Phosphatases modulate transmission and serotonin facilitation at synapses: Studies with the inhibitor okadaic acid

We examined the role of phosphatases in synaptic transmission using the permeant phosphatase inhibitor okadaic acid (OA). In the crayfish neuromuscular junction (NMJ), postsynaptic effects including increases in input resistance occurred at doses greater than 5 μM OA. At lower doses (0.5–5 μM) the effects were solely presynaptic and transmitter release increased over three-fold despite small reductions in amplitude and duration of presynaptic action potentials. Potentiating effects of serotonin on transmitter release, Which depend on phosphorylation, were increased by OA. Frequency facilitation was reduced but its decay was not affected. In frog NMJs, OA increased spontaneous and evoked release two-fold through presynaptic mechanisms. An inactive analog of OA, OA tetra-acetate, had no effect on transmitter release at frog and crayfish NMJ. Therefore, phosphatases have a strong modulating influence on synaptic transmission.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

神经营养因子对神经肌肉接头传递的调制作用

运动单位由运动神经元及其支配的肌纤维组成。神经肌肉接头(neuromuscular junction,NMJ)传递受到严密的调节,因而能和运动单位的活动协调一致。在NMJ,神经调制物质的释放与运动单位的活动有关,并能决定突触传递的效能。脑源性神经营养因子(brain—derived neurotrophic factor,BDNF)和神经营养因子4(neurotrophin-4,NT-4)由运动神经末梢和肌纤维产生。肌肉释放营养因子受肌肉活动调节。在NMJ,BDNF和NT-4通过激活酪氨酸激酶B受体(tyrosine kinase receptor B,TrkB),能加强自发性和诱导性的突触活动。突触前Ca^2 量的迅速增加或突触胞吐过程的易化,都能增加突触囊泡的释放,从而改善NMJ的突触传递。事实上,BDNF能促进突触前细胞内Ca^2 的释放,TrkB的激活也能通过有丝分裂活化蛋白激酶,引起突触素I(synapsinI)的磷酸化,进而增加可释放的突触囊泡的数量。在NMJ,神经营养因子还能通过影响神经调节素(neuregulin)或其他神经源性调制物质的局部释放,对接头传递进行调节。本文对近年来在NMJ突触传递的调节,运动单位的NMJ特性以及神经营养因子对突触传递效能的影响等方面的研究进展做一综述。     

Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction

4-Aminopyridine markedly potentiates transmitter release at the frog pectoris neuromuscular junction by increasing the quantal content even when applied at low concentrations (5-20 microM). This enhancement of transmitter release is associated with greater minimum synaptic latency, but the dispersion of the synaptic latencies does not appear much affected. This is in contrast with the action of tetraethylammonium (0.2-0.5 mM) in which case similar enhancement of transmitter release results not only in larger minimum synaptic latency but also in greater dispersion of the synaptic latencies. The time course of transmitter release associated with enhanced transmitter output is hence much more prolonged in the presence of tetraethylammonium than 4-aminopyridine, at least for low concentrations of 4-aminopyridine (5-20 microM). This indicates that their presynaptic actions differ significantly. This conclusion is further strengthened by the finding that unlike tetraethylammonium, 4-aminopyridine induces bursts of release, presumably by producing multiple action potentials in the nerve terminal. Tetraethylammonium probably acts by blocking the delayed potassium conductance, but the blockade of Ca2+-activated K+ conductance cannot be excluded. 4-Aminopyridine, however, probably blocks the fast inactivating (IA) K+ current, but it also may be acting directly on the voltage-dependent Ca2+ conductance or on the intracellular Ca2+ buffering.     

Octopamine action on the contractile system of crustacean skeletal muscle

1. In the opener muscle of walking legs of crayfish (Astacus leptodactylus) octopamine (OA) greatly enhances the contractions resulting from brief applications of L-glutamate or of elevated K-concentrations. Synephrine is as effective as OA. 2. In the case of potentiation of responses to high-K applications a presynaptic component of the OA action was excluded by first desensitising the muscle fibres to the action of the natural transmitter, using a high concentration (1 mM) of glutamate. 3. The Ca-antagonists Co, Ni and Mn (1 mM) reduced the effects of glutamate and of elevated K to about one-half. In preparations treated with OA, the same Ca-antagonists also depressed the potentiated contractural responses to glutamate and to elevated K, again to about one-half. 4. OA also enhanced contractions resulting from the application of caffeine. 5. With 5-hydroxytryptamine (5-HT) application, the same postsynaptic effects were obtained as described for OA, except that the 5-HT actions were much weaker. 6. With OA, maximal effects were obtained with concentrations of 5 x 10(-6)-10(-5) M; maximally effective concentrations of 5-HT were around 10(-5) M. 7. The lowest effective concentrations of OA were around 10(-8) M; those of 5-HT were around 10(-7) M. 8. In the same preparation, 5-HT is far more effective in enhancing transmitter release (presynaptic action) than OA, the lowest effective concentration being around 10(-11) M while no presynaptic effects of OA were seen at concentrations below 10(-8) M, in some cases even below 10(-5) M.     

Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses

The localization of Ca2+ channels relative to the position of transmitter release sites was investigated at the frog neuromuscular junction (NMJ). Ca2+ channels were labeled with fluorescently tagged omega-conotoxin GVIA, an irreversible Ca2+ channel ligand, and observed with a confocal laser scanning microscope. The Ca2+ channel labeling almost perfectly matched that of acetylcholine receptors which were labeled with fluorescent alpha-bung-arotoxin. This indicates that groups of Ca2+ channels are localized exclusively at the active zones of the frog NMJ. Cross sections of NMJs showed that Ca2+ channels are clustered on the presynaptic membrane adjacent to the postsynaptic membrane.     

Effects of ethimizol on frog neuromuscular junction

The effects were studied of ethimizol, a substance activating memory processes, on features of synaptic transmission during experiments on frog cutaneous pectoris muscle. It was found that the presynaptic action of ethimizol consists of raising the frequency of miniature potentials, when used at a concentration of 0.5–10 mM, and modulating quantal content of synaptic transmission due to changes in binomial quantal release parameters p and n when 0.5–2 mM ethimizol was used. This substance facilitated transmission at synapses with a low initial level of transmitter release. This substance facilitated transmission at synapses with a low initial level of transmitter release. Ethimizol was also found to have a postsynaptic action, consisting of reducing amplitude at a concentration of 5–10 mM and prolonging synaptic currents and potentials when concentrations of 0.5–10 mM were used. The latter effect produced a considerable increase in the time integral of endplate potentials. The postsynaptic action of ethimizol is perhaps seen in its effects on features of postsynaptic ionic channels. The effects of ethimizol are discussed with a view to how it may act within the central nervous system as a nonspecific modulator.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 757–763, November–December, 1985.     

Presynaptic uptake blockade hypothesis for LSD action at the lateral inhibitory synapse in Limulus

We investigated the action of LSD at the putative indoleaminergic lateral inhibitory synapse in the lateral eye of Limulus polyphemus. We recorded extracellular and intracellular voltage responses from eccentric cells while producing inhibition either by light or by antidromic stimulation of the optic nerve in the presence of LSD, serotonin (5-HT), chlorimipramine, or a bathing medium whose high Mg++ and low Ca++ concentrations partially or completely blocked synaptic transmission. We found (a) light-evoked and antidromically stimulated lateral inhibition is enhanced during superfusion of low (1-5 microM) concentrations of LSD and suppressed by higher (5-20 microM) concentrations; (b) these actions of LSD are markedly reduced by bathing the retina in a medium high in Mg++ and low in Ca++; (c) very low concentrations of chlorimipramine, a putative uptake blocker of serotonin, appear to mimic actions of LSD both on eccentric cell firing rate and on lateral inhibition; (d) superfused 5-HT depresses lateral inhibition at all superthreshold concentrations (0.1-25 microM). These results suggest that LSD's action may require an intact inhibitory transmitter release and postsynaptic response mechanism, whereas serotonin exerts a direct postsynaptic effect. We propose that LSD blocks presynaptic uptake of transmitter at the lateral inhibitory synapse. The concentration dependence of LSD's action can be accounted for as follows: low concentrations partially restrict transmitter reuptake, thereby prolonging the lifetime of the transmitter in the synaptic cleft and thus increasing the magnitude and duration of postsynaptic inhibition. Higher concentrations cause more presynaptic uptake sites to be blocked; this causes accumulation of transmitter in the synaptic cleft, which causes a functional blockade of the synapse because of postsynaptic desensitization. As an alternative, we propose a hypothesis based on LSD action at presynaptic autoreceptors. Similar hypotheses can account for many aspects of LSD's action in mammalian brain.     

A metabotropic glutamate receptor regulates transmitter release from cone presynaptic terminals in carp retinal slices.

The role of group III metabotropic glutamate receptors (mGluRs) in photoreceptor-H1 horizontal cell (HC) synaptic transmission was investigated by analyzing the rate of occurrence and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in H1 HCs uncoupled by dopamine in carp retinal slices. Red light steps or the application of 100 microM cobalt reduced the sEPSC rate without affecting their peak amplitude, which is consistent with hyperpolarization or the suppression of Ca(2+) entry into cone synaptic terminals reducing vesicular transmitter release. Conversely, postsynaptic blockade of H1 HC AMPA receptors by 500 nM CNQX reduced the amplitude of sEPSCs without affecting their rate. This analysis of sEPSCs represents a novel methodology for distinguishing between presynaptic and postsynaptic sites of action. The selective agonist for group III mGluRs, l-2-amino-4-phosphonobutyrate (L-APB or L-AP4; 20 microM), reduced the sEPSC rate with a slight reduction in amplitude, which is consistent with a presynaptic action on cone synaptic terminals to reduce transmitter release. During L-APB application, recovery of sEPSC rate occurred with 500 microM (s)-2-methyl-2-amino-4-phosphonobutyrate (MAP4), a selective antagonist of group III mGluR, and with 200 microM 4-aminopyridine (4-AP), a blocker of voltage-dependent potassium channels. Whole-cell recordings from cones in the retinal slice showed no effect of L-APB on voltage-activated Ca(2+) conductance. These results suggest that the activation of group III mGluRs suppresses transmitter release from cone presynaptic terminals via a 4-AP-sensitive pathway. Negative feedback, operating via mGluR autoreceptors, may limit excessive glutamate release from cone synaptic terminals.     

Cyclic AMP, 5-HT, and the modulation of transmitter release at the crayfish neuromuscular junction

In this study it was found that several agents which elevate cAMP levels in cells also increase dramatically the quantity of transmitter released from crayfish excitatory nerve terminals in response to a stimulus. With respect to time course and magnitude, the increase produced by one of these agents, the cyclic nucleotide phosphodiesterase inhibitor Squibb 20,009 (SQ 20,009), is unlike any reported for such a drug at a synapse. Additionally, SQ 20,009 potentiated the facilitation of transmitter release produced by serotonin (5-HT) at this synapse. These results establish a possible role for cAMP in the control and modulation of transmitter release at the crayfish neuromuscular junction (NMJ). They further suggest that 5-HT functions here by activation of a presynaptically located adenylate cyclase.     

Effects of pyrocatechol on neuromuscular transmission

Effects of pyrocatechol on neuromuscular transmission were studied both in the frog pectoral-cutaneous muscle and in the mouse phrenic-diaphragmatic preparation by means of extracellular microelectrode recording of synaptic signals. Pyrocatechol applied in a concentration of 0.05 mM increased the frequency of miniature end-plate currents (MEPC) and the amplitude of end-plate current (EPC) by increasing its quantum content. Pyrocatechol also increased the duration of presynaptic response. When voltage-dependent potassium channels had been blocked, pyrocatechol affected neither the EPC quantum content nor the duration of presynaptic response. It is suggested that the pyrocatechol-induced enhancement of transmitter release results from modulatory effects of pyrocatechol on voltage-dependent potassium current in the membrane of a nerve terminal.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 405–408, November–December, 1993.     

The action of the venom of the spider Eresus niger on the neuromuscular synapses of the frog

The effect of Eresus niger spider venom on frog neuromuscular preparations has been studied using the intracellular microelectrodes. The spider venom has been found to block both spontaneous and elicited transmitter release and possesses the phospholipase activity. It has been suggested that the venom blocking synaptic transmission results from its phospholipase activity by analogy with the action of snake presynaptic neurotoxins.     

Investigating the safety factor at an invertebrate neuromuscular junction

Fidelity of synaptic transmission is essential at the neuromuscular junction (NMJ). To ensure that transmission does not fail, vertebrate motoneurons often release more neurotransmitter than is required for muscle contraction. This safety factor allows some loss of synaptic function without failure of muscle contraction. It is not known whether a similar mechanism operates at the invertebrate neuromuscular junction. In our study of the Drosophila NMJ, we find that glutamate receptor mutants can exhibit a substantial decrease in synaptic function while maintaining muscle contraction. The persistence of neuromuscular function in these mutants is not explained by synaptic facilitation, temporal summation of high frequency stimuli, or a hyperpolarizing shift in the activation range of muscle calcium channels. Instead, the attenuated synaptic response is sufficient to drive muscle contraction. Quantitative analysis of the decrease in synaptic transmission in these mutants implies that at the wild-type NMJ there is an approximately five- to ninefold excess in released transmitter. Hence, the presence of a synaptic safety factor is a conserved feature of neuromuscular organization in both invertebrates and vertebrates.     

Effect of carbacholine on proximal and distal regions of motor nerve terminals in the frog]

Carbacholine depressed postsynaptic currents in the frog m. sartorius leaving intact presynaptic currents in proximal and distal portions of the motor nerve ending. The carbacholine depressing action was followed by an increase in the time gap between the beginning of presynaptic depolarisation and subsequent quantal release. This effect was considerably more obvious in the distal portions of the nerve endings. Effect of extracellular potassium was evident in a diminishing of presynaptic currents due to membrane depolarisation. The data obtained suggest that carbacholine presynaptically depresses synaptic transmission via metabotropic cholinergic receptors controlling the time course of the transmitter release.     

Effects of adenosine on Ca2+ entry in the nerve terminal of the frog neuromuscular junction

This study aimed to test whether nerve-evoked and adenosine-induced synaptic depression are due to reduction in Ca2+ entry in nerve terminals of the frog neuromuscular junction. Nerve terminals were loaded with the fluorescent Ca2+ indicator fluo 3 (fluo 3-AM) or loaded with dextran-coupled Ca2+ green-1 transported from the cut end of the nerve. Adenosine (10-50 microM) did not change the resting level of Ca2+ in the presynaptic terminal, whereas it induced large Ca2+ responses in perisynaptic Schwann cells, indicating that adenosine was active and might have induced changes in the level of Ca2+ in the nerve terminal. Ca2+ responses in nerve terminals could be induced by nerve stimulation (0.5 or 100 Hz for 100 ms) over several hours. In the presence of adenosine (10 microM), the size and duration of the nerve-evoked Ca2+ responses were unchanged. When extracellular Ca2+ concentration was lowered to produce the same reduction in transmitter release as the application of adenosine, Ca2+ responses induced by nerve stimulations were reduced by 40%. This indicates that changes in Ca2+ responsible for the decrease in release should have been detected if the mechanism of adenosine depression involved partial block of Ca2+ influx. Ca2+ responses evoked by prolonged high frequency trains of stimuli (50 Hz for 10 or 30 s), which caused profound depression of transmitter release, were sustained during the whole duration of the stimulation, and adenosine had no effect on these responses. These data indicate that neither adenosine induced synaptic depression nor stimulation-induced synaptic depression are caused by reductions in Ca2+ entry into the presynaptic terminal in the frog neuromuscular junction.     

Presynaptic mechanisms of zinc effects on the neuromuscular transmission]

The effect of zinc ions on presynaptic currents and transmitter release was studied at the neuromuscular junction of the frog cutaneous pectoris muscle preparation with using an extracellular microelectrode. It has been shown that zinc (100 mkM) amplified MEPP frequency at first, but suppressed it later. Zinc affected the presynaptic spike waveform and transmitter release in a concentration-dependent manner. Depending on concentration and time of exposure zinc increased or suppressed transmitter release. Increase of transmitter release was shown to be resulted by blockade voltage gated and calcium activated potassium channels in nerve ending, leading to broad of both presynaptic spike and action potential. Strong change of presynaptic spike waveform after high concentration zinc treatment supposed that under this condition zinc depressed voltage gated calcium and sodium channel leading to decrease of transmitter release. It was concluded that the final and irreversible depression of acetylcholine release by zinc was due to alteration of whole ion conductances in nerve ending and to change of configuration of proteins included in structure of ion channels. It is discussed possible mechanisms of various effects of zinc ions at the neuromuscular synapse.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

Action of D-alpha aminoadipic acid on the sensory organs of the inner ear in the frog

Following the suggestions in the literature that glutamate or aspartate may be the transmitter at the primary afferent synapses of acoustico-lateralis organs, we have employed the "selective" excitatory amino acid antagonist. D-alpha amino adipate (DAA) as a tool with which to shed further light on this problem in the labyrinthine organs of the frog. DAA produces a dose-responsive, reversible depression of spontaneous activity in the afferent nerves of the posterior semicircular canal, saccule and basilar papilla. These structures are examples of ampullar, otolithic and auditory organs, respectively. The drug effect seems qualitatively the same throughout the labyrinth. The most interesting finding was that of a presynaptic (hair cell) effect of DAA on the semicircular canal. The means of recording did not permit detection of a presynaptic effect in the other organs examined. All the observed effects of DAA could be explained by a presynaptic action to inhibit transmitter release. Therefore, the ability of DAA to reduce transmission at primary afferent synapses of the frog labyrinth must not necessarily be interpreted to imply that the transmitter is an excitatory amino acid. A presynaptic action to reduce the release of a transmitter (of unknown structure) could explain all our results.