted to more positive potentials and its slope reduced.

Membrane and biochemical alterations after denervation and during reinnervation of mouse skeletal muscle.

Denervation of the extensor digitorum longus (EDL) muscle of the mouse by either nerve crush or nerve section produced: a reduction of the resting membrane potential (Em), alterations in the properties of muscle fibre action potentials and the development of tetrodotoxin (TTX)-resistant action potentials. These changes in membrane electrical properties were accompanied by an increase in the endocytic activity of the muscle and an increase in the activities of the lysosomal enzymes cathepsin D and N-acetylglucosaminidase (NAGA). Reinnervation of muscle was indicated at 9 days after nerve crush by the presence of miniature end-plate potentials. The recovery of membrane electrical properties, beginning with the onset of reinnervation, were not temporally related. The Em increased in two stages: an early rapid repolarization and a later slower repolarization. The muscle fibers were sensitive to the blocking action of TTX by 12 days after nerve crush, whereas the rate of rise (dV/dt) of the action potential did not approach values of innervated muscles until 21 days. Reinnervation resulted in a decrease in endocytosis and a decrease in the activities of cathepsin D and NAGA toward innervated values by 21 days after nerve crush. The results suggest that membrane alterations after denervation and during reinnervation may occur by endo- and exocytosis of membrane constituents and that the lysosomal system may play a role in the breakdown and/or recycling of these structures.

Alterations in membrane electrical properties during long‐term denervation of rat skeletal muscle

Some membrane electrical properties of the extensor digitorum longus muscle of the rat were examined up to 21 days after denervation. The resting potential was significantly more depolarized at 3 days after denervation than it was at later times. The rate of rise (dV/dt) of the action potential decreased throughout the time course of the study but approached a steady value between 14 and 21 days after denervation. In addition, the dV/dt of tetrodotoxin (TTX)-resistant action potentials increased up to and including 7 days after nerve section, but declined thereafter. When expressed as % of control, the dose-response to TTX was similar throughout denervation. It is suggested that the large depolarization observed early in denervation may be related to the turnover of membrane constituents occurring at this time. The results also suggest that denervation produces a reduction in the number of fast TTX-sensitive Na+ channels with the appearance of a new population of slow TTX-resistant channels. However, long term denervation results in a reduction in the density of both types of channels.

Studies on neurotrophic regulation of murine skeletal muscle.

1. A quantitative comparison was made of the effects of the paralysis caused by botulinum toxin (BoTx) type A with those of surgical denervation on the development of tetrodotoxin (TTX) resistant action potentials and of extrajunctional acetylcholine (ACh) receptors in rat and mouse skeletal muscle.2. After surgical denervation, TTX resistant action potentials were present in all fibres on the third day and their rate of rise and amount of overshoot reached peak values at the fifth day. BoTx poisoning failed, despite causing complete paralysis, to induce TTX resistant action potentials in all fibres and their average rate of rise was at all times (4-12 days) only about half that in denervated fibres. Similarly BoTx poisoning induced a smaller increase than surgical denervation in the number of extrajunctional ACh receptors, measured as (3)H-labelled Naja naja siamensis alpha-neurotoxin binding sites.3. Surgical denervation of BoTx poisoned muscles induced TTX resistant action potentials in all fibres and their rate of rise and amount of overshoot were 2-3 times those in BoTx poisoned muscles only. Denervation also significantly increased the binding of labelled alpha-neurotoxin. These effects of denervation were prevented by the administration of actinomycin D, a blocker of protein synthesis.4. Administration of the alpha-neurotoxin to BoTx poisoned animals resulted in the appearance of TTX resistant action potentials in all fibres and in a significant increase in their rate of rise and overshoot.5. The results show that, despite causing complete paralysis, BoTx is less effective than surgical denervation in inducing denervatory changes in skeletal muscle. This suggests that the BoTx poisoned nerve has an influence which suppresses the appearance of denervation signs. Since the alpha-neurotoxin blocked this influence remaining release of ACh, quantal or non-quantal, may be responsible for this neurotrophic action.

Effects of different dosages of vincristine on the component conductances of rat skeletal muscle fibers

Summary Membrane potentials, cable parameters and component resting conductances, were measured in extensor digitorum longus (EDL) muscle fibers from adult rats, in vitro at 25°–28°C, at 6 to 33 days following chronic application of 0.05% and 0.1% vincristine sulfate to the peroneal nerve. All the vincristine treated fibers were depolarized; from 6 to 14 days of treatment tetrodotoxin resistant action potentials, were present. Membrane resistance in normal solution in the 0.05% vincristine treated fibers did not change significantly until the 23th day of t reatment; for longer period of 0;05% and in the 0;1% vincristine treated preparations it increased significantly. Membrane resistance in chloride free medium showed a large decrease in all the preparations. In the 0.05% vincristine treated fibers, mean chloride conductance GC1 did not change significantly, until the 23th day of treatment, while mean potassium conductances Gk increased significantly from a mean control value of 163±17 to 454±68 μmhos/cm2; at 33 day of treatment GC1 dropped significantly while Gk was still high. In the 0.1% vincristine treated fibers, mean Gk showed a tendency to rise, while GC1 decreased significantly from a control value of 3427±114 to 1699±108 μmhos/cm2. In accordance with the literature it was concluded that factors transported by the microtubular and may be by the microfilament system are important for the mainteinance of the high resting GCl and the low resting Gk of mammalian skeletal muscle fibers and therefore for the integrity of the neuromuscular function.

Control of ACh sensitivity in temporarily unconnected (“decentralized”) segments of diaphragm-muscle fibres of the rat

1. After a local lesion of the diaphragm muscle, which produced a segment of innervated muscle fibres connected with an intact nerve-free segment by a region of crushed muscle fibres, the sensitivity to ACh, the presence of tetrodotoxin resistant action potentials (AP) and the transmission of AP along the muscle fibres were studied. 2. Three days after local injury of the diaphragm muscle ACh sensitivity and TTX resistance appeared in the crushed and nerve-free segments between the place of injury and the tendineous attachment. 5-7 days after injury transmission of action potentials through the damaged to the undamaged ("decentralized"), nerve-free part of the fibres is restored. High ACh sensitivity and TTX resistance of the latter segment, however, are completely lost only 20 days after the local injury. During this period contractility of the muscle remains practically unchanged. Enzymatic activities (SDH, ATPase and phosphorylase) of the damaged part were lost 3 days after crushing and recovered slowly between 7-10 days of regeneration of the diaphragm muscle fibres. 3. The experiments suggest that during regeneration of damaged muscle fibres supersensitivity to ACh remains high inspite of normal AP activity and that intracellular mechanisms may be involved in the induction and disappearance of ACh hypersensitivity.

Some electrophysiological properties of isolated extensor digitorum longus muscles from normal and genetically-distrophic hamsters

Experiments have been made on isolated extensor digitorum longus muscles of normal and genetically dystrophic hamsters. The dystrophic muscle fibers exhibited low resting membrane potentials and reduced maximum rate of rise and overshoot amplitude of the action potentials. The mean “input-resistance” of the dystrophic fibers was the same as normal, but the variation from fiber to fiber was much greater than normal. Fibrillation was not uncommon in the dystrophic fibers, but extrajunctional acetylcholine sensitivity and tetrodotoxin resistant action potentials were rare. Miniature end-plate potentials were apparently present in all fibers. In some muscles, the abnormal properties of the fibers could be correlated with the macroscopic appearance of the muscles. The implications of the results are discussed in terms of the etiology of muscular dystrophy in the hamster

Studies on Tetrodotoxin Resistant Action Potentials in Denervated Skeletal Muscle

AbstractIn this study it is shown that action potential generation in rat, mouse and cat muscles is blocked by tetrodotoxin (TTX, 10‐6 M) but that the chronically denervated muscles of these species are partially resistant to TTX. In denervated rat muscles the development of the TTX resistant action potential during the first four days of denervation is paralleled by the spread of extrajunctional cholinergic sensitivity. However, the extrajunctional cholinergic receptor and the TTX resistant action potential can be selectively blocked by drugs, indicating that the action potential site and the cholinergic receptor are not identical structures. Denervated muscles are also partially resistant to saxitoxin, although less so than to TTX.

Action potential generation in denervated rat skeletal muscle. II. The action of tetrodotoxin.

AbstractAction potential generation was studied in the presence of tetrodotoxin (TTX) up to one week after denervation in individual muscle fibres of the extensor digitorurn longus and diaphragm muscles of the rat. TTX 10‐6 M completely blocked spike generation in innervated inuscle and in niuscle denervated for one day. On the second and subsequent days after denervation, action potentials resistant to TTX were recorded in the muscle fibres. In the extensor digitorum longus muscle denervated for 5 days, action potentials recorded in the presence of TTX 10‐6 M had a mean rate of rise 250 V/sec, and they exceeded the zero membrane potential by an average of 28 mV. This TTX resistant action potential generation was impaired by reducing the external sodium concentration, and abolished in the absence of that cation. A correlation was obsrrved between the presence of TTX resistant action potentials and the acetylcholine sensitivity of the muscle membrane.

Tetrodotoxin resistant action potentials in denervated rat skeletal muscle

It is shown that, following denervation, the action potential generating mechanism becomes resistant to the blocking action of tetrodotoxin. Five days after denervation, action potentials with an average overshoot of 28 mV and an average maximal rate of rise of 252 V/sec were recorded in the presence of tetrodotoxin (10 −6M).