Termination of the presynaptic effect of carbacholine by tubocurarine]

The presynaptic action of carbacholine (Cch) was studied in experiments on the frog sartorius muscle neuromuscular preparation. Cch proved to decrease the quantum content of the end plate potential (EPP); this effect was induced by a direct Cch action on the motor nerve endings. d-tubocurarine decreased the sensitivity of the nerve endings to Cch significantly. Both d-tubocurarine and Cch had concurrent antagonistic interrelations in respect to their action on the quantum content of EPP. Atropine in low concentrations had no influence on the presynaptic effect of Cch. It was concluded that Cch decreased the quantum content of EPP through the N-cholinergic structures of the motor nerve endings.     

Effect of armin on synaptic transmission in frog neuromuscular preparations

The action of armin, an organophosphorus inhibitor of cholinesterases, on synaptic transmission parameters was studied by means of intracellular registration of end plate potentials and currents (EPP and EPC) in the frog. On 10-minute exposure the increase in the temporary parameters became manifest provided the drug was administered at a concentration of 10(-6) g/ml and over. EPC reversal potential and cholinoreceptor sensitivity to armin did not change substantially. At a concentration of 10-(-8) g/ml armin exerted a potentiating effect on the frequency of miniature EPP and quantum composition of EPP, while that effect was not related to armin anticholinesterase activity. The presynaptic acetylcholine release was suppressed by high concentration of armin (10(-5) g/ml). Under the conditions cited there was a decrease in the depot of the available transmitter quanta in nerve terminals.     

Mediator liberation at the myoneural junctions of the frog sartorius muscle when axoplasmic transport has been disrupted with colchicine]

Colchicine application to the nerve innervating the frog sartorius muscle leads to reduction of the differences in functional condition of the myoneural synapses of this muscle by the signs of quantum content of the end plate potential (EPP) and by the changes of the EPP amplitudes in high frequency stimulation. Along with this there occurs a rarefaction of the frequency of miniature potential of the end plate, and reduction of its amplitude. Apparently, the latter is caused by reduction of effective resistance of the postsynaptic membrane. Changes of the induced and spontaneous secretion of the mediator are connected with disturbances of the axoplasmic transport just in the somatic nerve fibers.     

Location and functioning of transmitter release sites in frog neuromuscular junction

End-plate potentials (EPP) and miniature EPP (MEPP) were recorded in a single neuromuscular synapse of the frog sartorius muscle by means of two microelectrodes with a resistance of 0.5–2.0 M. Groups of signals (fields), reflecting transmitter secretion in spatially distinct release sites were identified by extracellular recording on MEPP amplitude scatter diagrams. Release sites in the nerve ending were found to be unevenly distributed, to be grouped in certain areas, and to differ in their probability of secretion of a quantum of transmitter. Comparison of fields on MEPP and uniquantal EPP amplitude scatter diagrams in solution with low Ca⁺⁺ concentration (0.2–0.4 mM) showed that ability to induce evoked and spontaneous transmitter release at the release site differs, and that sometimes a release site does not participate in evoked secretion. The results of simultaneous recording of synaptic potentials using extra- and intracellular electrodes indicate that transmitter secretion in spatially distinct groups of release sites leads to the appearance of polymodality in the distribution of amplitudes of intracellularly recorded MEPP and uniquantal EPP.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 152–160, March–April, 1985.     

Effect of dimedrol on the function of the neuromuscular synapse and the generation of action potentials by motor nerve fibers

Microelectrode registration of synaptic potentials in the frog cutaneous-pectoris muscle has shown dimedrol (7.9 X 10(-5) M) to act on synaptic transmission decreasing the quantal content, estimated by mean EPP amplitude to mean miniature EPP amplitude ratio, the quantal content calculated by variation coefficient of EPP amplitude being unaffected. The data suggest possible transmitter release and depletion of mediator stock. The experiments on isolated motor nerve fibers have demonstrated dimedrol to cause the increase in transmitter release probability by widening the action potentials in the terminals and thus enhancing Ca2+ influx.     

Incorporation of [14C]histidine into homocarnosine and carnosine of frog brain in vivo and in vitro

1. Homocarnosine, carnosine and histidine were determined in brain from several species and the results compared with values in the literature. 2. [(14)C]Homocarnosine and [(14)C]carnosine were isolated from frog brain after intracerebral injection of [(14)C]histidine in vivo. 3. Whole frog brain, incubated in vitro with l-[(14)C]histidine, formed labelled homocarnosine and carnosine. 4. A frog brain homogenate or supernatant fraction catalysed the incorporation of l-[(14)C]histidine into homocarnosine and carnosine. 5. The results indicate that brain tissue can synthesize homocarnosine and carnosine.     

Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction

In a frog neuromuscular preparation of m. sartorius, glutamate had a reversible dose-dependent inhibitory effect on both spontaneous miniature endplate potentials (MEPP) and nerve stimulation-evoked endplate potentials (EPP). The effect of glutamate on MEPP and EPP is caused by the activation of metabotropic glutamate receptors, as it was eliminated by MCPG, an inhibitor of group I metabotropic glutamate receptors. The depression of evoked EPP, but not MEPP frequency was removed by inhibiting the NO production in the muscle by L-NAME and by ODQ that inhibits the soluble NO-sensitive guanylyl cyclase. The glutamate-induced depression of the frequency of spontaneous MEPP is apparently not caused by the stimulation of the NO cascade. The particular glutamate-stimulated NO cascade affecting the evoked EPP can be down-regulated also by adenosine receptors, as the glutamate and adenosine actions are not additive and application of adenosine partially prevents the further decrease of quantal content by glutamate. On the other hand, there is no obvious interaction between the glutamate-mediated inhibition of EPP and inhibitory pathways triggered by carbacholine and ATP. The effect of glutamate on the evoked EPP release might be due to NO-mediated modulation (phosphorylation) of the voltage-dependent Ca2+ channels at the presynaptic release zone that are necessary for evoked quantal release and open during EPP production.     

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, <Emphasis Type="Italic">Cyclorana alboguttata</Emphasis>

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.     

Elucidation of the mechanism and site of action of quinuclidinyl benzilate (QNB) on the electrical excitability and chemosensitivity of the frog sartorius muscle

The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate of rise and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration.     

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca<Superscript>2+</Superscript>-channels and ryanodine receptors

L-type Ca²⁺-channel blockers, verapamil (5 μM) and nifedipine (10 μM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca²⁺-channel activator BAY K 8644 (1 μM) has a decreased mediator secretion level. Nifedipine (10 μM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 μM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 μM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 μM).     

Extra-pair paternity is not repeatable between years in Pied Flycatchers (Ficedula hypoleuca): a role for social context

Extra-pair paternity (EPP) is the consequence of the interactions between a social pair and extra-pair males during the female's fertile phase in a specific social context and ecological environment. Although EPP occurs in many avian species, there is a lack of understanding of how environmental factors may impact its frequency. Accordingly, the relative importance of individual characteristics on the one hand, and of their immediate social environment on the other, may affect EPP repeatability and thereby the capacity of this trait to respond to selection. If EPP is an individual genetically based trait that may respond to selection, we expect it to be repeatable across breeding seasons. To check this possibility, we analysed the within-individual repeatability in EPP of breeding males and females in two natural populations of Pied Flycatchers Ficedula hypoleuca in central Spain during three field seasons. We then studied the relationship between EPP and individual male and female traits (only some of which were themselves repeatable), as well as key context variables such as breeding synchrony and population density. Our results showed no repeatability for EPP in either sex. We found a positive association with laying date and stronger associations of EPP with male plumage and morphological traits than with female characteristics. We suggest that the variable social environment is an important modulator of EPP patterns, and that differences between field EPP studies in this respect may explain contradictory results in the same species, even in the same populations in different years.     

Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation- induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation- induced changes in MEPP frequency and EPP amplitude have similar time- courses.     

Differential effects of Ba2+, Sr2+, and Ca2+ on stimulation-induced changes in transmitter release at the frog neuromuscular junction

Endplate potentials (EPP) were recorded from the frog sartorius neuromuscular junction under conditions of low quantal content to study the effect of Ba2+, Sr2+, and Ca2+ on the changes in evoked transmitter release that occur during and after repetitive stimulation. The addition of 0.1-1 mM Ba2+ or Sr2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with 0.8-1.4 mM Sr2+, led to a greater increase in EPP amplitudes during and immediately after repetitive stimulation. These changes in release were analyzed in terms of the four apparent components of increased transmitter release that have previously been distinguished on the basis of their kinetic properties. The Ba2+-induced increase in EPP amplitudes was associated with an increase in the magnitude but not the time constant of decay of augmentation. Ba2+ had little effect on potentiation or the first and second components of facilitation. The Sr2+-induced increase in EPP amplitudes was associated with an increase in the magnitude and the time constant of decay of the second component of facilitation. Sr2+ had little effect on potentiation, augmentation, or the first component of facilitation. The selective effects of Ba2+ on augmentation and of Sr2+ on the second component of facilitation were reversible and could be obtained in the presence of the other ion. The addition of 0.1-0.3 mM Ca2+ to the bathing solution had little effect on potentiation, augmentation, or the two components of facilitation. These results provide pharmacological support for the proposal that there are four different components of increased transmitter release associated with repetitive stimulation and suggest that the underlying factors in the nerve terminal that give rise to these components can act somewhat independently of one another.     

Extrapair paternity in a flock-living passerine, the vinous-throated parrotbill Paradoxornis webbianus

Understanding the factors that affect the occurrence of extrapair paternity (EPP) is one of the central issues in sexual selection. We investigated genetic parentage and the ecological factors affecting patterns of EPP in the vinous-throated parrotbill, Paradoxornis webbianus , a flock-living species with double broods. Using microsatellite DNA fingerprinting, we determined parentage of 246 offspring in 50 broods over two years (2005 and 2006). Nineteen offspring (8%) from 13 broods (26%) were sired by extrapair males and one offspring (0.4%) was probably the result of intraspecific brood parasitism. The prevalence of EPP varied significantly through the breeding season: 95% of broods with EPP (12/13) occurred in the first of two laying peaks. Parentage assignment revealed that half of extrapair males (6/12) were adjacent neighbours. The distribution of EPP was not significantly related to the ecological factors including breeding density and breeding synchrony. Instead, we suggest that social characteristics such as flocking and weak territoriality may determine the observed pattern of EPP in this study.     

Metabolism of intracerebrally administered histidine, histamine and imidazoleacetic acid in mice and frogs

Abstract— After intracerebral administration of [¹⁴C]histidine to mice the major labelled substance found in the brain extracts was histidine itself; small amounts of labelled carnosine and homocarnosine were detected. No other labelled substances were detected on radio- autographs of two-dimensional TLC's of the extracts. In the case of the frog, radioactive histidine, N-acetylhistidine, carnosine and homocarnosine were found in the brain extracts at various times after intracerebral injection of the labelled histidine. With time, approximately 90 per cent of the radioactivity in the extracts was found in the N-acetylhistidine. In neither the mouse nor frog could we find unequivocal evidence for the formation either of histamine or imidazoleacetic acid from intracerebrally administered histidine, but our analytical procedures may have lacked sufficient sensitivity to pick up extremely low activities of histamine and imidazoleacetic acid. Experiments with [¹⁴C]histamine administered intracerebrally into mice demonstrated the major pathway of metabolism in brain to be histamine → methylhistamine → methylimidazoleacetic acid. No detectable label appeared in inlidazoleacetic acid. In the frog intracerebral administration of the labelled histamine led to the formation of methylhistamine and imidazoleacetic acid, but at most only traces of methylimidazoleacetic acid were found. The injection of [¹⁴C]imidazoleacetic acid intra- cerebrally into mice and frogs resulted in virtually no loss of the label in the form administered in the frog brain over a period of 4 h and in a slow rate of decrease in the mouse brain. No radioactive metabolites of imidazoleacetic acid were found in either species. The limitations of trying to determine natural functions of substances in brain by following the fate of exogenously administered materials is discussed.     

Effects of dantrolene and ryanodine on the evoked activity of neuromuscular synapses in mice

Ryanodine was shown to potentiate in a frequency-dependent manner a late phase of initial facilitation increasing the EPP amplitude for 15%-40% in a fixed interval 140-400 ms after the first EPP in the discharge. Dantrolene prevented the ryanodine induced increase of the late phase of facilitation, decreased 2-fold the amplitude and quantum content of single EPPs, augmented initial depression, blocked facilitation in 50% of synapses. Only in presence of dantrolene, the ryanodine's ability to increase the EPPs quantum content and early phase of the EPP facilitation in rhythmically active synapses, was revealed.     

Characterization of neuromuscular transmission in mice with progressive motoneuronopathy

Progressive motoneuronopathy (PMN) is an autosomal recessive mouse disease, which is characterized by the development of hind limbs paralysis rapidly progressing to the anterior parts of the body, muscular atrophy, respiratory depression, and death at 6-7 postnatal weeks. Here, we recorded the resting membrane potential (RMP), spontaneous miniature endplate potentials (MEPPs), and quantum content of endplate potentials (EPP) at the diaphragm muscle fibers in controls and PMN mice aged 18 to 43 days. In control animals, there was a progressive increase in RMP, MEPP frequency and EPP quantum content, as well as a decrease in mean MEPP amplitude. In PMN mice, the developmental increase in frequency and decrease in the amplitude of MEPPs was practically stopped at the postnatal day 18, whereas RMP increased but only until the age of 31 days and then progressively decreased. The distribution histogram of RMP in PMN mice older than 35 days revealed the existence of two subpopulations of muscle fibers: one showing a denervation-like decrease in RMP and the second, which was matching controls. In addition, EPP quantum content was significantly attenuated in older PMN animals. These results indicate that neurotransmission is severely affected in advanced, but not in early stage of disease, which is apparently due to a partial denervation of the muscles.     

Interstitial sodium nuclear magnetic resonance relaxation times in perfused hearts.

Separation of intracellular and extracellular sodium nuclear magnetic resonance (NMR) signals would enable nondestructive monitoring of intracellular sodium. It has been proposed that differences between the relaxation times of intracellular and extracellular sodium be used either directly or indirectly to separate the signal from each compartment. However, whereas intracellular sodium relaxation times have been characterized for some systems, these times were unknown for interstitial sodium. In this study, the interstitial sodium NMR relaxation times have been measured in perfused frog and rat hearts under control conditions. This was achieved by eliminating the NMR signal from the extracardiac (perfusate) sodium, and then quantifying the remaining cardiac signal. The intracellular signal was measured to be 8% (frog) or 22% (rat) of the cardiac signal and its subtraction was found to have a negligible effect on the cardiac relaxation times. Therefore this cardiac signal is considered to provide a good estimate of interstitial relaxation behavior. For perfused frog (rat) hearts under control conditions, this signal was found to have a T1 of 31.6 +/- 3.0 ms (27.3 +/- 1.6 ms) and a biexponential T2 of 1.9 +/- 1.0 ms (2.1 +/- 0.3 ms) and 25.2 +/- 1.3 ms (26.3 +/- 3.2 ms). Due to the methods used to separate cardiac signal from perfusate signal, it is possible that this characterized only a part of the signal from the interstitium. The short T2 component attributable to the interstitial signal indicates that separation of the NMR signals from each compartment on the basis of relaxation times alone may be difficult.     

Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.