Regulated spacing of synapses and presynaptic active zones at larval neuromuscular junctions in different genotypes of the flies Drosophila and Sarcophaga

Synapses at larval neuromuscular junctions of the flies Drosophila melanogaster and Sarcophaga bullata are not distributed randomly. They have been studied in serial electron micrographs of two identified axons (axons 1 and 2) that innervate ventral longitudinal muscles 6 and 7 of the larval body wall. The following fly larvae were examined: axon 1--wild-type Sarcophaga and Drosophila and Drosophila mutants dunce(m14) and fasII(e76), a hypomorphic allele of the fasciclin II gene; and axon 2--drosophila wild-type, dunce(m14), and fasII(e76). These lines were selected to provide a wide range of nerve terminal phenotypes in which to study the distribution and spacing of synapses. Each terminal varicosity is applied closely to the underlying subsynaptic reticulum of the muscle fiber and has 15-40 synapses. Each synapse usually bears one or more active zones, characterized by dense bodies that are T-shaped in cross section; they are located at the presumed sites of transmitter release. The distribution of synapses was characterized from the center-to-center distance of each synapse to its nearest neighbor. The mean spacing between nearest-neighbor pairs ranged from 0.84 microm to 1.05 microm for axon 1, showing no significant difference regardless of genotype. The corresponding values for axon 2, 0.58 microm to 0.75 microm, were also statistically indistinguishable from one another in terminals of different genotype but differed significantly from the values for axon 1. Thus, the functional class of the axon provides a clear prediction of the spacing of its synapses, suggesting that spacing may be determined by the functional properties of transmission at the two types of terminals. Individual dense bodies were situated mostly at least 0.4 microm away from one another, suggesting that an interaction between neighboring active zones could prevent their final positions from being located more closely.     

Postsynaptic abnormalities at the neuromuscular junctions of utrophin-deficient mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophin-deficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (alpha-bungarotoxin [alpha-BgTx] binding reduced to approximately 60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

An early stage in sodium channel clustering at developing rat neuromuscular junctions

Voltage-gated sodium channels (VGSCs) are concentrated in the postsynaptic membrane at the adult rat neuromuscular junction (NMJ). We have used immunolabelling to determine the pattern of initial VGSC accumulation during development. At birth, but not 3 days before, VGSC labelling is detectable at the NMJ and in the perijunctional (periJ) membrane but not elsewhere. A much higher density cluster of VGSCs forms at the NMJ itself 1-2 weeks later. If the nerve is cut 2 days after birth, VGSC labelling persists in the periJ region for at least 4 weeks but the clustering of VGSCs at the NMJ fails to develop. Thus an early, stable accumulation of VGSCs develops near the NMJ at least a week before high density postsynaptic VGSC clusters form.     

Evoked quantal currents at neuromuscular junctions of wild type Drosophila larvae

Malignant infantile osteopetrosis is a rare disease but can be clinically unequivocally diagnosed. Normal bone formation in the presence of decreased bone breakdown leads to the typical symptoms. The only proven curative approach, bone marrow transplantation, can reverse most of the symptoms and prevent progression to irreversible nerve damage when done early in infancy. Therefore, early diagnosis is decisive. We present a case report of an infant with osteopetrosis and discuss pathogenesis and therapeutical options.     

Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.     

New evidence that AV node slow pathway conduction directly influences fast pathway function

INTRODUCTION: Shortening of the AV node fast pathway effective refractory period (ERP) following successful slow pathway ablation may be a nonspecific effect of energy application at the AV junction or may be due to elimination of a direct effect of slow pathway conduction on the fast pathway. METHODS AND RESULTS: Twenty-six consecutive patients (20 women and 6 men; mean age 45 +/- 3 years) with typical AV nodal reentrant tachycardia who underwent successful slow pathway ablation (defined as complete elimination of dual AV node physiology) were studied. The fast pathway ERP (at a drive train cycle length of 600 msec) was determined prior to ablation (baseline) and following unsuccessful and successful ablation attempts. Successful slow pathway ablation shortened the fast pathway ERP significantly (317 +/- 9 msec; P < 0.001) compared to baseline (386 +/- 12 msec), whereas unsuccessful ablations had no effect (376 +/- 11 msec). Sinus cycle length, the AH interval, and blood pressure were unchanged following successful ablation. Shortening of the fast pathway ERP did not correlate with the number of energy applications or with two measures of the proximity between the slow and the fast pathway. CONCLUSION: These results support the hypothesis that shortening of the fast pathway ERP following slow pathway ablation is due to elimination of a direct effect of slow pathway conduction on fast pathway function rather than a nonspecific effect of repeated energy delivery at the AV junction.     

Regulation of presynaptic NMDA responses by external and intracellular pH changes at developing neuromuscular synapses

NMDA receptors play important roles in synaptic plasticity and neuronal development. The functions of NMDA receptors are modulated by many endogenous substances, such as external pH (pHe), as well as second messenger systems. In the present study, the nerve-muscle cocultures of Xenopus embryos were used to investigate the effects of both external and intracellular pH (pHi) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents (SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the SSC frequency via the activation of presynaptic NMDA receptors. A decrease in pHe from 7.6 to 6.6 reduced NMDA responses to 23% of the control, and an increase in pHe from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency. The effect of NMDA on intracellular Ca2+ concentration ([Ca2+]i) was also affected by pHe changes: external acidification inhibited and alkalinization potentiated [Ca2+]i increases induced by NMDA. Intracellular pH changes of single soma were measured by ratio fluorometric method using 2,7-bis (carboxyethyl)-5, 6-carboxyfluorescein (BCECF). Cytosolic acidification was used in which NaCl in Ringer's solution was replaced with weak organic acids. Acetate and propionate but not methylsulfate substitution caused intracellular acidification and potentiated NMDA responses in increasing SSC frequency, intracellular free Ca2+ concentration, and NMDA-induced currents. On the other hand, cytosolic alkalinization with NH4Cl did not significantly affect these NMDA responses. These results suggest that the functions of NMDA receptors are modulated by both pHe and pHi changes, which may occur in some physiological or pathological conditions.     

ATP and adenosine inhibit transmitter release at the frog neuromuscular junction through distinct presynaptic receptors

1. The effects of exogenous ATP or adenosine on end-plate currents (e.p.cs; evoked by simultaneous action of a few hundred quanta of ACh) or on miniature e.p.cs (m.e.p.cs) were studied under voltage clamp conditions on frog sartorius muscle fibres. 2. ATP or adenosine (100 microM(-1) mM) reduced the e.p.c. amplitude but did not affect m.e.p.c. amplitude, decay time constant and voltage-dependence of m.e.p.c., suggesting that e.p.c. depression induced by these purines had presynaptic origin only. 3. The action of ATP, unlike that of adenosine, was prevented by the P2-purinoceptor antagonist suramin (100 microM). The stable ATP analogue alpha,beta-methylene ATP (100 microM), known to be desensitizing agent on P2X receptors, also abolished the depressant effect of ATP while sparing the action of adenosine. Concanavalin A, an inhibitor of ecto-5'-nucleotidase, did not affect the presynaptic action of exogenously applied ATP. 4. The presynaptic action of adenosine was prevented by theophylline (1 mM), a blocker of adenosine receptors, while the effect of ATP was not changed under these conditions. The selective blocker of A1 adenosine receptors, 8-cyclopentyl-1,3,dipropylxanthine (DPCPX; 0.1 microM), abolished the presynaptic action of adenosine but did not prevent the depressant effect of ATP. 5. The effects of ATP and adenosine (at nearly saturating concentration) were additive suggesting that these purines activated not only distinct receptors but also different intracellular signalling mechanisms. 6. In contrast to the hypothesis that at the neuromuscular junction ATP reduces transmitter release via enzymatic degradation to presynaptically active adenosine, our data suggest that ATP (through its own presynaptic receptors) directly inhibits ACh release. Thus, ATP and adenosine might be almost equipotent as endogenous prejunctional neuromodulators at the neuromuscular junction.     

Endurance training increases acetylcholine receptor quantity at neuromuscular junctions of adult rat skeletal muscle

The aim of the study was to test the hypothesis that a 16 week endurance training program would alter the abundance of endplate-associated nicotinic acetylcholine receptors (nAChR) in various rat skeletal muscles. We found a 20% increase in endplate-specific [125I]alpha-bungarotoxin binding in several muscles of trained rats, accompanied by equal susceptibility of toxin binding to the inhibitory effect of D-tubocurarine in sedentary and trained muscles. We conclude that the neuromuscular junction adaptations that occur with increased chronic activation include an increase in nAChR number. Results of experiments designed to determine nAChR turnover also suggest that this effect is mediated by an alteration in the receptor's metabolic state. The potential implications and mechanisms of this adaptation are discussed.     

The influence of skeletal muscle incubation medium on fatigue of neuromuscular preparation and on transmitter release at neuromuscular junctions in the frog

The effect of frog skeletal muscle incubate on fatigue was studied in frog sciatic nerve, sartorius muscle preparation. Fatigue was produced by prolonged repetitive (1 s-1) stimulation of motor nerve or of curarized muscle. The incubate partially restored isometric contraction amplitudes of muscle fatigued by nerve stimulation. This effect of partial recovery from fatigue (PRF effect) was exerted mainly by a relatively low-molecular fraction (LMF; < 10 kDa) of the incubate. The incubate and its fractions failed to produce the PRF effect in experiments with directly stimulated muscle. The action of LMF on synaptic transmission in unfatigued cutaneous-pectoris muscle was examined using binomial analysis of quantal transmitter release. LMF produced an increase in the end-plate potential quantal content (m) at synapses with low initial m values. In contrast, it produced a decrease i n m at synapses with higher m values. Both effects were due to respective changes in binomial parameter n. It is assumed that the stimulatory presynaptic action of the incubate on synapses the effectiveness of which was lowered during fatigue, could account for the PRF effect. A possible contribution of low- and high-molecular components of the incubate is discussed.     

Strength of synaptic transmission at neuromuscular junctions of crustaceans and insects in relation to calcium entry

Crustacean and insect neuromuscular junctions typically include numerous small synapses, each of which usually contains one or more active zones, which possess voltage-sensitive calcium channels and are specialized for release of synaptic vesicles. Strength of transmission (the number of quantal units released per synapse by a nerve impulse) varies greatly among different endings of individual neurons, and from one neuron to another. Ultrastructural features of synapses account for some of the physiological differences at endings of individual neurons. The nerve terminals that release more neurotransmitter per impulse have a higher incidence of synapses with more than one active zone, and this is correlated with more calcium build-up during stimulation. However, comparison of synaptic structure in neurons with different physiological phenotypes indicates no major differences in structure that could account for their different levels of neurotransmitter release per impulse, and release per synapse differs among neurons despite similar calcium build-up in their terminals during stimulation. The evidence indicates differences in calcium sensitivity of the release process among neurons as an aspect of physiological specialization.     

Distribution of neuromuscular junctions in the bowel affected by hypoganglionosis

Using monoclonal antibody 171B5 against synaptic vesicle proteins, neuromuscular junctions and synapses in the muscle layers of the bowel affected by hypoganglionosis were labeled and their distribution analysed. In the hypoganglionic bowel, there were less synapses in the myenteric plexus and few neuromuscular junctions compared to normal bowels. The bowel dysmotility of the patient with hypoganglionosis appears to be due to inadequate innervation between ganglion cells and smooth muscle cells.     

Postnatal development of Schwann cells at neuromuscular junctions, with special reference to synapse elimination

The neuromuscular junctions (NMJs) of postnatal rat soleus muscles were examined by immunohistochemical staining for S100, a marker of Schwann cells (SCs), and for protein gene product 9.5, a neuronal marker, to elucidate the involvement of SCs in synapse elimination. The morphological maturation of S100-immunoreactive terminal SCs at NMJs proceeded with the gradual increase in their number. The number of terminal SCs per NMJ was one or two at postnatal day (P) 7, reaching the adult number at P28, when it became three or four. Confocal laser scanning microscopic analysis of multi-innervated NMJs, whose number decreased between P7 and P14, revealed a change in the ratio between terminal SCs and axons with age. At P7, the ratio between axons and terminal SCs per NMJ was > or = 2:1, which was exactly the reverse of that in adults, while at P14 this had changed to 2:2. A structural change appeared to occur at the same time at the preterminal region, this being prior to the establishment of a 1:1 relationship between axon and SC sheath which was detected at P14, with the > or = 2:1 relationship seeming to occur at P7. Thus, synapse elimination seems to proceed, at least for one week, with the gradual loss of axons which are at different stages of maturation with respect to their spatial relationship with SCs. From our results it seems unlikely that SCs play an active role in selecting a single axon to survive.     

Acetylcholine receptor bars and transmitter release in frog neuromuscular junctions

After labelling with rhodaminated alpha-bungarotoxin, acetylcholine receptors in cutaneous pectoris muscles of normal adult frogs (Rana temporaria) appear as brightly fluorescent straight bars, usually extending over the whole gutter. Here we investigated first whether receptor bars can undergo changes and secondly whether they would provide a structural correlate for the strength of a junction. Bars of low fluorescence intensity, as well as short or discontinuous receptor bars consisting of two or three segments, suggest plasticity at the receptor/active zone level. In order to elucidate this notion, receptor bars were studied at different seasons which have previously been shown to be associated with structural changes. In two groups of frogs kept under laboratory conditions simulating wintertime and summertime, respectively, the length and number of receptor bars and the amount of discontinuous bars were investigated. Synaptic contact length, which is the summed length of labelled synaptic branches, and the number and total length of receptor bars did not differ significantly. A clear difference between Group I ("winter" frogs) and Group II ("summer" frogs) was found in the number of discontinuous bars, which was almost twice as high in Group I compared with Group II (6.4 +/- 3.3% S.D. vs 3.4 +/- 1.3% S.D., n = 8 and 7 muscles, respectively, P < 0.05). In addition, the average length of individual bars was slightly longer in Group I frogs (2.16 +/- 0.7 micron S.D. vs 2.07 +/- 0.12 microns S.D., 0.1 < P < 0.05). Transmitter release has been shown to be different in these two groups--as determined from endplate potential measurements in tubocurarine-containing bathing solutions--although it was equal when measured in low Ca2+/high Mg2+ [Dorl?chter M. et al. (1991) Pflügers Arch. 418, Suppl. 1, R31]. We also investigated whether receptor bars would be a reasonable structural correlate of synaptic function by comparing different measures of transmitters release with different structural parameters in 19 identified junctions. The mean quantal content (m) of a junction was positively correlated with the number and total length of receptor bars, but not with synaptic contact area or length. Amplitudes of the first, maximum, and plateau endplate potentials (corrected for a common resting potential and apparent input resistance) at tetanic nerve stimulation (40 Hz for 2 s) in tubocurarine block were strongly correlated with both synaptic contact length and total receptor bar length (r = 0.90 for maximum endplate potential); correlations between m and any structural measure were significantly worse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that a combined activator-repressor protein regulates Dictyostelium stalk cell differentiation

The ecmA gene is expressed in Dictyostelium prestalk cells and is inducible by differentiation-inducing factor (DIF), a low-molecular-weight lipophilic substance. The ecmB gene is expressed in stalk cells and is under negative control by two repressor elements. Each repressor element contains two copies of the sequence TTGA in an inverted relative orientation. There are activator elements in the ecmA promoter that also contain two TTGA sequences, but in the same relative orientation. Gel retardation assays suggest that the same protein binds to the ecmB repressor and the ecmA activator. We propose that DIF induces prestalk cell differentiation by activating this protein and that the protein also binds to the promoters of stalk-specific genes, acting as a repressor that holds cells in the prestalk state until culmination is triggered.     

Depression of synaptic efficacy at intermolt in crayfish neuromuscular junctions by 20-hydroxyecdysone, a molting hormone

This report demonstrates that ecdysteroids can reduce synaptic transmission at an intermolt stage of a crustacean tonic neuromuscular junction by acting at a presynaptic site. The steroid molting hormone, 20-hydroxyecdysone (20-HE), appears to act through a rapid, nongenomic mechanism that decreases the probability of synaptic vesicle release and reduces the number of release sites. Quantal analysis revealed that fewer vesicles were released for a given stimulus when 20-HE was present, and this in turn accounted for the reduced synaptic efficacy. Reduced synaptic efficacy produced smaller evoked postsynaptic currents and smaller excitatory postsynaptic potentials (EPSPs) across the muscle fiber membrane. The reduction in EPSPs was observed among muscle fibers that were innervated by high- or low-output terminals. The behavior of crustaceans/crayfish during the molt cycle, when 20-HE is high, may be explained by the reduction in synaptic transmission. Crustaceans become quiescent during the premolt periods as do insects. The effects of 20-HE can be reversed with the application of the crustacean neuromodulator serotonin, which enhances synaptic transmission.     

Affect influences false memories at encoding: Evidence from recognition data.

Memory is susceptible to illusions in the form of false memories. Prior research found, however, that sad moods reduce false memories. The current experiment had two goals: (1) to determine whether affect influences retrieval processes, and (2) to determine whether affect influences the strength and the persistence of false memories. Happy or sad moods were induced either before or after learning word lists designed to produce false memories. Control groups did not experience a mood induction. We found that sad moods reduced false memories only when induced before learning. Signal detection analyses confirmed that sad moods induced prior to learning reduced activation of nonpresented critical lures suggesting that they came to mind less often. Affective states, however, did not influence retrieval effects. We conclude that negative affective states promote item-specific processing, which reduces false memories in a similar way as using an explicitly guided cognitive control strategy. (PsycINFO Database Record (c) 2011 APA, all rights reserved)     

Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, we have used this preparation to investigate the role of NO. We examined LTP following intra- or extracellular application of an NO scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.     

Evidence that 3-aminopicolinate stimulates glutamine metabolism by rat renal cortical tissue in vitro

The hyperglycemic agent 3-aminopicolinate used at concentrations of 0.05 and 0.1 mM stimulates the removal of 5 mM glutamine. It also stimulates the accumulation of glutamate and the formation of ammonia, glucose and aspartate by isolated rat kidney-cortex tubules. These effects are consistent with a stimulation of glutaminase and an inhibition of phosphoenolpyruvate carboxy-kinase by this compound. Higher concentrations (0.5 and 1 mM) of 3-aminopicolinate fail to affect the removal of 5 mM glutamine, but greatly alter the fate of both glutamine carbon and amino nitrogen. Similar effects of 3-aminopicolinate are observed when glutamine is used at a near-physiological concentration.