Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions

1. Frog cutaneous pectoris nerve-muscle preparations were incubated with collagenase and protease and examined with electrophysiological and electron microscopic techniques.2. The physiological properties and intracellular ultrastructural appearance of individual muscle and nerve cells were not affected by the enzyme treatment. However, neuromuscular transmission and the morphology of the nerve-muscle junction were altered.3. Collagenase produced an irreversible loss of activity of end-plate cholinesterase and a partial loss of stainable ;synaptic cleft material'.4. Protease produced these changes and, in addition, the entire basement membrane was digested, which led to ;synaptic disjunction' of nerve terminals and muscle end-plates.     

Effect of Blockers of Potential-Dependent and Calcium-Activated K+-Channels on Facilitation of Neuromuscular Transmission

Rhythmic stimulation of nerve-muscle preparation of frog sternal muscle bathed in low-Ca(2+) saline increased the release of neurotransmitter (facilitation) and modified the shape of extracellular response of nerve terminal (decreased phase III amplitude). Iberiotoxin and 4-aminopyridine modified the dynamics these processes. We conclude that inactivation of potential-dependent K(+)-channels and activation of calcium-dependent K(+)-channels in frog motor nerve terminals during rhythmic activity modulate Ca(2+) influx into nerve terminals and contribute into facilitation of neurotransmitter secretion. The degree of these mechanisms depends on the rate of synaptic rhythmic activity.     

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.     

Ephaptic influence of the electrical activity of muscle on the neighboring nerve.

Experiments were performed on isolated nerve-muscle and leg preparations of frog in order to investigate the excitatory influence of the electrical activity of muscle on the contiguous nerve under various conditions. The results show that the active muscles are able to induce multiple transmission of excitation from muscle to nerve and to modify the excitation travelling in the nerve being in contact with the muscle. It is concluded that under some special circumstances the ephaptic influence of the electrical activity of muscle on the nerve in its neighborhood can play an occasional role in the processes related with the nerve and muscle excitation.     

Muscle reinnervation--I. Restoration of transmitter release mechanisms

Following sciatic nerve crush the restoration of neuromuscular transmission in the extensor digitorum longus muscle of rat proceeds in a well defined manner: (a) as soon as the nerve-muscle contact is reformed, a subthreshold end-plate potential is recorded; no 'non-transmitting stage' is observed; (b) 24 hours later muscle action potentials are induced by nerve stimulation; (c) miniature end-plate potentials are absent or very rare at the newly reinnervated end-plates; their frequency returns to normal in about 4 weeks; (d) the frequency is also very much reduced in 30 mM K+ and hypertonic solutions and recovers slowly, in 4 and 5 weeks, respectively, while black widow spider venom is from the beginning as powerful as in normal neuromuscular junctions; (e) at the early stages of reinnervation the Ca2+-dependent release mechanisms are much stronger than control cases, while the Ca2+-independent mechanisms are weaker and recover in 5 weeks. The gradual reassembly and restoration of neurotransmitter release mechanisms of the extensor digitorum longus nerve terminal indicate the complexity of pre-synaptic ending organization.     

脱细胞异体神经移植物桥接大鼠坐骨神经缺损促进神经-肌结构重建和功能恢复的实验研究

目的 探讨脱细胞同种异体神经移植物桥接大鼠坐骨神经缺损对神经-肌结构重建和功能恢复的作用。方法用脱细胞同种异体神经移植物桥接大鼠坐骨神经10mm缺损;称量术后12周、24周实验组手术侧胫骨前肌湿重,并与对照组术侧该肌湿重进行比较;用电生理学方法检测再生神经传导速度及其对胫骨前肌的再支配作用;用AChE和AChE结合镀银染色观察胫骨前肌内神经和运动终板的再生。结果 术后12周、24周实验组胫骨前肌湿重与对照组相比无显差异;再生神经传导速度与对照组相比,也无显差异;术后12周肌内见有AChE阳性反应的运动终板;24周运动终板AChE阳性反应加深,并整齐地排列于胫骨前肌的中上部形成终板带,经结合镀银染色后可见再生的神经束及其发出的分支与运动终板相连;肌电显示再生神经已支配胫骨前肌的运动。结论 脱细胞同种异体神经移植物桥接大鼠坐骨神经缺损具有促进神经一肌结构重建和运动功能恢复的作用。     

Suppression by elevated calcium of black widow spider venom activity at frog neuromuscular junctions

Summary Frog neuromuscular junctions were treated with both concentrated black widow spider venom (BWSV) and elevated extracellular calcium (5–50 mM). This procedure causes a dramatic increase in the frequency of spontaneous miniature endplate potentials (mepps) which persists for only a few minutes. In contrast, BWSV-induced mepp activity, the venom effect (VE), continues for 20 min–1 h at junctions in elevated calcium Ringer solutions treated with doses of dilute venom or at junctions in normal calcium (1.91 mM) Ringer solution treated with concentrated venom. Following the disappearance of the VE in elevated extracellular calcium, only a few normal amplitude mepps and a few giant amplitude mepps are observed. The disappearance of the VE in these preparations is irreversible and occurs whether exposure to elevated extracellular calcium precedes or follows exposure to BWSV.Electron microscopy indicates that the major structural alterations produced by exposure to concentrated BWSV and 20 mM calcium Ringer solution are the swelling of nerve terminal mitochondria and the clumping of synaptic vesicles, large numbers of which remain in the terminals. Exposure to 20 mM calcium Ringer solution alone produces no ultrastructural modifications in these preparations.These observations can best be explained if one of the effects of BWSV is to increase the permeability of the nerve terminal membrane to calcium. Only doses of concentrated venom can sufficiently elevate intracellular calcium to a concentration at which synaptic vesicles clump together, thus interrupting the transmitter release process.     

Transmitter Secretion in the Frog Neuromuscular Synapse after Prolonged Exposure to Calcium-Free Solutions

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5–6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings. At initially low extracellular Ca ion concentrations (0.15–0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.     

Paralyzing and myotoxic effects of a recombinant bothropstoxin-I (BthTX-I) on mouse neuromuscular preparations

As a first step to investigate the structure-function relationship of bothropstoxin-I (BthTX-I), a myotoxin from Bothrops jararacussu snake venom, our group previously cloned a recombinant toxin (rBthTX-I) in Escherichia coli. The aim of this work was to characterize the biological activities of this rBthTX-I (1.0 microM) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-I (1.0 microM) was used as a standard. The influence of heparin (27.5 microg/ml) upon the biological activities of both toxins was also investigated. rBthTX-I had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-I was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-I was expressed in a fully active form, and presents a biological profile similar to the native toxin.     

Ultrastructural studies of normal and degenerating mouse neuromuscular junctions

Summary A quantitative study of the ultrastructural changes occurring at degenerating motor axon terminals of the mouse hemidiaphragm in the first 26 h following unilateral phrenicotomy has been made. Several ultrastructural characteristics of axon terminals from normal diaphragms and phrenicotomized and control hemidiaphragms of phrenicotomized preparations were analyzed. Considerable swelling and disruption of mitochondria occurred in terminals of control hemidiaphragms at 3, 6 and 9 h post-phrenicotomy after which no more damage than that seen in normal terminals after fixation for electron microscopy was observed. At terminals of the phrenicotomized hemidiaphragm, much mitochondrial damage was observed from 3 h post-phrenicotomy onwards. In phrenicotomized hemidiaphragms all terminals had completely degenerated by 26 h post-phrenicotomy. Reduced synaptic vesicle population densities occurred during degeneration. At many axon terminals on phrenicotomized hemidiaphragms the population densities of synaptic vesicles were reduced compared with controls and vesicle aggregates were noted in many engulfed or partially engulfed nerve terminals.These results are discussed with respect to the vesicle hypothesis for nerve-muscle transmission. The mechanisms underlying Schwann cell hyperactivity are also considered.     

Isolation of a polypeptide from the venom of Bungarus multicinctus that binds to ganglia and blocks the ganglionic transmission in mammals

A 15,000 dalton polypeptide purified from Bungarus multicinctus venom (which normally copurifies with alpha-bungarotoxin) was characterized biochemically and its biological effects were studied. This polypeptide, P15, had an aminoacid composition and molecular weight different from those of both alpha- and beta-bungarotoxin. It inhibited the ganglionic transmission in the guinea-pig hypogastric nerve-vas deferens preparation and did not block, even at very high concentrations, the neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. In the same preparations alpha-bungarotoxin was unable to block the response at the ganglionic synapse while it was fully active in blocking the neuromuscular transmission. However, a pretreatment of the vas deferens preparation with alpha-bungarotoxin prevented the inhibitory effect of P15. 125I-Labeled P15 showed a specific and saturable binding to rat superior cervical ganglia homogenate and to a Torpedo postsynaptic membrane fraction. The binding of P15 to ganglia was inhibited by curare. The binding was Ca2+ dependent. The density of binding sites was of 300 fmol/mg of protein in the ganglion and 500 fmol/mg of protein in Torpedo membranes. The amount of P15-binding sites in ganglia was not modified by denervation, indicating that P15 binds to postsynaptic receptors. The binding of 125I-labeled P15, both in ganglia and Torpedo membranes, was inhibited by alpha-bungarotoxin. P15 had a Ca2+-dependent phospholipase A2 activity. Lowering Ca2+ concentration in incubation media affected the phospholipase A2 activity more than binding properties and inhibition of phospholipase activity with p-bromophenacyl bromide did not affect the activity of P15 on vas deferens preparation, suggesting that the phospholipase activity is not necessary for the activity of P15 on nicotinic receptors. Our results suggest that P15 toxin may be a specific and valuable probe for studying the ganglionic nicotinic receptor.     

Empty synaptic vesicles recycle and undergo exocytosis at vesamicol-treated motor nerve terminals.

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.     

Electrophysiological studies of neuromuscular transmission in hereditary `motor end-plate disease' of the mouse

1. Studies have been made on isolated nerve-muscle preparations from mice with hereditary ;motor end-plate disease'.2. Spontaneous fibrillation was observed in the isolated preparation.3. Muscles gave only a weak twitch or failed to contract in response to nerve stimulation. Direct stimulation of muscles caused a twitch response which had a slower time course than normal. Peripheral nerve conduction was normal.4. Intracellular recording from single muscle fibres showed that with longer survival of the animal an increasing proportion of fibres failed to show end-plate potentials or action potentials in response to nerve stimulation.5. Miniature end-plate potentials (m.e.p.p.s) were recorded in almost all muscle fibres including those in which neuromuscular transmission had failed. The frequency of m.e.p.p.s was greater than normal, was not increased by tetanic stimulation of the nerve but was increased by a raised external potassium concentration.6. Muscle fibres were supersensitive to acetylcholine.7. The results suggest that the muscular weakness in this disease is due to the failure of nerve action potentials to invade motor nerve terminals so that muscle fibres become ;functionally denervated'.     

Block of synaptic vesicle exocytosis without block of ca2+-influx. An ultrastructural analysis of the paralysing action of habrobracon venom on locust motor nerve terminals

The venom of the wasp Habrobracon hebetor presynaptically blocks excitatory but not inhibitory neuromuscular transmission at locust skeletal muscle. Its mode of action on excitatory motor nerve terminals has been studied at the retractor unguis muscle of Schistocerca by means of ultrastructural stereology. Paralysed and unparalysed preparations, either resting or stimulated for 7 min at 20 Hz, were compared. Paralysis does not cause structural damage to the nerve terminals but prevents the depletion of vesicles occurring upon nerve stimulation in the controls. Prolonged paralysis leads to an increase in the number and the size of vesicles resulting in an increase of total membrane per terminal cross-section by about 33% after 2 days. Stimulation causes swelling of mitochondria both in controls and in paralysed preparations, resulting from a rise of intraterminal [Ca²⁺] as is indicated by the absence of the swelling if extracellular Ca²⁺ is replaced by Mg²⁺. In addition, stimulation leads to a reduction of vesicle size, an increase in the area of axolemma and in the number of cisternae and of profiles of the smooth endoplasmic reticulum in controls but not in paralysed preparations. However, neither in controls nor in paralysed preparations is the total amount of membrane per teminal cross-section affected by stimulation. Under paralysis, vesicles tend to stick to the presynaptic membrane.It is concluded that Habrobracon venom does not block the depolarization-dependent Ca²⁺-influx into the nerve terminal and that it is unlikely to interfere with some transmitter-related process. Rather, the venom seems to block vesicle exocytosis itself. The results lend further support to the view that in insect neuromuscular synapses exocytosis is the mechanism whereby transmitter quanta are released.     

Structure and distribution of neuromuscular junctions on slow muscle fibers in the frog

Neuromuscular junctions on slow fibers in frog cruralis muscle have been examined with light and electron microscopy. The slow fiber bundle of this muscle is approximately half slow fibers and half fast twitch fibers. Each slow fiber has two-five clusters of nerve terminals in its central region. In contrast, fast fibers in the same bundle have single anastomosing neuromuscular junctions. The average length of terminal clusters on slow fibers is half that of the terminals on fast fibers. Less cholinesterase activity is associated with nerve terminals on slow muscle fibers. The two types of muscle fiber were identified in freeze-fracture replicas by characteristic patterns of the sarcolemmal caveolae and square arrays. Presynaptic membranes of terminals on fast fibers have long, paired double rows of intramembrane particles which lie along the sides of ridges aligned above each fold in the muscle. Ridges are less prominent or absent in terminals on slow fibers and the associated membrane particles are more often in single rows. The lengths of the particle rows also tend to be shorter and rows tend to branch and to lie at various orientations with respect to the longitudinal axis of the terminal. The average length of the particle rows per unit length of nerve terminal on slow fiber terminals is half that of particle rows on fast fiber terminals. It is concluded that the total length as well as the concentration of these active zone specializations is less at terminals on slow fibers. The lack of parallel orientation of the presynaptic active zones at slow fiber terminals corresponds to a lack of postsynaptic folds. Postsynaptic specializations seen in thin sections through slow fibers are either on flat expanses of sarcolemma or on low sarcolemmal bulges. Patches of large intramembrane particles, similar to those on fast fibers, mark regions of postsynaptic specialization in freeze-fracture replicas of slow fibers.     

Quantal secretion and loss of vesicles induced by veratridine at the crayfish neuromuscular junction

Experiments were conducted in nerve-muscle preparations of small young crayfish (Austropotamobius torrentium, Astacus astacus). Application of veratridine in the superfusate induced strong quantal release of transmitter. After about 5 min when quantal release had declined to a low level preparations were fixed for electron microscopy. Unlike control preparations, veratridine-treated preparations revealed nerve terminals which were largely depleted of their synaptic vesicles. Our findings suggest that in the presence of veratridine the decline of quantal secretion results from the loss of vesicles caused by tonic nerve terminal depolarization. Moreover, our results indicate that during or after excessive quantal release triggered by veratridine synaptic vesicles may fuse with both the presynaptic membrane and each other.     

Involvement of axonal phospholipase A2 activity in the outgrowth of adult mouse sensory axons in vitro.

The effect on axonal outgrowth of inhibition of phospholipase A2 activity was studied in a recently developed in vitro model, where dorsal root ganglia with attached spinal roots and nerve stumps from young adult mice were cultured in an extracellular matrix material (Matrigel). The phospholipase A2 inhibitors 4-bromophenacyl bromide and oleyloxyethyl phosphorylcholine dose-dependently reduced axonal outgrowth from the sciatic nerve stump. A similar inhibitory effect was seen when only the cut nerve end was exposed to the inhibitors in a compartmental culture system. The local effect of phospholipase A2 inhibition was further investigated on axons established in culture, using time-lapse recording. Exposure to phospholipase A2 inhibitors caused the retraction of filopodia extensions and a reduction in growth cone motility within a few minutes. After removal of inhibition, normal growth cone motility and axonal growth were regained. Nerve cell bodies and axons, in contrast to Schwann cells, showed immunoreactivity after staining with an antiserum against secretory phospholipase A2, and elevated levels of the enzyme could be detected after culture for 24 h. The immunoreactive protein was of approximately 170,000 molecular weight (phospholipase A2-170) as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. The localization of phospholipase A2-170 in axons growing into the Matrigel was also demonstrated by use of a whole-mount technique. The results of this study show the importance of continuous phospholipase A2 activity for growth cone motility and axonal outgrowth in the mammalian peripheral nerve, and suggest the involvement of an axonally localized enzyme.     

The effects of chronic stimulation on the morphology of the frog neuromuscular junction

Summary A quantitative study was made of the effects of 24 h continuous stimulation on the morphology of the frog neuromuscular junction. The synaptic vesicle concentration in the nerve endings of frog sartorius muscles stimulatedin vitro for 24 h at 2 Hz was the same as that in controls stimulated for only 0.3 h at 2 Hz. The control preparations were either freshly dissected or maintained at restin vitro for 23 h prior to stimulation. Chronically stimulated terminals differed from their controls only in having more cisternae and fewer dense cored vesicles. Varying the lengths of the nerves to both chronically stimulated andin vitro control muscles had little effect on the morphology of the nerve endings.Continuous recording of muscle twitch tension demonstrated that neurotransmission was effective throughout the 24 h period of stimulation. Additional evidence that nerve failure or degeneration was not a factor in the results came from a second set of control and chronically stimulated preparations that were tetanized at 30 Hz for 0.3 h before fixation. Changes attributable to rapid stimulation were evident in 87 to 100% of their nerve terminals.Although the distribution of membrane among various membrane organelles differed from one treatment group to another, the total amount of measurable membrane in the nerve terminals was the same in all of the treatment groups; that is, the total amount of membrane was not altered by maintenancein vitro, chronic stimulation at 2 Hz, rapid stimulation at 30 Hz, reduced nerve length, or any tested combination of these treatments. This conservation of total membrane suggests that membrane exchange between axon and nerve terminal occurs at a relatively slow rate which is unaffected by synaptic activity, and that the local mechanism for recycling synaptic vesicle membrane in frog neuromuscular junctions is more autonomous and durable than has been suspected.     

Discrete and discontinuous action of brown widow spider venom on the presynaptic nerve terminals of frog muscle.

1. A study was made of the effects of the venom of the brown widow spider (Latrodectus geometricus) on end-plates of the frog sartorius muscle. 2. The increase in the frequency of the minature end-plate potentials (m.e.p.p.s), elicited by the venom in normal-"Ca2+" Ringer solution, occurs in discrete volleys having a sharp onset and end. The frequency of the m.e.p.p.s is high (up to 300 sec-1) and relatively constant during the volley. 3. The volleys recur at intervals during a period from 5 to 10 min after addition of the venom until the onset of electrical silence, up to 4 hr later. The activity occurs in groups containing volleys of loing and short duration. 4. Simultaneous intracellular and extracellular recording from single end-plates indicates that the volleys originate at highly localized areas of the nerve terminals. The high-frequency release of m.e.p.p.s in hypertonic sol solutions, which was studied for comparison purposes, occurs randomly over the entire end-plate. Volleys originating simultaneously at different sites are often superimposed in the intracellular recordings. 5. In high-"Ca2+" Ringer solution, the initial frequency of the m.e.p.p.s in a volley is comparatively higher. However, the frequency drops to one half its value in a few seconds. The volley then terminates or else the frequency of m.e.p.p.s remains high for some time and the volley has no sharp end. Activity occurs in groups containing both long and short volleys. Many more short (less than 5 sec) and long (greater than 30 sec) volleys occur in high-"Ca2+" solution than in normal-"Ca2+" solutions. 6. In low-"Ca2+", high-"Mg2+" Ringer solution, the volleys of m.e.p.p.s are fewer in number and much longer in duration. Intra- and extra-cellular recording of uninterruped activity during long periods suggests that in this solution the m.e.p.p.s originate diffusely rather than at discrete areas of the nerve terminals. 7. Implications of the above data on possible modes of action of the venom are discussed.     

The spontaneous release of transmitter from insect nerve terminals as predicted by the negative binomial theorem

1. In the retractor unguis muscles of Periplaneta americana and Blaberus giganteus, the rate of spontaneously occurring miniature potentials from nerve-muscle synapses associated with the `white' muscle fibres was greater than that observed in the `red' muscle fibres.

2. In 5% of the intracellular recordings, particularly from the `white' fibres, short `bursts' of relatively high (20-100 sec^-1) rates of m.e.p.p.s occurred infrequently in an unpredictable manner. This bursting phenomenon was not observed in extracellular recordings of the miniature potential discharge.

3. Rigorous statistical analyses indicated that the spontaneous mode of transmitter release at cockroach retractor unguis muscle fibres was predictable by the negative binomial theorem with a 90% reliability.

4. Perfusion of nerve-muscle preparations with high Mg²⁺ salines resulted in low spontaneous discharge rates which were characterized by a negative binomial distribution.

5. The fit of the data to the negative binomial theorem implied some mutual interaction between those processes responsible for the spontaneous release of transmitter from cockroach nerve terminals.