Mammalian Motor Nerve Terminals Research Articles

Quantal release of acetylcholine in mice with reduced levels of the vesicular acetylcholine transporter

Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

Ultrastructural evidence for the uptake of a neurotoxic snake venom phospholipase A 2 into mammalian motor nerve terminals

A mutant form of ammodytoxin A, a neurotoxic phospholipase A 2 from the venom of the long nosed viper Vipera ammodytes ammodytes, was prepared by site-directed mutagenesis, conjugated to a nanogold particle and inoculated into the antero-lateral aspect of one hind limb of female mice. Eight hours later the mice were killed, the soleus muscles of both ipsi- and contra-lateral hind limbs were removed, exposed to a silver enhancing medium and then prepared for transmission electron microscopy. Silver-enhanced particles were subsequently found concentrated in the peri-synaptic area, particularly within the synaptic gutter and the deep synaptic folds, and in many cases had been taken up into the cytoplasm of the terminal boutons of the motor axon. The results suggest that the presynaptic neurotoxicity of snake venom phospholipases A 2 involves several components of the neuromuscular apparatus, including intracellular organelles of the motor nerve terminal.

Effect of purines on calcium-independent acetylcholine release at the mouse neuromuscular junction

At the mouse neuromuscular junction, activation of adenosine A1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca2+ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca2+-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A1 receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) and the P2Y12-13 agonist 2-methylthio-adenosine 5′-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A1 and P2Y12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5′-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A1 and P2Y12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca2+-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related.

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals.

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-bungarotoxin-induced sprouting.

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48-169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedure permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxin-induced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization.

P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals.

The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (IK(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of IK(Ca) were also examined. The calcium channel blockers of the P/Q family, omega-agatoxin IVA (omega-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on IK(Ca). In contrast, nitrendipine and omega-conotoxin GVIA (omega-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the IK(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates IK(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and slow Ca2+ chelators.

Decreased calcium currents in motor nerve terminals of mice with Lambert-Eaton myasthenic syndrome.

1. The effects of immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome on Ca2+ currents in mammalian motor nerve terminals are unknown. Therefore, we recorded these currents in phrenic nerves of mice injected with serum from either LEMS patients, myasthenia gravis patients, or healthy control individuals. 2. In control preparations, the endplate currents induced by repetitive stimulation at > or = 20 Hz were depressed as expected. However, in the LEMS animals quantal content decreased and either depression did not occur or synaptic facilitation occurred. 3. Ca2+ currents were smaller in LEMS animals. At 0.5 Hz stimulation frequency, normalized Ca2+ currents in LEMS animals were 57 +/- 14% of those in control. At higher frequencies, Ca2+ currents become smaller in control but not in LEMS animals. 4. Ca2+ currents in controls were unaffected by addition of nifedipine but were reduced by 37% upon addition of omega-conotoxin GVIA. In LEMS animals, however, the currents were depressed by 43% by nifedipine but were unaffected by omega-conotoxin GVIA. Thus, LEMS is associated with reduced Ca2+ currents and a shift to dihydropyridine sensitivity.

Effect of Pinellia ternata Lectin on Membrane Currents of Mouse Motor Nerve Terminals

Pinellia ternata lectin (PTL) extracted from the fresh juice of rhizome of pinellia ternata used as a traditional Chinese medicine facilitated the quantal release of acetylcholine (ACh) in the mouse motor nerve terminals and formed cation channels in artificial lipid bilayer. Here we report the action of PTL on presynaptic membrane currents of motor nerve terminals.The experiments were performed on the intercostal nerve triangularis sterni muscle preparations. By means of the perineurial recording, the effects of PTL on the sodium current in the preterminal part , three potassium currents and two calcium currents generated from the nerve terminals were investigated. The results show that PTL increases voltage-dependent fast Ca2+ current (ICa,f), Na+ current (INa) and Ca2+-acti-vated K+ current (IK,Ca) without action on either the voltage-dependent fast K+ current (IK,f) or the slow K+ current (IK,S). These effects are irreversible, but can be reversed by mannan, the specific binding sugar for PTL.The total action of PTL can be considered to raise [Ca2+], by promoting Ca2+ influx, and then facilitate the neurotransmitter release. To our knowledge, this is the first report about the influence of lectin on presynaptic membrane currents at mammalian motor nerve terminals.

Sprouting of mammalian motor nerve terminals induced by in vivo injection of botulinum type-D toxin and the functional recovery of paralysed neuromuscular junctions

Paralysis of the mouse levator auris longus muscle by in vivo injection of Clostridium botulinum type-D neurotoxin (BoNT/D) triggered a marked outgrowth of the motor nerve from the original terminal arborization. The increase in total nerve terminal length was due to both increase in the number of terminal branches and in average branch length. Asynchronous quantal transmitter release in response to nerve impulses was a prominent feature in paralysed junctions that started 24 h after poisoning and lasted for about 15 days. The functional recovery of poisoned junctions occurred 25–30 days after poisoning and was characterized by the synchronous quantal transmitter release upon nerve stimulation that triggered synaptically evoked action potentials and muscle fibre contraction.

Species specific morphology of mammalian motor nerve terminals

Motor nerve terminals in the diaphragm, extensor digitorum longus and soleus muscles of young and adult rat, hamster and guinea pig were studied with the light microscope after staining with methylene blue. In adult animals the nerve terminals are smaller in the diaphragm than in the two other muscles. In the rat and hamster the extensor digitorum longus and soleus terminals are of similar area, but the terminals in extensor digitorum longus are shorter. In the guinea pig the terminals are smaller and shorter in soleus than in extensor digitorum longus. The density of nerve terminal varicosities is lowest in the diaphragm in all three species. In the rat and hamster the density is higher in extensor digitorum longus than in soleus. In the guinea pig the converse is found. In all three muscles the density of varicosities is higher in the rat than in the hamster and guinea pig. The nerve terminal branches in the diaphragm are mostly organized in one group. In the rat and hamster the soleus terminal branches are more separated in groups than the extensor digitorum longus terminal branches. In the guinea pig the number of groups is almost the same in the two muscles. These muscle and species-specific differences appear already in very young animals.

Light chain of botulinum neurotoxin is active in mammalian motor nerve terminals when delivered via liposomes

Liposomal encapsulation of the individual light and heavy chain of botulinum neurotoxin A was used to investigate their intra-cellular effects on synaptic transmission at the murine neuromuscular junction. Bath-application to phrenic nerve-hemidiaphragms of liposomes containing heavy chain (up to 75 nM) caused no alteration in neurally-evoked muscle tension. In contrast, liposomes with entrapped light chain (9–20 nM final concentration) gave a pre-synaptic blockade of neuromuscular transmission that could be relieved temporarily by 4-aminopyridine, as for the dichain toxin. Any contribution from contaminating intact toxin was excluded both by the purity and minimal toxicity in mice of the light chain preparations used, and by the lack of neuromuscular paralysis seen with liposomes containing the maximum amount of native toxin that could have been present in the light chain liposomes. As bath-application of high concentrations of light chain in the absence of liposomes failed to affect neurotransmiter release, it is concluded that this chain alone can mimic the action of the whole toxin inside mammalian motor nerve endings, its predominant site of action. Thus, light chain could provide a more effective probe for an intra-cellular component concerned with Ca 2+-dependent secretion.

Multiple potassium conductances at the mammalian motor nerve terminal.

The possibility that multiple K+ conductances are present in mammalian motor nerve terminals was investigated by measuring the differential effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on transmitter release and on nerve terminal excitability in mouse phrenic nerve-diaphram preparations. Neither 4-AP nor TEA alter the spontaneous frequency of miniature end-plate potentials (fmepp) and therefore evidently do not affect calcium channels directly. Both 4-AP and TEA increased the quantal content of end-plate potentials (epps) evoked by nerve stimulation but the effects were not mutually exclusive; TEA continued to act in the presence of a maximally effective concentration of 4-AP. The increase in transmitter release evoked by focal polarisation of the terminal was not affected by 4-AP, whereas TEA exerted an effect consistent with a reduction in membrane conductance. Similarly, threshold for nerve terminal action potential generation was not affected by 4-AP, but TEA caused a reduction in threshold and alteration of the 'accommodation curve' indicative of a reduction of membrane conductance. Under conditions where one would predict no contribution of calcium K+ conductance, i.e., when release was evoked by Ba2+ in the absence of Ca2+, the different and non-competing effects of TEA and 4-AP were still apparent. It is concluded, therefore, that the motor nerve terminal possesses at least two K+ conductances, not including calcium activated gK, which may be distinguished pharmacologically.

Possible functional role of substance P on the mammalian motor nerve terminals

The effect of substance P (sP) on mammalian skeletal myoneural transmission was studied employing innervated and denervated isolated rat diaphragm preparations. sP at a concentration of 3.7 nM facilitated the indirect twitch responses of the rat diaphragm and antagonised the paralytic effect of d-tubocurarine (d-Tc). sP failed to affect the direct twitch responses as well as the contractures induced by acetylcholine (ACh) and potassium chloride (KCl) in the denervated diaphragm. The amount of ACh released into the bathing medium in response to tetanic stimulation of the phrenic nerve was doubled in presence of sP. The study illustrates a presynaptic facilitatory involvement of sP on mammalian myoneural transmission.

Enhancement by Anemonia sulcata toxin II of spontaneous quantal transmitter release from mammalian motor nerve terminals

J. Molgo, M. Lemeignan and F. Tazieff-Depierre. Enhancement by Anemonia sulcata toxin II of spontaneous quantal transmitter release from mammalian motor nerve terminals. Toxicon 24, 441 – 450, 1986. — The action of Anemonia sulcata toxin II (ATX-II) on spontaneous quantal transmitter release from motor nerve terminals was investigated by recording miniature end-plate potentials (MEPPs) from isolated mouse phrenic nerve - hemidiaphragm nerve - muscle preparations. ATX-II (3.2 μM) when applied for 30 – 40 min to junctions bathed in a normal ionic medium enhanced about one hundred fold the rate of spontaneous MEPPs. Concomitantly, ATX-II depolarized the muscle fiber. The effect of the toxin on MEPP frequency was markedly reduced when junctions were exposed to Na-deficient solutions or pre-treated with dantrolene sodium (10 μM). ATX-II (0.24 – 3.2 μM) increased MEPP rate in junctions exposed to a Ca-free medium containing 2 mM EGTA and 2 mM Mg 2+ in a dose- and time-dependent manner. Tetrodotoxin (0.2 – 1 μM) prevented the effects of ATX-II on MEPP frequency and on the resting membrane potential of muscle fibers. Tetrodotoxin also antagonized the acceleration of MEPP induced by ATX-II. The experimental findings suggest that ATX-II acts to increase quantal transmitter output from motor nerve terminals by enhancing Na + influx through tetrodotoxin-sensitive presynaptic channels, since ATX-II action does not appear to depend upon entry of Ca 2+ from the extracellular medium. It is likely that ATX-II, by increasing intraterminal Na + concentration, may trigger calcium release from internal stores.

Studies on a False Transmitter at the Neuromuscular Junction

Monoethylcholine (MECh) has been shown to be taken up, acetylated, and released as a false transmitter, acetylmonoethylcholine (AMECh) by mammalian motor nerve terminals. AMECh can be distinguished electrophysiologically from ACh by its briefer postsynaptic action, which results in a shortening of the decay time constant of miniature end-plate currents recorded with a voltage clamp. This shortening corresponds with the mean channel lifetime for AMECh, which is 44% shorter than that of ACh. This chapter investigates, whether at intermediate stages of exchange, two populations of quanta (corresponding to preexisting ACh quanta and newly synthesized AMECh quanta) can be detected. No evidence was found for two distinct populations, but a clear increase in variability of quanta, indicative of some heterogeneity of transmitter composition. A possible interpretation of these results is that the major part of the presynaptic transmitter store is in the form of a free pool and only a small part is in the immediately releasable vesicular store.

Factors affecting the rate of incorporation of a false transmitter into mammalian motor nerve terminals.

1. The incorporation of acetylmonoethylcholine (AMECh) into transmitter store at the mammalian neuromuscular junction has been studied using electrophysiological techniques. 2. Incubation of rat muscle in the presence of 0.1 mM-monoethylcholine (MECh) and 40 mM-K+ for 60-90 min produced a maximal reduction in the time constant of decay of synaptic currents and potentials, indicating that acetylcholine (ACh) had been replaced by AMECh in the released quanta. 3. Under resting conditions, muscles incubated for up to 24 hr in the presence of MECh showed no incorporation of AMECh into the released transmitter. In contrast, muscles pre-loaded with AMECh and then incubated in choline-containing medium showed a substantial reversion to ACh in the released transmitter within 4 hr. 4. It is suggested that this difference results from the rate of synthesis or packaging of transmitter being considerably slower with MECh than with choline, so that stimulation in the presence of MECh causes an over-all depletion of transmitter stores that does not occur with choline as the precursor. Measurements of m.e.p.c. amplitude following K+-evoked release in the presence of MECh or choline confirmed this interpretation. 5. In order to test whether newly formed AMECh is incorporated into a single homogeneous pool of transmitter from which the released quanta are derived, the rate of incorporation of AMECh into the released transmitter was measured as a function of the number of quanta released when transmitter output was increased by various methods. 6. The number of quanta released before 63% conversion of the released transmitter to AMECh was brought about varied from 1.3 X 10(5) (nerve stimulation at 3 Hz) to about 4 X 10(5) (release by high-K+ solution). With nerve stimulation in Mg2+-blocked muscles the value was 2.5 X 10(5). 7. Incorporation of AMECh into the quanta released by nerve stimulation appeared to take place more rapidly than its incorporation into spontaneously released quanta. 8. These results are discussed in terms of the compartmentation of transmitter stores in the nerve terminals.

Glucocorticoid effects on the edrophonium responsiveness of normal and degenerating mammalian motor nerve terminals

An intensive short-term triamcinolone regimen in cats preserves the prejunctional actions of edrophonium in degenerating motor nerves. These edrophonium actions include the induction of a stimulus-dependent afterdischarge and the initiation of fasciculations. The relationship between fasciculations and stimulus-dependent afterdischarge is discussed. The glucocorticoid preservation of these edrophonium effects is like that previously reported for the preservation of posttetanic facilitation in motor nerves equally compromised. The results therefore show that glucocorticoid and facilitatory drug actions synergize to increase facilitation in degenerating but still functional motor nerves. This drug synergy is comparable to that which occurs in normal motor nerves. This interaction may provide a basis for effectively combining glucocorticoid and facilitatory drugs in the treatment of myasthenia gravis.

The effect of curare on the release of acetylcholine from mammalian motor nerve terminals and an estimate of quantum content.

Curarized and non-curarized rat hemidiaphragm muscles were indirectly stimulated in vitro. 2. The fluid bathing the active curarized muscles was eluted through a dextran gel (Sephadex G-10), effecting a complete separation of ACh from curare. The acetylcholine fraction was then assayed on an isometric leech muscle preparation. 3. Prostaglandin (PGE1) in a concentration fifteen times that estimated to be released from the skeletal muscle preparation did not affect the response of leech muscle to ACh. 4. The amount of ACh released by curarized muscles (4-9 X 10(-18) mole/impulse-junction) was not significantly different from that released by non-curarized muscles (4-6 X 10(-18) mole/impulse-junction). These quantities are similar to those obtained by previous workers. It is concluded that curare in a paralytic dose does not affect the output of ACh from motor nerve terminals stimulated at low frequencies. 5. Spontaneous release of ACh from non-curarized muscles was estimated at 0-45-0-65 p-mole/min. hemidiaphragm. It is calculated that only 2% of this amount could give rise to post-synaptic electrical events, the remainder having a non-synaptic source. 6. The number of molecules of 'quantal' ACh released by stimulated muscle is calculated as 2-5 X 10(6)/impulse-junction, taking account of the non-synaptic release. The number of ACh molecules in one quantum was estimated to be 6250, an amount that could be easily accommodated in one synaptic vesicle.

On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals.

1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mM) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 muM or 1 mM) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine (ACh). 3. Stimulation of isolate phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0-1 mM or 'calcium-free'), stimulation failed to release adenine nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 times 10-minus 5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.

β-Bungarotoxin inhibition of calcium accumulation by rat brain mitochondria

Electrophysiological studies have shown that β-bungarotoxin modifies the release of neurotransmitter from mammal ian motor-nerve terminals. In this paper we demonstrate that β-bungarotoxin can also inhibit calcium accumulation into sub-cellular fractions from rat brain at very low concentrations (2–8 pmoles toxin/mg protein). Since the calcium uptake which is inhibited has the characteristics of mitochondrial calcium accumulation (DNP-sensitivity, succinate stimulation), we conclude that the toxin affects the mitochondria. We suggest that the electrophysiological observations are consistent with direct or indirect inhibition by toxin of mitochondrial calcium uptake.