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.

Regenerating crayfish motor axons assimilate glial cells and sprout in cultured explants.

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2-3-day-old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial-section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve. Both internal and external sprouts have a simple construction: a cytoskeleton of microtubules and populations of mitochondria, clear synaptic vesicles, membranous sacs, and extrasynaptic active zone dense bars, features reminiscent of motor nerve terminals. Close intermingling of the sprouts of several axons give rise to a neuropil-like arbor within the nerve. Thus, extensive sprouting is an intrinsic response of crayfish motor axons to transection. Second, an equally dramatic remodeling feature is the appearance of nuclei, which resemble those of adjacent glial cells, within the motor axons. These nuclei often appear where the adjoining membranes of the axon and glial cell are disrupted and where free-standing lengths of the double membrane are present. These images signify a breakdown of the dividing membranes and assimilation of the glial cell by the axon, the nucleus being the most visible sign of such assimilation. Thus, crayfish motor axons respond to transection by assimilating glial cells that may provide regulatory and trophic support for the sprouting response.

Allotransplanted nerves regenerate inhibitory synapses on a crayfish muscle: Possible postsynaptic specification.

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.

Remodeling of the proximal segment of crayfish motor nerves following transection.

Transected crustacean motor axons consist of a soma-endowed proximal segment that regenerates and a soma-less distal segment that survives for up to a year. We report on the anatomical remodeling of the proximal segment of phasic motor nerves innervating the deep flexor muscles in the abdomen of adult crayfish following transection. The intact nerve with 10 phasic axons and its two branches with subsets of 6 and 7 of these 10 axons undergo several remodeling changes. First, the transected nerve displays many more and smaller axon profiles than the 6 and 7 axons of the intact nerve, approximately 100 and 300 profiles in the two branches of a preparation transected 8 weeks previously. Serial images of the transected nerve denote that the proliferation of profiles is due to several orders of axon sprouting primary, secondary, and tertiary branches. The greater proliferation of axon sprouts, their smaller size, and the absence of intervening glia in the one nerve branch compared with the other branch denote that sprouting is more advanced in this branch. Second, the axon sprouts are regionally differentiated; thus, although in most regions the sprouts are basically axon-like, with a cytoskeleton of microtubules and peripheral mitochondria, in some regions they appear nerve terminal-like and are characterized by numerous clear synaptic vesicles, a few dense-core vesicles, and dispersed mitochondria. Both regions possess active zone dense bars with clustered synaptic vesicles found opposite other sprouts, glia, hemocytes, and connective tissue, but because the opposing membranes are not differentiated into a synaptic contact, the active zones are extrasynaptic. Third, some of the transected axons display a glial cell nucleus denoting assimilation of an adaxonal glial cell by the transected axons. Fourth, within the nerve trunk are a few myocytes and muscle fibers. These most likely originate from adjoining and intimately connected hemocytes, because such transformation occurs during muscle repair. In a crustacean nerve, however, where muscle is clearly misplaced, its presence implies an instructive role for motor nerves in muscle formation.

Structural definition of the neuromuscular system in the swimming-paddle opener muscle of blue crabs.

Blue crabs are excellent swimmers, using their highly modified last pereiopods as sculling paddles. Hence, the hypertrophied paddle opener muscle was examined for adaptations of its motor innervation by an excitor and a specific inhibitor axon. The muscle has a uniform composition of slow fibers with long (6-12 microm) sarcomere lengths. Individual fibers are richly innervated with approximately two-thirds excitatory and one-third inhibitory innervation. The profuse excitatory innervation reflects the high activity levels of this motoneuron in swimming. Adaptation to sustained activity associated with swimming is also reflected in the motor nerve terminals by a high concentration of energy source, which is equally divided between glycogen granules and mitochondria, the former providing a more rapid source of energy. The excitor axon makes predominantly neuromuscular synapses, but also a few synapses onto the inhibitor axon. The location of these excitatory axoaxonal synapses suggests regional modulation of the inhibitor axon. The specific inhibitor axon makes less than two-thirds of its synapses with the muscle fiber, regulating contraction via postsynaptic inhibition. The remaining inhibitory synapses are onto the excitor axon, signaling very strong presynaptic inhibition. Such presynaptic inhibition will effectively decouple the opener muscle from the stretcher muscle even though both are innervated by a single excitor axon.

Regeneration of phasic synapses on a crayfish slow muscle following allotransplantation of a mixed phasic-tonic nerve.

Separate phasic or tonic nerves allotransplanted to reinnervate a denervated slow superficial flexor muscle (SFM) in the abdomen of adult crayfish regenerate synaptic nerve terminals with phasic or tonic properties. To test competitive interactions between tonic and phasic axons, we allotransplanted the sixth abdominal ganglion with its third nerve root containing a mixture of phasic and tonic axons onto the denervated SFM. The resulting reinnervation of the SFM was compared to the normal innervation on the contralateral intact SFM, which receives innervation only from tonic motoneurons. Variable sizes of excitatory postsynaptic potentials indicated that 2-3 axons innervated each muscle fiber of the SFM in both the allotransplant and normal preparations. Compared to the normal tonic terminals on the intact contralateral side, the allotransplanted synaptic terminals had more phasic-like properties; specifically, they gave rise to larger synaptic potentials, had a lower mitochondrial content and contained a higher density of active zone dense bars per synapse. Moreover, prolific sprouting of the axons in the regenerated nerve, typical of phasic axons, points to more vigorous regeneration of phasic rather than tonic axons to the denervated SFM. In keeping with this prolific axon sprouting, there was both a much higher density of innervation in the allotransplanted SFM compared to the normal SFM, and a higher frequency of extrasynaptic active zones in regenerated terminals of the mixed nerve compared to those of the tonic nerve. Thus, an allotransplanted mixed nerve regenerates mainly phasic axons and synapses on the slow denervated SFM, demonstrating the instructive nature of the neuron in synapse specification, as well as the permissive nature of the target muscle.

Dichotomy in phasic-tonic neuromuscular structure of crayfish inhibitory axons.

Crustacean muscles are unique in their innervation by both excitatory and inhibitory neurons; therefore, they exhibit polyneuronal and multiterminal innervation. Because excitatory motoneurons are broadly divided into phasic and tonic types, we hypothesized that inhibitory neurons would follow a similar dichotomy. The abdominal extensor muscles in crayfish are separated into parallel deep and superficial bundles; the former has fast muscle fibers innervated by phasic excitatory motoneurons, and the latter has slow fibers supplied by tonic excitatory motoneurons. Each muscle also is innervated by a single, separate inhibitory neuron that uses gamma-aminobutyric acid (GABA) as the inhibitory neurotransmitter. The pattern of axonal branching by the separate inhibitory axons in phasic and tonic abdominal extensor muscles was visualized with confocal microscopy in preparations labeled for GABA-like immunoreactivity. Initial observations indicated that the phasic muscle was covered by extensive GABAergic, filiform axon terminals, whereas innervation of the tonic muscle was comprised of more localized and varicose terminals. With quantitative analyses, we found that the phasic axon has a more highly branched nature than the tonic in first- and second-order branches. The phasic axon branches also were significantly longer than the tonic branches in the second- and third-order branches. Synaptic varicosities in the phasic branches were smaller and less frequent than those in the tonic branches. The fine structure of the inhibitory nerve terminals near synaptic contacts examined with thin-serial-section electron microscopy revealed distinct differences between the phasic system and the tonic system. The phasic terminals were smaller in cross-sectional area than the tonic terminals, and they had smaller synapses and fewer mitochondria. The presynaptic active zone dense bodies were similar in length and number between phasic and tonic synapses. However, their number per synaptic area was two-fold higher in phasic synapses compared with tonic synapses because of the smaller size of the phasic synapses. Thus, within the same neuromuscular system, inhibitory synaptic terminals revealed unique phasic and tonic identities similar to those observed for the excitatory axons.

Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa.

Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N:-terminal signal sequence and the C:-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both alpha helical and beta sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization.

Synaptic differentiation between two phasic motoneurons to a crayfish fast muscle.

Synaptic differentiation among crustacean phasic motoneurons was investigated by characterizing the synaptic output and nerve terminal morphology of the two axons to the adductor exopodite muscle in the crayfish uropod. The muscle is of the fast type with short sarcomeres (2-3 micro m) and a low thin to thick filament number (6:1). On single muscle fibers, excitatory postsynaptic potentials generated by the large-diameter axon are significantly larger than those by the small-diameter axon suggesting a presynaptic origin for these differences. Nerve terminals arising from these two axons have typical phasic features, filiform shape and a low (6-8%) mitochondrial density. Synaptic contacts are similar in size between the two axons as is the length and number of active zone dense bars at these synapses. The large-diameter axon, however, exhibits a twofold larger area of nerve terminal than the small-diameter axon resulting in a higher density of synapses per muscle fiber. Hence, differences in synaptic density may in part account for differences in synaptic output between these paired phasic axons.

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons.

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.

Neurotransmitter levels and synaptic strength at the Drosophila larval neuromuscular junction are not altered by mutation in the sluggish-A gene, which encodes proline oxidase and affects adult locomotion.

The sluggish-A (slgA) gene of Drosophila melanogaster has been shown to encode for the enzyme proline oxidase, a mitochondrial enzyme which catalyzes the first step in the conversion of L-proline to L-glutamate. The slgA transcript is expressed in both larval and adult Drosophila melanogaster. Mutations in this gene lead to reduced proline oxidase activity and an elevation of free proline levels. Adult mutant flies show a striking reduction of motor activity. Since proline oxidase may contribute to the supply of the neurotransmitter glutamate in the nervous system, a reduction in proline oxidase activity could reduce neural glutamate pools and affect synaptic transmission in neurons utilizing glutamate as a transmitter, including peripheral motor neurons. We tested the hypothesis that glutamate, and synaptic transmission mediated by glutamate, are reduced at synapses of glutamatergic motor neurons in slgA mutants. Levels of glutamate and proline in different cell compartments, and functional properties of synaptic transmission were compared in slgA and control specimens. Proline is elevated in muscle cells of slgA mutants, indicating that the slgA gene regulates tissue proline levels. In nerve terminal varicosities, proline levels were low in both mutants and controls. Glutamate levels in nerve terminal varicosities of slgA mutants and controls were similar. In addition, we found that glutamatergic synaptic transmission at individual nerve endings and at the whole-cell level was similar in slgA mutants and controls. Thus, proline oxidase does not play a major role in generating neuronal glutamate pools at the Drosophila larval neuromuscular junction, and larval neuromuscular performance is not altered significantly in slgA mutants. Metabolic pathways other than that involving proline oxidase are able to sustain glutamatergic synaptic function in Drosophila larvae.

Crab stomach pyloric muscles display not only excitatory but inhibitory and neuromodulatory nerve terminals.

Movements of the foregut in crustaceans are produced by striated muscles that are innervated by motor neurons in the stomatogastric ganglion (STG). Firing of the STG motor neurons generates excitatory junctional potentials (EJPs) in the stomach muscles. We now provide evidence for the existence of separate inhibitory and neuromodulatory innervations of some pyloric muscles in the foregut of several crabs, Callinectes sapidus, Cancer magister, and Cancer borealis. Electron microscopic examination of several pyloric muscles revealed three distinct types of nerve terminals. Excitatory terminals were readily identified by the spherical shape of their small, clear synaptic vesicles. These terminals also housed a few large dense core vesicles. Inhibitory nerve terminals were recognized by the elliptical shape of their small, clear synaptic vesicles, and contacted the muscles at well-defined synapses equipped with dense bar active zones. Bath application of GABA reduced the amplitudes of EJPs in a pyloric muscle of C. borealis, consistent with the presence of GABAergic inhibitory innervation. Neuromodulatory terminals were characterized by their predominant population of large dense and dense core vesicles. These terminals formed synapses with presynaptic dense bars on the muscle, as well as on the excitatory and inhibitory nerve terminals. The presence of the inhibitory and neuromodulatory terminals creates a functional context for previously described reports of neuromodulatory actions on stomach muscles and suggests that the transfer function from STG motor patterns to pyloric movement may be orchestrated by a complex innervation from sources outside of the STG itself.

Failure of female baboons (Papio anubis) to conceive following immunization with recombinant non-human primate zona pellucida glycoprotein-B expressed in Escherichia coli.

Progress in the development of an immunocontraceptive vaccine based on zona pellucida glycoproteins has been hampered due to observed ovarian dysfunction associated with immunization using these as immunogens. In this study four female baboons (Papio anubis) were immunized with recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (r-bmZPB) expressed in Escherichia coli and conjugated to diphtheria toxoid (DT) using Arlacel-A and Squalene as adjuvants. All the immunized animals elicited a good antibody response against r-bmZPB, continued to have ovulatory cycles and showed no disturbance in the cyclicity. In presence of high titres of circulating anti-bmZPB antibodies (>2x10(3) antibody units), the immunized animals failed to conceive following mating with males of proven fertility. Pregnancy was observed in the immunized animals subsequent to the decline in anti-r-bmZPB antibody titres. These results, though preliminary, suggest that immunization with ZPB may be used for immunocontraception without obvious ovarian dysfunction.

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Anatomy and physiology of neurons composing the commissural ring nerve of the cricket, Acheta domesticus.

The commissural ring nerve (RN) of the cricket Acheta domesticus links together the two cercal motor nerves of the terminal abdominal ganglion. It contains the axons of almost 100 neurons including two bilateral clusters of eight to 13 ventrolateral neurons and approximately 75 neurons with midline somata within the terminal abdominal ganglion. The ventrolateral neurons have an ipsilateral dendritic arborization within the dorsal neuropil of the ganglion and their axons use the RN as a commissure in order to enter the contralateral nerves of the tenth ganglionic neuromere. In contrast, most midline neurons have bifurcating axons projecting bilaterally into the neuropil of the ganglion as well as into the RN where they often branch extensively before entering the contralateral tenth nerves. Most RN neurons have small, non-spiking somata with spike initiation zones distant from the soma. Many midline neurons also produce double-peaked spikes in their somata, indicative of multiple spike initiation zones. Spontaneous neuronal activity recorded extracellularly from the RN reveals several units, some with variable firing patterns, but none responding to sensory stimuli. The RN is primarily composed of small (50 nm diameter) axon profiles with a few large (0.5-1 microm diameter) profiles. Occasionally, profiles of nerve terminals containing primarily small clear vesicles and a few large dense vesicles are observed. These vesicles can sometimes be clustered about an active zone. We conclude that the primary function of the RN is to serve as a peripheral nerve commissure and that its role as a neurohemal organ is negligible. J. Exp. Zool. 286:350-366, 2000.

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding.

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.

Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses.

Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release.

Visible evidence for differences in synaptic effectiveness with activity-dependent vesicular uptake and release of FM1-43.

Activity-dependent uptake and release of the fluorescent probe FM1-43 were used to compare synaptic performance (rates of transmitter release and synaptic vesicle turnover) at different frequencies in phasic and tonic motor neurons innervating the crayfish leg extensor muscle and in the tonic motor neuron of the opener muscle. The phasic extensor motor neuron, which has a high quantal content of transmitter release, accumulated and released FM1-43 more rapidly than the tonic motor neuron, especially at low frequencies of stimulation. Individual bright spots appeared on the varicosities of the junctional terminals during stimulation in FM1-43; these spots corresponded to zones of immunostaining for the synaptic vesicle associated protein synaptotagmin, but they were larger and less numerous than synapses identified by electron microscopy and appear to represent one to several synapses with their associated clusters of synaptic vesicles. The number of bright spots observed on varicosities of the tonic terminal after stimulation at >/=20 Hz is generally similar to values for responding units (n) calculated from binomial distributions derived from quantal analysis. At frequencies of

Human zona pellucida glycoproteins: characterization using antibodies against recombinant non-human primate ZP1, ZP2 and ZP3.

Characterization and classification of human zona pellucida glycoproteins is essential to understand the functions of these components during fertilization. To achieve this, antibodies were raised in rabbits against recombinant non-human primate [Bonnet Monkey (Macaca radiata)] zona pellucida proteins, bmZP1, bmZP2 and bmZP3 expressed in Escherichia coli. Antibodies against the three recombinant zona proteins reacted with human zonae as revealed by indirect immunofluorescence. Such antibodies were used as specific probes to further characterize human zona pellucida glycoproteins in Western blot of heat solubilized human zonae pellucidae (hSIZP) resolved by one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Under non-reduced conditions human (h) hZP1, hZP2 and hZP3 resolved as 60, 100 and 53 kDa bands respectively. Under reduced conditions, dominant reactivity of hZP1, hZP2 and hZP3 was localized to 63, 65 and 58 kDa and faint reactivity to 53, 96 and 138 kDa bands respectively. In two-dimensional SDS-PAGE, hZP1 was shown to comprise two chains at 63-58 and 55-45 kDa, each consisting of multiple isomers. hZP2 was less acidic when compared with hZP1 and hZP3 and comprised a major component of 65 kDa and a minor component of approximately 96 kDa. The 65 kDa component displayed a higher degree of charged isomers in comparison with the 96 kDa component. hZP3 comprised a broad band in the range 68-58 kDa. These studies show conclusively that the hZP1 heavy train overlaps with hZP3 and that in previous studies, hZP2 was likely to have been misinterpreted as being hZP1. Our studies failed to distinguish two distinct species of hZP3, unlike previous reports. These studies will further help in our understanding of the nature of human zona pellucida glycoproteins.