Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.     

Desensitization and recovery at the frog neuromuscular junction

The time course of carbachol-induced desensitization onset and recovery of sensitivity after desenitization have been compared at the frog neuromuscular junction. The activation-desensitization sequence was determined from input conductance measurements using potassium-depolarized muscle preparations. Both desensitization onset and recovery from desensitization could be adequately described by single time constant expressions, with tauonset being considerably shorter than taurecovery. In nine experiments, tauonset was 13+/-1.3 s and taurecovery was 424+/-51 s with 1 mM carbachol. Elevating the external calcium or carbachol concentration accelerated desensitization onset without changing the recovery of sensitivity after equilibrium desensitization. Desensitization onset was accelerated by a prior activation-desensitization sequence to an extent determined by the recovery interval that followed the initial carbachol application. The time course of return of tauonset was closely parallel to, but slower than the time course of recovery of sensitivity. These results are consistent with a cyclic model in which intracellular calcium is a factor controlling the rate of development of desensitization.     

Large dense-core vesicles at the frog neuromuscular junction: characteristics and exocytosis

The population of large dense-core vesicles (LDCVs) in motor nerve terminals of the frog cutaneous pectoris muscle was analysed after various experimental protocols leading to large acetylcholine release. Three types of LDCVs classified according to their size and the core density were detected. Vesicles, 100–150 nm in diameter, with a large and very dense core (type 1) or with an irregular and diffuse dense core (type 2) were present in similar proportions (45 and 50% respectively) in controls. Smaller vesicles, 50–80 nm in diameter, with a very dense core (type 3) were rare, representing around 5% of the cored vesicles. The relative proportion of type 1 and type 2 LDCVs was not modified after prolonged treatment with 25 mM K+. In contrast, the proportion of type 2 LDCVs significantly increased whereas that of type 1 LDCVs decreased after two or three series of 20 Hz electrical stimuli applied to the nerve at 5 s intervals. These changes suggest that type 2 LDCVs are newly recycled LDCVs in the process of reloading. Images of fusion of LDCVs with the axolemma in regions facing Schwann cell digitations were observed both in K+- and in electrically stimulated preparations. They indicate that exocytosis of LDCVs at the frog neuromuscular junction takes place preferentially away from the active zones. The presence of a clathrin-like coat on large pockets still containing a core and of both type 1 and type 2 LDCVs in the vicinity of coated pockets strongly suggests that LDCVs might undergo a combined process of exo–endocytosis at the same site.     

Nuclear labelling after prolonged H-uridine incorporation as visualized by high resolution autoradiography

Localization of radioactive labelling over the nuclei of BSC₁ cells is visualized after long periods of ³H-5-uridine incubation followed or not followed by periods of postincubation in nonradioactive medium for up to several days, using high resolution autoradiography combined with a preferential staining method for ribonucleoproteins.It is shown that when cells are labelled for 1 or 6 h with ³H-uridine and postincubated with a non-radioactive medium up to several days, there is always some radioactivity present in the nucleolus and nucleoplasm. When sections of cells fixed after 1 h of labelling followed by 24 h of postincubation are treated with RNase, part of the radioactivity found in the nucleus disappears almost completely only after a succeeding DNase digestion.The majority of interchromatin granules are weakly labelled after most incubation times, with the label localized rather at the periphery of clusters of granules, or are unlabelled.The results are discussed in the context of recent biochemical findings. It is proposed that interchromatin granules might represent a structure containing a limited quantity of slowly labelled nuclear RNA.     

Psychosine-induced changes in the neuromuscular transmission and structure of the neuromuscular junction in the frog

A decrease in the amplitude of the miniature and evoked end-plate potentials, as well as a change in the course of facilitation and depression of the end-plate potentials under rhythmic stimulation, were observed in psychosine-treated preparations of the cutaneous-pectoral muscle of the frog. The results of electron microscopic investigations indicate changes in the structure of synaptic Schwann cells enveloping the motor terminals and disturbances of the inner mesaxon structure of the myelinated axons.A. A. Ukhtomskii Institute of Physiology, Saint Petersburg University. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 482–490, July–August, 1992.     

Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction

Three nicotinic agonists, suberyldicholine, acetylcholine and carbachol, have been investigated by single channel recording at the endplates of adult frog muscle fibres. All three agonists can block the channels that they open. Suberyldicholine is the most potent blocker; it has an equilibrium constant for binding to the open channel of about 6 microM and blockages last for about 5 ms on average, at -105 mV. A plot of the mean number of blockages per unit open time against concentration ('blockage frequency plot') suggests that suberyldicholine does not produce long-lived blocked states such as might occur, for example, if it could be trapped within a shut channel. The characteristics of the 'blockage frequency plot' are analysed in Appendix 2. Block by acetylcholine and carbachol has much lower affinity (the equilibrium constants being a few millimolar for both), and blockages are much briefer, so that blockage appears to produce noisy single channel currents of reduced amplitude. A method based on the spectral density of the excess 'open' channel noise has been used to investigate the rate of blocking and unblocking. The basis of this method is discussed in Appendix 1. It is estimated that the mean duration of a blockage is about 18 microseconds for acetylcholine and 9 microseconds for carbachol.     

Nonquantum release of acetylcholine in frog neuromuscular junction after blocking of axoplasmic transport with colchicine

Standard microelectrode techniques were used to evaluate the effect of d-tubocurarine chloride on membrane potential of junctional and extrajunctional areas of muscle fibers in a potassium-free Ringer solution. The experiments were made on frogs after inactivation of acetylcholinesterase. d-Tubocurarine chloride hyperpolarized the membrane of muscle fibers only in the junctional area. Blockade of axoplasmic transport with colchicine did not affect the magnitude of the hyperpolarization response of the membrane end plate to the presence of d-tubocurarine chloride, but at the same time it significantly reduced the membrane rest potential of muscle fibers, and gave rise to the appearance of extrajunctional sensitivity to acetylcholine. It is concluded that the blockade of axoplasmic transport does not affect the pattern of non-quantum acetylcholine release from nerve terminals. Therefore, this is unlikely to cause denervation-like changes in the muscle under the conditions described.     

The rising phase of the miniature endplate current at the frog neuromuscular junction

(1) The rising phase of minature endplate currets was recorded at the frog's neuromuscular junction using both the two electrode voltage clamp and a single external electrode, or Strickholm, voltage clamp. (2) The $\text{Q}$(10) of the miniature endplate current rising phase was 2.3 in a variety of solutions selected to alter presynaptic behavior. (3) Increasing the solution's viscosity by an amount sufficient to slow the diffusion coefficient of acetylcholine by a third has no effect on the duration of the rising or the decay phase. This solution does seem to further slow the miniature endplate current decay phase, but not the rising phase, after inhibition of the acetylcholinesterase. (4) As the membrane potential is made more positive, the miniature endplate current rising phase is prolonged, with an $\text{e-}\text{fold}$ slowing per 170 mV change. (5) It is concluded that neither presynaptic nor subsynaptic events determine the rising phase of miniature endplate currents at the frog neuromuscular junction. Rather, the limiting step occurs within the membrane and is most likely a change in the binding constant of the receptor for the acetylcholine molecule.     

Development and ultrastructure of the neuromuscular junction in bronchial smooth muscle tissue

At various stages of pre- and postnatal ontogenesis ultrastructure of contacts of smooth myocytes and nervous terminals in the white mice bronchial wall has been investigated. Nervous fibers grow into the forming tissue of the lung beginning from the 11th day of embryogenesis. By the end of the prenatal development the nervous fibers fasciculi with varicosites and having vesicles are localized at the distance of 100-300 nm from the developing myocytes. Formation of dense neuromuscular junctions with the distance of 35-60 nm between the axonal membranes and the myocyte is observed on the 10-15 day after birth. In mature animals combination of various types of neuromuscular connections is revealed; they ensure local and distant neurotrophic regulation. In the bronchial smooth musculature afferent connections are revealed, as well as connections of myocytic processes with the effectors. Terminals of the cholinergic type predominate, adrenergic effectors occur very seldom. There are terminals, in which combination of vesicles having various structure and diameter are observed.     

Effect of the "calcium ionophore" A-23187 on transmitter release at the frog neuromuscular junction.

The ionophore A-23187 when added to the usual Ca²⁺-Ringer at the frog neuromuscular junction has almost no effect on the frequency of miniature end-plate potentials (min.e.p.p.s). The ionophore does increase the rate of Ca²⁺ efflux from frog muscle, so it is in effective concentrations in the Ringer. When added to Ringer containing Ni²⁺ instead of Ca²⁺, the ionophore increases the min.e.p.p. frequency. We suggest that the ionophore can carry divalent cations into the terminal, but there are mechanisms to keep the Ca²⁺ low.Apparently these mechanisms are unable to rapidly eject or sequester Ni²⁺.     

The human SOX18 gene: cDNA cloning and high resolution mapping

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY and these genes play key roles during animal development. We report here cloning and characterisation of the human SOX18 gene. SOX18 gene is expressed in foetal brain as well as in a wide range of foetal and adult tissues indicating its function is not restricted to early development. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3, 27.29 cR distal from the marker D20S173.     

The effect of sodium ions of MEPP frequency at the frog neuromuscular junction.

MEPP frequency at the frog neuromuscular junction maintained in saline with normal [K]_o was insensitive to reductions in [Na]_o at 130°C. However, at 23°C, decreasing [Na]_o causes a progressive rise in MEPP frequency; there is an approximately linear relationship between log [Na]_o and the rate of spontaneous release. The effect of reducing [Na]_o is dependent on [Ca]_o; thus MEPP frequency is unaffected, even at 23°C, by changes in [Na]_o when [Ca]_o is reduced to the low level of 5 × 10^?7M. It is suggested that: (i) MEPP frequency is determined by [Ca]_i at the presynaptic terminals (ii) Reductions in [Na]_o cause an increase in Ca-influx. At 13°C the presynaptic terminals are able to maintain [Ca]_i constant when challenged whereas, at 23°C, there is a mobilization of Ca from intracellular storage sites and under these conditions [Ca]_i is not maintained constant in the face of a rise in Ca-influx (associated with a reduction in [Na]_o) and MEPP frequency consequently rises. The ways in which both extracellular and intracellular Na affect [Ca]_i and MEPP frequency are discussed.     

Desensitization onset and recovery at the potassium-depolarized frog neuromuscular junction are voltage sensitive

The influence of voltage on the time-course of desensitization onset and recovery has been studied at the frog neuromuscular junction. The activation-desensitization sequence was determined from carbachol- induced end-plate currents in potassium-depolarized fibers voltage- clamped either to -40 mV or +40 mV. The time-course of both desensitization onset and recovery developed exponentially, with onset occurring more rapidly than recovery. Desensitization onset was voltage dependent, the onset time constant being 8.3 +/- 1.3 s (11 fibers) at - 40 mV and 19.3 +/- 3.4 s (15 fibers) at +40 mV. Recovery from desensitization was also influenced by voltage. The extent of recovery after 2 min was 80.4 +/- 6.3% in those fibers voltage-clamped to -40 mV and 57.4 +/- 3.6% in those fibers voltage-clamped to +40 mV. The voltage dependence of desenistization onset and recovery did not result from a difference in ability to control voltage at these two levels of membrane potential. These results demonstrate that in the potassium- depolarized preparation the processes controlling both desensitization onset and recovery of sensitivity from the desensitivity from the desensitized state are influenced by membrane voltage.     

Atypical endplate miniature potentials in the frog neuromuscular junction after modification of the intercellular matrix and osmotic exposures]

In frog cutaneous-pectoris muscles the frequency of slowly rising atypical miniature endplate potentials (MEPPs) was significantly enhanced after collagenase (0.1%) treatment. Treatment with trypsin, hyaluronidase, hyper- and hypoosmotic solutions caused no changes in slowly rising MEPP (frequency in muscle fibers with intact acetylcholinesterase (AChE). Inhibition of AChE caused appearance of giant MEPPs. Acceleration of acetylcholine diffusion from synaptic cleft after treatment with hyaluronidase decreased giant MEPP frequency demonstrating their dependence upon nonhydrolyzed acetylcholine in synaptic cleft. The relation between slowly rising MEPPs and activity of synaptic Schwann cells in discussed.     

Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion

Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.