Facilitation at the sexually differentiated laryngeal synapse of Xenopus laevis

Under physiological conditions, the laryngeal synapse of male Xenopus laevis exhibits marked facilitation during repetitive nerve stimulation. The male laryngeal synapse is weak and requires facilitation to produce muscle action potentials and ultimately sound. The female laryngeal synapse is strong: muscle contractions are produced to single nerve stimuli. We sought to determine if laryngeal synapses of males and females also differ in their ability to facilitate. To measure facilitation, laryngeal muscle action potentials were suppressed either postsynaptically by bathing the preparation in saline containing curare or presynaptically by bathing the preparation in reduced calcium/elevated magnesium saline. Facilitation of postsynaptic potential amplitude or quantal content in response to paired pulses was measured in male and female larynges: there is no sex difference in paired pulse facilitation. Facilitation in response to trains of stimuli, in curare-blocked preparations, increased and reached plateau values more rapidly in females than in males, although the facilitation between the last and first pulses in the train was the same in the sexes. Thus, the sexually differentiated behavior of this synapse is controlled more by a sex difference in synaptic strength than by a sex difference in the ability to facilitate. Accepted: 14 June 1997     

Attaining and maintaining strong vocal synapses in female Xenopus laevis

Synaptic efficacy at the laryngeal neuromuscular synapse differs markedly in adult male and female Xenopus laevis. Here, we examined the relation between circulating estrogen and synapse strength in developing and adult female frogs. Circulating estrogen levels in males and females during juvenile and adult stages were measured using radioimmunoassays. Synaptic strength was determined by quantal analysis in isolated female larynges. In males, estrogen levels are low (<40 pg/mL) throughout development. In females, estrogen levels are similar to those in males until 9 months after metamorphosis is complete and then increase throughout development. Female laryngeal synapses have low quantal contents until 24 months; quantal content increases significantly between 24 and 26 months, and high quantal contents are maintained thereafter. Measures of reproductive maturation, ovary, and oviduct weights, are strongly and positively correlated with estrogen level in 16- to 26-month females, while oocyte maturation is age dependent. Estrogen level and quantal content are not well correlated in these females. Ovariectomy at 24 months prevents the expected increase in quantal content and ovariectomy at 28 months results in a decrease in quantal content. Thus, the sex difference in efficacy of the laryngeal synapse develops under the influence of the ovary and requires the ovary for maintenance of strong synapses in adulthood. While the influence of the ovary is most likely due to estrogen secretion, the pattern of estrogen secretion required for maturation of the synapse in females is not known. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 441–448, 1998     

Sex differences in play behavior in juvenile tufted capuchin monkeys (Cebus apella)

According to the motor training hypothesis, play behavior in juvenile primates improves motor skills that are required in later adult life. Sex differences in juvenile play behavior can therefore be expected when adult animals assume distinct sexually dimorphic roles. Tufted capuchin monkeys show sexually dimorphic levels of physical antagonism in both inter- and intra-group encounters. Accordingly, it can be predicted that juvenile capuchins also show sex differences in social play behavior. To test this hypothesis, the play behavior of nine juvenile and two infant capuchins was examined. As predicted, juvenile males showed significantly higher levels of social play (wrestle, chase) than juvenile females, but no differences were found in nonsocial play (arboreal, object). Levels of infant play behavior were comparable to that of juveniles. These results lend support to the motor training hypothesis and highlight the need for more detailed investigations of individual differences in play behavior. An erratum to this article can be found at     

Nonhomogeneous excitatory synapses of a crab stomach muscle

Neuromuscular synapses of pyloric muscle P1 in the blue crab Callinectes sapidus were examined using electrophysiological and electron microscopic methods. The muscle is innervated by a single excitatory axon of the stomatogastric ganglion. Excitatory postsynaptic potentials show striking facilitation at very low frequencies of stimulation, indicating very slow decay of the facilitation process after a single nerve impulse. Quantal content of transmitter release at a low frequency of stimulation averaged 1.5. Evidence was obtained that not all synapses on a muscle fiber are equivalent. This was particularly evident at the morphological level in serially sectioned nerve terminals. On each nerve terminal examined, a wide range of synapse sizes was found. Synaptic contact areas ranged from less than 0.5 micron2 to almost 10 micron2; the latter value is large compared with those obtained for other crustacean neuromuscular synapses. Most of the smaller synapses lacked the presynaptic dense bodies which are putative release sites for the transmitter substance. The larger synapses all had presynaptic dense bodies, and some showed evidence of splitting apart into smaller subunits. It is postulated that about half the morphologically identified synapses are relatively inactive.     

Fast-axon synapses of a crab leg muscle

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.     

Strength of synaptic transmission at neuromuscular junction 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.     

Ultrastructure of synapses with different transmitter-releasing characteristics on motor axon terminals of a crab,Hyas areneas

Synaptic terminals on branches of an excitatory motor axon in a spider crab (Hyas areneas) were examined by electron microscopy to determine whether differences in size, structure, and number of synapses could be correlated with differences in transmitter release. Terminals releasing relatively large amounts of transmitter during low frequencies of nerve impulses ("high-output" terminals) had larger synapses, more prominent presynaptic dense bodies (active zones), and fewer synapses per unit length than terminals releasing relatively small amounts of transmitter ("low-output" terminals). Neither the difference in synaptic area, nor the quantitative differences in the active zones, were sufficient in themselves to explain the difference in synaptic efficacy, and it is postulated that a non-linear relationship may exist between structural features of the synapse and release of transmitter by a nerve impulse, and that differences other than those apparent from the ultrastructure could be involved. Greater facilitation at low-output terminals with high frequencies of nerve impulses may be due to greater reserves of "immediately available" transmitter, and to recruitment or activation of more individual synaptic contacts.     

Growth of inhibitory innervation in a lobster muscle

The fine structure of inhibitory innervation to a limb muscle was examined in larval, juvenile, and adult lobsters. The innervation is essentially similar in qualitative features among these different stages, although there are some marked quantitative changes associated with growth. From being localized to discrete regions in the larval muscle, the inhibitory innervation spreads to groups of muscle fibers in the early juvenile muscle and to single fibers in the late juvenile and adult muscles. Concurrently, its neuromuscular synapses enlarge in area, become perforated, and acquire more active sites of transmitter release. Inhibitory nerve terminals occur in close proximity to their excitatory counterparts in the muscles of larval and early juvenile stages, although in later stages this juxtaposition occurs preferentially in some muscle fibers but not others. The inhibitory innervation is, nevertheless, much more restricted in occurrence than is the excitatory innervation.     

Differential Responses of Crab Neuromuscular Synapses to Cesium Ion

Excitatory postsynaptic potentials (EPSP's) generated in crab muscle fibers by a single motor axon, differ in amplitude and facilitation. Some EPSP's are large at low frequencies of stimulation and show little facilitation; others are smaller and show pronounced facilitation. When K⁺ is replaced by Cs⁺ in the physiological solution, all EPSP's increase in amplitude, but small EPSP's increase proportionately more than large ones. Quantal content of transmission, determined by external recording at single synaptic regions, undergoes a much larger increase at facilitating synapses. The increase in quantal content of transmission is attributable to prolongation of the nerve terminal action potential in Cs⁺. After 1–2 h of Cs⁺ treatment, defacilitation of synaptic potentials occurs at synapses which initially showed facilitation. This indicates that Cs⁺ treatment drastically increases the fraction of the "immediately available" transmitter store released by each nerve impulse, especially at terminals with facilitating synapses. It is proposed that facilitating synapses normally release less of the "immediately available" store of transmitter than poorly facilitating synapses. Possible reasons for this difference in performance are discussed.     

The molecular mechanisms of quantum mediator secretion in the synapse

The modern condition of knowledge about the molecular mechanisms underlying the quantal transmitter release in the central and the peripheric synapses is analysed. The data about the synaptic vesicles types, their forming, transporting to the sites of release at the nerve endings, exo- and endocytosis processes are presented. Ultrastructural and molecular organization of active zone of nerve ending and transmitter release morphofunctional unit--secretosome, which includes synaptic vesicle, exocytosis protein complex and calcium channels, are described. The basic proteins involved in the exo- and endocytosis and their interactions during transmitter release are examined. The role of the intracellular buffer systems, calcium micro- and macrodomains in the quantal transmitter secretion are considered. The reasons of the active zones functional non-uniformity and plasticity and factors reduced transmitter release in the active zone to the single quantum are analysed.     

A Monte Carlo model reveals independent signaling at central glutamatergic synapses

We have developed a biophysically realistic model of receptor activation at an idealized central glutamatergic synapse that uses Monte Carlo techniques to simulate the stochastic nature of transmission following release of a single synaptic vesicle. For the a synapse with 80 AMPA and 20 NMDA receptors, a single quantum, with 3000 glutamate molecules, opened approximately 3 NMDARs and 20 AMPARs. The number of open receptors varied directly with the total number of receptors, and the fraction of open receptors did not depend on the ratio of co-localized AMPARs and NMDARs. Variability decreased with increases in either total receptor number or quantal size, and differences between the variability of AMPAR and NMDAR responses were due solely to unequal numbers of receptors at the synapse. Despite NMDARs having a much higher affinity for glutamate than AMPARs, quantal release resulted in similar occupancy levels in both receptor types. Receptor activation increased with number of transmitter molecules released or total receptor number, whereas occupancy levels were only dependent on quantal size. Tortuous diffusion spaces reduced the extent of spillover and the activation of extrasynaptic receptors. These results support the conclusion that signaling is spatially independent within and between central glutamatergic synapses.     

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.     

Aberrant morphology and residual transmitter release at the Munc13-deficient mouse neuromuscular synapse

In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and gamma-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.     

Active zones and receptor surfaces of freeze-fractured crayfish phasic and tonic motor synapses

Deep and superficial flexor muscles in the crayfish abdomen are innervated respectively by small populations of physiologically distinct phasic and tonic motoneurons. Phasic motoneurons typically produce large EPSP's, releasing 100 to 1000 times more transmitter per synapse than their tonic counterparts, and exhibiting more rapid synaptic depression with maintained stimulation. Freeze-fracturing the abdominal flexor muscles yielded images of phasic and tonic synapse-bearing terminals. The two types of synapse are qualitatively similar in ultrastructure, displaying on the presynaptic membrane's P-face synaptic contacts recognized by relatively particle-free oval plaques which are often framed by the muscle fiber's E-face leaflet with its associated receptor particles. Situated within these presynaptic plaques are discrete clusters of large intramembrane particles, forming active zone (AZ) sites specialized for transmitter release. AZs of phasic and tonic synapses are similar: 80% had a range of 15–40 large particles distributed in either paired spherical clusters or in linear form, with a few depressions denoting sites of synaptic vesicle fusion or retrieval around their perimeters. The packing density of particles is similar for phasic and tonic AZs. The E-face of the muscle membrane displays oval-shaped receptor-containing sites made up of tightly packed intramembranous particles. Phasic and tonic receptor particles are packed at similar densities and the measured values resemble those of several other crustacean and insect neuromuscular junctions. Overall, the similarity between phasic and tonic synapses in the packing density of particles at their presynaptic AZs and postsynaptic receptor surfaces suggests similar regulatory mechanisms for channel insertion and spacing. Furthermore, the findings suggest that morphological differences in active zones or receptor surfaces cannot account for large differences in transmitter release per synapse.     

Transmitter release in proximal and distal parts of a nerve terminal of the frog sartorius muscle

Evoked synaptic potential were recorded extracellularly in experiments on a nervemuscle preparation of the frog sartorius muscle. A decrease in evoked transmitter release was found from the proximal to the distal parts of the nerve ending, due to a decrease in the probability of transmitter quantum release. The terminal portions of the synapse are less sensitive than the proximal parts to changes in Ca⁺⁺ concentration, they show less marked facilitation of transmitter release during paired and repetitive stimulation, and exhibit deeper and more rapidly developing depression. It is concluded that differences in transmitter release in the terminal parts of the synapse are due to the low reserves of transmitter and the lower premeability of the presynaptic membrane to Ca⁺⁺.     

Fine structure of comparable synapses in a mature and larval lobster muscle

Neuromuscular synapses from the single excitor axon to the proximal accessory flexor muscle (PAFM) was studied by serial section electron microscopy in a 1st stage larval (< 0.1 g) and a large adult (6.8 kg) lobster. The adult innervation of a lateral and a medial fiber, physiologically identified as low- and high-output respectively, was similar in the number and mean size of synapses but had significantly larger pre-synaptic dense bars for the high-output synapses. This correlation between quantal transmitter output and pre-synaptic dense bars and the appearance of exocytotic profiles along the dense bars strongly implicates the bars as active sites of transmitter release. Moreover the mature innervation is differentiated on the basis that the percentage of dense bar area to synaptic area is 9% for the low-output type compared to 22% for its high-output counterpart. In the larval PAFM the excitatory axon has not proliferated many branches and the innervation is therefore localized to groups of fibers in the lateral, medial and central regions of the muscle rather than to individual fibers. The lateral and medial sites of innervation representing putative low- and high-output types respectively (because of their location) do not differ in the size and number of pre-synaptic dense bars thereby suggesting a similarity in quantal synaptic transmission. However the percentage of dense bar area to synaptic area is 40% for the lateral site compared to 67% for the medial site. Since this is a trend mimicking the mature innervation it shows an early stage in the differentiation of low-and high-output synapses. Furthermore the main axon provides half of the total innervation in the larval PAFM but none in the adult thereby demonstrating a restructuring of multiterminal innervation.     

Neuroeffectors for vocalization in Xenopus laevis: hormonal regulation of sexual dimorphism

South African clawed frogs use sex-specific vocalizations during courtship. In the male, vocalizations are under the control of gonadal androgen. Though females have moderate levels of circulating androgen, they do not give male-typical mate calls. Both muscles of the vocal organ and neurons of the central nervous system (CNS) vocal pathway are sexually dimorphic and androgen-sensitive. Recent studies suggest that the failure of androgen to masculinize adult females results from a male-specific, androgen-regulated developmental program. At metamorphosis the larynx is sexually monomorphic and feminine in morphology, muscle fiber number and androgen receptor content. During the next six months, under the influence of increasing androgen titers and high receptor levels, myoblasts proliferate in the male and muscle fibers increase at an average rate of 100/day. Females have much lower hormone levels, receptor values decline and they display no net addition of fibers. At metamorphosis, both males and females have approximately 4000 muscle fibers. By adulthood, males have eight times the female fiber number. In the CNS, adult laryngeal motor neurons are more numerous with larger somata and dendritic trees in males than in females. Certain connections of neurons in the vocal pathway are also less robust in females. Unlike the periphery, motor neuron number does not appear to be established by androgen-induced proliferation. Our current hypothesis is that androgen acts at the level of laryngeal muscle to produce more muscle fibers and thus provide more target for motor neurons in the male. This process could regulate cell number by ontogenetic cell death. In the CNS, androgen-target neurons become capable of accumulating hormone shortly before metamorphosis. Androgen receptor in laryngeal motor neurons may permit the dendritic growth characteristic of males by increasing sensitivity to afferent stimuli. Such a process could account for the observed differences in CNS vocal "circuitry" in X. laevis and thus behavioral differences between the sexes.     

Androgen regulation of neuromuscular junction structure and function in a sexually dimorphic muscle of the frog Xenopus laevis

Specific forelimb muscles in anurans are sexually dimorphic and underlie the androgen-dependent clasping response of males during amplexus. Previous studies have reported that androgen treatment slows the contractile properties of these sexually dimorphic forelimb muscles. In amphibians, the expression of functionally distinct acetycholine (ACh) receptors, the levels of acetylcholinesterase (AChE), the extent of multiple innervation, and the structure of individual end plates vary with the contractile properties of the muscle fibers. In higher vertebrates, androgens have been reported to alter the expression of ACh receptors, AChE, and the neuromodulator, calcitonin gene-related peptide (CGRP). To determine whether the known androgen-dependent changes in contraction of androgen-sensitive forelimb muscles are accompanied by concomitant changes in synaptic structure or function, we have compared functional neuromuscular transmission, the pattern of innervation, and CGRP immunoreactivity in nerve or muscle preparation from castrated (C) and castrated and testosterone-treated (CT) adult male Xenopus laevis. CGRP expression in androgen receptor (AR)-immunopositive neurons was increased in CT animals. However, no significant differences were found in ACh-mediated single channel or macroscopic currents, the extent of multiple end plates, or end plate morphology for forelimb fibers isolated from C and CT Xenopus. In contrast, analysis of forelimb fibers from gonadally intact adult females and juvenile animals of both sexes revealed that macroscopic synaptic currents were significantly shorter in these animals than in either C or CT adult males. Our data suggest that forelimb fibers in sexually dimorphic muscles of Xenopus do show significant differences in synaptic transmission; however, neither end-plate organization nor functional neuromuscular transmission are subject to activational effects of androgens in adult male frogs. © 1995 John Wiley & Sons, Inc.     

Sexual dimorphism and species differences in the neurophysiology and morphology of the acoustic communication system of two neotropical hylids

We examined auditory tuning and the morphology of the anatomical structures underlying acoustic communication in female Hyla microcephala and H. ebraccata and compared our findings to data from a previous study (Wilczynski et al. 1993) in which we showed species differences in the traits that in males relate to differences in the species-typical calls. Female species differences in the best excitatory frequency (BEF) of the basilar papilla (BP) were similar to the differences seen in males, and females had a significantly lower BEF in H. ebraccata, but not H. microcephala. In both species, females had lower BP thresholds. Snout-vent length, head width, and tympanic membrane diameters were sexually dimorphic in both species and larger in females, whereas laryngeal components were sexually dimorphic and larger in males. Middle and inner ear volumes were not sexually dimorphic. Despite the significant species differences in laryngeal morphology seen in males, female larynges are not significantly different. Furthermore, the interaction of species and sex differences resulted in significantly different degrees of sex dimorphism in the species, particularly for the larynx, which is more sexually dimorphic in H. microcephala, and measures of body size, which are more dimorphic in H. ebraccata. Accepted: 6 December 1996     

Quantitative comparison of low- and high-output neuromuscular synapses from a motoneuron of the lobster (Homarus americanus)

Summary Representative examples of lowand high-output neuromuscular synapses between motoneuron and distal accessory flexor muscle of the lobster were selected on the basis of their mean quantal content, and subsequently analysed by serial section electron microscopy. The high-output terminal has twice as many synapses as the low-output terminal. However, since the mean surface area of synapses is significantly smaller in the high-output terminal than in the low-output one, the total synaptic surface area between the two types of terminals is similar. Also, though the high-output terminal possesses a greater number of presynaptic dense bodies than its low-output counterpart, the mean number per synapse is similar for the two terminals. The terminals, however, differ significantly in the size of their dense bodies. Thus both the mean and total surface area of these bodies is greater in the high-output terminal than in the low-output one. Moreover, the mean ratio of dense body area to synaptic area is significantly greater for the high-output terminal than for its low-output counterpart. This difference in dense body area parallels the difference in quantal content of synaptic transmission between the lowand high-output terminals and supports the hypothesis that presynaptic densities represent the ultrastructural correlates of transmitter mobilization and/or release.Supported by grants from the National Research Council and Muscular Dystrophy Association of Canada to C.K. Govind. D.E. Meiss is a post-doctoral fellow of the Muscular Dystrophy Association of Canada. We thank Eva Yap-Chung for her expert and unfailing technical assistance