Ultrasound-induced changes in synaptic processes with different transmitters in smooth muscles

The effect of therapeutic-intensity ultrasound on neuromuscular transmission and spontaneous electrical and contractile activity in smooth muscles of the gastrointestinal tract of guinea pig was studied by a modified sucrose-gap technique. The action of ultrasound was found to facilitate the acetylcholinergic neuromuscular transmission (mainly by increasing the amplitude of excitatory postsynaptic potentials). The higher efficiency of the nonadrenergic neuromuscular transmission was manifested as an increase (nearly twofold) in the total duration, but not in the amplitude, of inhibitory postsynaptic potentials. Modulations of the first and second components of the potentials caused respectively by the action of ATP and of nitric oxide as possible transmitters, were different. Concurrently with enhancing the synaptic transmission efficiency, ultrasound exerted an opposite, inhibitory, effect on generation of spontaneous action potentials and contraction of smooth muscles. All the ultrasound effects were fully reversible. The findings permit assuming a special mechanism of modification of the synaptic transmission in smooth muscles under the action of ultrasound.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 297–302, July–August, 1993.     

Slowing of synapse elimination by alpha-bungarotoxin superfusion of the neonatal rabbit soleus muscle

To examine the role of postsynaptic activity in regulating the rate of neuromuscular synapse elimination, contractile activity of neonatal rabbit soleus muscles was decreased by chronic superfusion of alpha-bungarotoxin (alpha-BGT) over their surfaces. Superfusion was begun at 6 days postnatal and continued for a variable duration (2 to 5 days) before muscles were analyzed. The percentage of polyinnervated fibers was assessed both physiologically and anatomically for alpha-BGT-treated muscles and their contralateral muscles, in addition to normal and control muscles of the same age. Within muscles exposed to alpha-BGT, polyinnervation was significantly greater than that for muscles from each of the control groups. The anatomical assay further revealed that the retention of polyinnervation in alpha-BGT-treated muscles was most pronounced near the muscle's surface, although end plates at the center were also affected. This finding, coupled with evidence that only a small percentage of the muscle fibers were completely inactivated, suggests that the activity block was also most pronounced near the surface and relatively low at the muscle's center. The percentage of end plates at which synapse elimination was delayed was greater than the estimated percentage whose activity was completely blocked, suggesting that synapse loss was slowed even in muscle fibers retaining some postsynaptic activity. These observations indicate that the rate of synapse elimination depends on the levels of functional acetylcholine receptors. This process could be mediated in a graded fashion by changes in postsynaptic activity (subthreshold or suprathreshold) or by a nonelectrical effect of blocking postsynaptic receptors.     

Dystrophin is a component of the subsynaptic membrane

A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300-kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy.     

Study of role of inhibitory interneurons in mechanisms of regulation of sensory synapses formed by thalamic and cortical inputs on pyramidal cells of the dorsolateral amygdala nucleus

The work deals with study of role of inhibitory interneurons in the process of regulation of sensory currents converging on soma of pyramidal cells of the dorsolateral amygdala nucleus as well as of role of these interneurons in mechanism of regulation of plasticity of amygdala synapses. It has been shown that the part of the spontaneous inhibitory postsynaptic currents recorded on the dorsolateral amygdala pyramidal cells is relatively high and amounts to about a half of the total amount of the recorded events. Analysis of the evoked postsynaptic responses has shown the interneurons to regulate activity and duration of these responses due to the postsynaptic membrane hyperpolarization as a result of activation of GABA_A-receptors. Also studied was role of interneurons in providing mechanisms of the long-term potentiation of the synaptic responses evoked by stimulation of cortical and thalamic inputs. Block of effect of interneurons with help of picrotoxin has been shown to lead to an increase of evoked potentiation of synaptic responses.     

Conditioned reactions to time of hypothalamic neurons. The perifornical nucleus]

In 40% of the 52 neurones of the hypothalamic perifornical nucleus in alert rabbits conditioned trace reactions of the activational (52%) and inhibitory (48%) type were recorded in the course of elaboration of a conditioned motor reflex to time. The sign and pattern of the trace responses were determined by the nature of cell reactions to actual paired stimuli. After 50 to 70 pairings, the unit trace conditioned reaction to time persisted for a period of 10 to 15 successive omissions. Trace responses were observed most frequently in the 5th of 8th omissions. In some cases conditioned enhancement of cell activity coincided with the conditioned motor response to time. This fact together with the maximal development of a summery trace cellular response at the moment of formation of conditioned motor reactions attests the participation of neurones of the perifornical nucleus in maintaining conditioned motor activity.     

Changes in the asynchronism of transmitter release in a neuromuscular synapse and the time course of evoke postsynaptic signals during growth and branching og frog nerve ending

The influence of both growth and branching of a nerve terminal on the asynchronism of transmitter release and the time-course of evoked postsynaptic responses was investigated using a model of a frog neuromuscular synapse in which the nerve terminal represents a population of spatially isolated active zones. It was shown that the appearance of additional branching in proximal parts of the nerve ending leads to decrease in the asynchronism of transmitter release, an increase in quantum content and the amplitude of the postsynaptic signal, and the shortening of its phase of growth. It was found that the asynchronism of transmitter release has a much stronger influence on the time-course of end plate currents compared with end plate potentials. The factors strengthening and weakening the asynchronism of transmitter release in a neuromuscular synapse and the reasons for various length and branching of vertebrate nerve terminals are considered.     

Effect of prolonged low-frequency stimulation on acetylcholine sensitivity of the postsynaptic membrane during blocking of neuromuscular transmission

Sensitivity of the postsynaptic chemoreceptive membrane of the frog sartorius muscle fiber to acetylcholine was studied during the development of a block to neuromuscular transmission in the course of prolonged indirect low-frequency stimulation. Calculation of the mean amplitude of miniature end-plate potentials, measurement of the input resistance of the electrogenic membrane of the muscle fiber, and application of acetylcholine to the postsynaptic membrane showed that sensitivity of the postsynaptic membrane to mediator is unchanged at the time of onset of the neuromuscular block. A decrease in amplitude of the end-plate potentials during development of fatigue is due to a reduction in their quantum composition, consequent upon negative antidromic influences from the muscle on motor nerve endings, with the participation of chemical agents formed in the muscle during the activity of its contractile system.     

Influence of forelimb afferents on the activity of lumbar spinal-cord motoneurons

Experiments were conducted on anesthetized cats with microelectrode recording to study the synaptic responses that develop in the lumbar motoneurons on stimulation of the afferent fibers of groups II and III in the nerves of the ipsilateral and contralateral forelegs. Stimulation of these afferents evoked predominantly inhibitory postsynaptic potentials (IPSP) in the extensor motoneurons and excitatory postsynaptic potentials (EPSP) in the flexor motoneurons. A basically inhibitory change in the rhythmic background activity developed under the influence of descending impulsation. The duration of the total inhibition of "spontaneous" motoneuron activity corresponded to the duration of the inhibitory influences exerted by the forelimb flexor-reflex afferents (FRA) on the interneurons. The interaction of the descending and segmental PSP resulted in inhibition and facilitation of the segmental responses in the motoneurons. The ultimate result of this interaction was determined by the shifts in the membrane potential of the motoneuron and by the effects created in the interneurons.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 3, No. 1, pp. 58–67, January–February, 1971.     

Maintained depolarization of synaptic terminals facilitates nerve-evoked transmitter release at a crayfish neuromuscular junction

Presynaptic and postsynaptic potentials were examined by intracellular recording at a crayfish neuromuscular junction. During normal synaptic transmission, the action potentials were recorded in the terminal region of the excitatory axon and postsynaptic responses were obtained in the muscle fibers. We found that it was possible to modify the synaptic transmission by applying depolarizing or hyperpolarizing currents through the presynaptic intracellular electrode. Typically, a 7-15 mV depolarization lasting longer than 50 msec leads to a large (500%) enhancement of transmitter release, even though the preterminal action potential is reduced in amplitude. Hyperpolarization increases the amplitude of the action potential, but slightly reduces the transmitter release. These results are different from those reported for other neuromuscular synapses and the squid giant synapse, but are similar in many respects to the results reported for several invertebrate central synapses. We conclude, first, that different synapses may have markedly different responses to conditioning by membrane polarization and, secondly, that maintained low-level depolarization may induce a potentiated state in the nerve terminal, perhaps brought about by slow entry of calcium.     

Increased neuromuscular activity causes axonal defects and muscular degeneration

Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor alpha-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

Statistical Estimation of Distribution of Quantal Release of Transmitter in Neuromuscular Synapse of the Lobster Homarus americanus

A new approach to estimation of quantal release distribution of transmitter under conditions of high synaptic activity is presented. Postsynaptic responses of neuromuscular excitatory synapse in muscle-opener of nipper of the lobster, which are obtained by focal extracellular recording, are used as original data set. Based on two data groups (value of evoked and spontaneous postsynaptic responses), the linear regression model is constructed. Parameters of this model describe completely the quantal release distribution. To evaluate the parameters, biased modifications of the least squares method—the penalized least squares method and the principal components method—were applied. As a result, it was possible to achieve estimations of the quantal release distribution with sufficiently low standard errors. Modeling studies have shown that the gain of accuracy of the estimation due to a decrease of the standard error exceeds considerably losses caused by its bias.     

Changes in the effect of acetylcholine on septo-hippocampal reactions after serotonin administration to rabbits

Impulse activity and focal evoked potentials appearing in the hippocampus in response to testing stimuli applied to the septum medial nucleus were recorded in nonimmobilized and unanaesthetized rabbits. The efficiency of acetylcholine (ACh) action on septo-hippocampal reactions was tested before and after microiontophoretic administration of the serotonin (5-OT) or stimulation of the raphe nuclei. The 5-OT and raphe stimulation produced significant changes in the ACh action on septo-hippocampal reactions. In most cases, after microiontophoretic administration of 5-OT, the efficiency of ACh diminished, independently of excitatory or inhibitory effect of the 5-OT. Thus, the modulating action of 5-OT consists not only in protracted trace changes of the magnitude of septo-hippocampal reactions but also in trace reduction of septo-hippocampal responses to ACh.     

Changes in membrane potential and postsynaptic potentials evoked by strychnine in neocortical neurons

Intracellular responses of neurons of the suprasylvian fissure to intracortical stimulation before and during topical cortical strychnine application was studied in experiments on immobilized, unanesthetized cats (a local anesthetic was used). Untreated cortical neurons responded to intracortical stimulation with a monosynaptic excitatory postsynaptic potential (EPSP) followed by an inhibitory postsynaptic potential (IPSP). Application of strychnine evoked epileptiform population activity and paroxysmal depolarizations of neuronal membrane potentials (MPs), followed by hyperpolarization. Increased hyperpolarizations, and the prolonged duration of their summation were responsible for an increased MP and reduced or abolished tonic spike activity. Intracellular application (as a result of diffusion from the microelectrode) of ethyleneglycoltetraacetate (EGTA) that blocked the calcium-dependent potassium membrane conductance (gK(Ca)) abolished the hyperpolarization. The development of epileptiform activity was accompanied by reduction of the IPSP, and an increase in the monosynaptic EPSP. The role of gK(Ca) and postsynaptic inhibition in epileptogenesis is discussed.I. I. Mechnikov State University, Odessa. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 684–691, November–December, 1992.     

Physiological activity-dependent ultrastructural plasticity in normal adult rat neuromuscular junctions

The major finding of the present study is that the ultrastructural organization of the neuromuscular synapse can be modified by a small, 4-week-long, physiological increase in the locomotor activity of the extensor digitorum longus muscle of normal adult rats trained to walk. This study measures these plastic adaptations using several synaptic morphological parameters. The observed changes in neuromuscular junctions affect both pre- and postsynaptic membranes. In particular, the presynaptic membrane densities in the active zones and the postsynaptic adaxonal membrane densities become larger, which shows that in the normal adult mammal neuromuscular junction, there is an activity-dependent modulation of the neurotransmission-related structures in response to slight physiologic functional demands. The nature and magnitude of these changes are discussed.     

Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.     

Neuromuscular synaptogenesis in wild-type and mutant zebrafish

Genetic screens for synaptogenesis mutants have been performed in many organisms, but few if any have simultaneously screened for defects in pre- and postsynaptic specializations. Here, we report the results of a small-scale genetic screen, the first in vertebrates, for defects in synaptogenesis. Using zebrafish as a model system, we identified seven mutants that affect different aspects of neuromuscular synapse formation. Many of these mutant phenotypes have not been previously reported in zebrafish and are distinct from those described in other organisms. Characterization of mutant and wild-type zebrafish, from the time that motor axons first arrive at target muscles through adulthood, has provided the new information about the cellular events that occur during neuromuscular synaptogenesis. These include insights into the formation and dispersal of prepatterned AChR clusters, the relationship between motor axon elongation and synapse size, and the development of precise appositions between presynaptic clusters of synaptic vesicles in nerve terminals and postsynaptic receptor clusters. In addition, we show that the mechanisms underlying synapse formation within the myotomal muscle itself are largely independent of those that underlie synapse formation at myotendinous junctions and that the outgrowth of secondary motor axons requires at least one cue not necessary for the outgrowth of primary motor axons, while other cues are required for both. One-third of the mutants identified in this screen did not have impaired motility, suggesting that many genes involved in neuromuscular synaptogenesis were missed in large scale motility-based screens. Identification of the underlying genetic defects in these mutants will extend our understanding of the cellular and molecular mechanisms that underlie the formation and function of neuromuscular and other synapses.     

Reproduction of a memory trace in amnesia and forgetting following alteration in the activity of pre- and postsynaptic dopamine receptors

The paper deals with analysis of the action of enantiomers 3-PPP on memory trace reproduction disturbed by amnestic effects and spontaneous forgetting in mice. A considerable antiamnestic effect is shown of (+)3-PPP and (-)3-PPP in 10 mg/kg doze changing the activity of postsynaptic dopamine receptors. The influence of drugs in 2 mg/kg doze changing the activity of presynaptic receptors consisted in recovery of conditioned habit only in situation of a weak amnestic effect and at forgetting, when the level of reproduction was like a weak amnesia. The range of enantiomers 3-PPP action on reproduction processes disturbed by amnesia or forgetting is determined by the possibility of specific activation of pre- and postsynaptic receptors at different depth of disturbances of memory trace reproduction causing differentiation of 3-PPP effects.