Localized Ca2+ and calcium-activated potassium conductances in terminals of a barnacle photoreceptor

Calcium channels are found in the presynaptic terminals of neurones, where they have a key role in synaptic transmission. They are also found in the somata of many cells, in dendrites and along a few axons. In no cell is the actual distribution of these channels known in detail, because there are no known toxins or other agents suitable for labelling calcium channels, and the current through these channels is usually too small to be quantified with extracellular electrodes. However, several experiments have suggested that the density of the channels is less in the axon than in the cell body or terminal region. Here we have used the indicator dye Arsenazo III in conjunction with an array of photodetectors to examine the spatial influx of calcium in the presynaptic terminal region of the giant barnacle, Balanus nubilus. In these cells, calcium entry occurs in a restricted region less than 50 micron in length, which corresponds closely to the region of synaptic contact with second-order cells. Outside this area the magnitude of calcium entry is reduced at least 50-fold. With reasonable assumptions it follows that the calcium channel density is equally localized. In addition, we demonstrate that these cells have a calcium-activated potassium conductance. Since calcium entry is restricted to the synaptic zone, this conductance must be effective only in this region.     

Synapsin I bundles F-actin in a phosphorylation-dependent manner

Synapsin I is a neuron-specific phosphoprotein localized to the cytoplasmic surface of synaptic vesicles. This phosphoprotein is a major substrate for cyclic AMP-dependent and calcium/calmodulin-dependent protein kinases. Its state of phosphorylation can be altered both in vivo and in vitro by a variety of physiological and pharmacological manipulations known to affect synaptic function. Recent direct evidence suggests that it may be involved in the regulation of neurotransmitter release from the nerve terminal. In the nerve terminal, synaptic vesicles are embedded in a cytoskeletal network, consisting in part of actin. We report here the ability of the dephospho-form of synapsin I to bundle F-actin. This bundling activity is reduced when synapsin I is phosphorylated by cAMP-dependent protein kinase and virtually abolished when it is phosphorylated by calcium/calmodulin-dependent protein kinase II or by both kinases. These results, demonstrating an interaction of synapsin I with actin in vitro, support the possibility that synapsin I is involved in clustering of synaptic vesicles at the presynaptic terminal and that the phosphorylation of synapsin I may be involved in regulating the translocation of synaptic vesicles to their sites of release.     

A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission.

The mammalian central nervous system (CNS) contains an abundance of the transition metal zinc, which is highly localized in the neuronal parenchyma. Zinc is actively taken up and stored in synaptic vesicles in nerve terminals, and stimulation of nerve fibre tracts that contain large amounts of zinc, such as the hippocampal mossy fibre system, can induce its release, suggesting that it may act as a neuromodulator. The known interaction of zinc with the major excitatory and inhibitory amino-acid neurotransmitter receptors in the CNS supports this notion. That zinc has a role in CNS synaptic transmission, however, has so far not been shown. Here we report a physiological role for zinc in the young rat hippocampus (postnatal, P3-P14 days). Our results indicate that naturally occurring spontaneous giant depolarizing synaptic potentials (GDPs) in young CA3 pyramidal neurones, mediated by the release of GABA (gamma-aminobutyric acid), are induced by endogenously released zinc. These synaptic potentials are inhibited by specific zinc-chelating agents. GDPs are apparently generated by an inhibitory action of zinc on both pre- and postsynaptic GABAB receptors in the hippocampus. Our study implies that zinc modulates synaptic transmission in the immature hippocampus, a finding that may have implications for understanding benign postnatal seizures in young children suffering with acute zinc deficiency.     

Endogenous electric field around muscle fibres depends on the Na+-K+ pump

We describe here experiments which reveal a new physiological specialization in the endplate (synaptic) region of skeletal muscle fibres. Using a vibrating microelectrode which can detect small currents flowing in extracellular fluid, we have found that the membrane in the endplate region behaves as though a steady positive current is generated in this location. Current re-enters the fibre in the extrajunctional region. Further experiments show that this current is dependent on the activity of the sodium pump. The electric field created by this current may be important for long-term interactions between muscle and nerve.     

Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix.

The primary sequence of two components of the dystrophin-glycoprotein complex has been established by complementary, DNA cloning. The transmembrane 43K and extracellular 156K dystrophin-associated glycoproteins (DAGs) are encoded by a single messenger RNA and the extracellular 156K DAG binds laminin. Thus, the 156K DAG is a new laminin-binding glycoprotein which may provide a linkage between the sarcolemma and extracellular matrix. These results support the hypothesis that the dramatic reduction in the 156K DAG in Duchenne muscular dystrophy leads to a loss of a linkage between the sarcolemma and extracellular matrix and that this may render muscle fibres more susceptible to necrosis.     

Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor

Basal lamina-rich extracts of Torpedo californica electric organ contain a factor that causes acetylcholine receptors (AChRs) on cultured myotubes to aggregate into patches. Our previous studies have indicated that the active component of these extracts is similar to the molecules in the basal lamina which direct the aggregation of AChRs in the muscle fibre plasma membrane at regenerating neuromuscular junctions in vivo. Because it can be obtained in large amounts and assayed in controlled conditions in cell culture, the AChR-aggregating factor from electric organ may be especially useful for examining in detail how the postsynaptic apparatus of regenerating muscle is assembled. Here we demonstrate that the electric organ factor causes not only the formation of AChR aggregates on cultured myotubes, but also the formation of patches of acetylcholinesterase (AChE). This finding, together with the observation that basal lamina directs the formation of both AChR and AChE aggregates at regenerating neuromuscular junctions in vivo, leads us to hypothesize that a single component of the synaptic basal lamina causes the formation of both these synaptic specializations on regenerating myofibres.     

Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin

Diverse glycoproteins of cell surfaces and extracellular matrices operationally termed 'adhesion molecules' are important in the specification of cell interactions during development, maintenance and regeneration of the nervous system. These adhesion molecules have distinct functions involving different cells at different developmental stages, but may cooperate when expressed together. Families of adhesion molecules which share common carbohydrate domains do exist, despite the structural and functional diversity of these glycoproteins. These include the Ca2+-independent neural adhesion molecules: N-CAM, myelin associated glycoprotein (MAG) and L1. L1 is involved in neuron-neuron adhesion, neurite fasciculation, outgrowth of neurites, cerebellar granule cell migration, neurite outgrowth on Schwann cells and interactions among epithelial cells of intestinal crypts. We show here that in addition to sharing carbohydrate epitopes with N-CAM and MAG, L1 is also a member of the immunoglobulin superfamily. It contains six C2 domains and also shares three type III domains with the extracellular matrix adhesion molecule fibronectin.     

Innervation of the ovary in amphioxus: ultrastructural and immunohistochemical study

The distribution of unmyelinated nerve fiber in the ovary of amphioxus was found with transmission electron microscopic technique for the first time. The fiber is located under the ovary coat, and in close contact with it. There are two types of synaptic vesicles in the terminals of nerve fiber: one is durse-cored vesicle, the other is clear vesicle. In addition, the nerve terminals contact with follicle cells of ovary can be seen. Using immunohistochemical method. it is further demonstrated that the unmyelited nerve fiber nlay be a noradrenergic nerve fiber which is located on the ovary coat and follicle cell.     

Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals

The vesicles that package neurotransmitters fall into two distinct classes, large dense-core vesicles (LDCVs) and small synaptic vesicles, the coexistence of which is widespread in nerve terminals. High resolution capacitance recording reveals unitary steps proportional to vesicle size. Measurements of capacitance steps during LDCV and secretory granule fusion in endocrine and immune cells have provided important insights into exocytosis; however, extending these measurements to small synaptic vesicles has proven difficult. Here we report single vesicle capacitance steps in posterior pituitary nerve terminals. These nerve terminals contain neuropeptide-laden LDCVs, as well as microvesicles. Microvesicles are similar to synaptic vesicles in size, morphology and molecular composition, but their contents are unknown. Capacitance steps of two characteristic sizes, corresponding with microvesicles and LDCVs, were detected in patches of nerve terminal membrane. Both types of vesicles fuse in response to depolarization-induced Ca(2+) entry. Both undergo a reversible fusion process commonly referred to as 'kiss-and-run', but only rarely. Fusion pores seen during microvesicle kiss-and-run have a conductance of 19 pS, 11 times smaller than LDCV fusion pores. Thus, LDCVs and microvesicles use structurally different intermediates during exocytosis.     

Molecular and biological characterization of a murine ligand for CD40.

The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.     

Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions

The basal lamina in the synaptic cleft of the vertebrate skeletal neuromuscular junction contains molecules that direct the formation of synaptic specializations in regenerating axons and muscle fibres. We have undertaken a series of experiments aimed at identifying and characterizing the molecules responsible for the formation of one of these specializations, the aggregates of acetylcholine receptors (AChRs) in the muscle fibre plasma membrane. We began by preparing an insoluble, basal lamina-containing fraction from Torpedo californica electric organ, a tissue which has a far higher concentration of cholinergic synapses than muscle, and showing that this fraction caused AChRs on cultured chick myotubes to aggregate. A critical step is learning whether or not the electric organ factor is similar to the receptor-aggregating molecule in the basal lamina at the neuromuscular junction. The importance of this problem is emphasized by reports that clearly non-physiological agents, such as positively charged latex beads, can cause AChR aggregation on cultured muscle cells. We have already shown that Torpedo muscle contains an AChR-aggregating factor similar to that of electric organ, although in much lower amounts. Here we demonstrate, using monoclonal antibodies, that the AChR-aggregating factor in our extracts of electric organ is, in fact, antigenically related to molecules concentrated in the synaptic cleft at the neuromuscular junction.     

First visualization of glutamate and GABA in neurones by immunocytochemistry

Immunocytochemical methods for peptides and serotonin have greatly advanced the study of neurones in which these substances are likely to be transmitters. Such direct techniques have not so far been available for the amino acid transmitter candidates. We report here the selective immunocytochemical visualization of the putative transmitters glutamate (Glu) and gamma-aminobutyrate (GABA) by the use of antibodies raised against the amino acids coupled to bovine serum albumin (BSA) with glutaraldehyde (GA). The tissue localizations of Glu-like and GABA-like immunoreactivities (Glu-LI and GABA-LI) matched those of specific uptake sites for Glu and GABA, and, in the case of GABA-LI, also that of the specific marker enzyme glutamic acid decarboxylase (GAD). Thus, GABA-LI was located in what are believed to be GABAergic inhibitory neurones, whereas Glu-LI was concentrated in excitatory, possibly glutamatergic neurones. Preliminary electron microscopic observations suggest that the transmitter amino acids are significantly concentrated in synaptic vesicles.     

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.     

Regulation of vertebrate left-right asymmetries by extracellular matrix.

The vertebrate body is organized along three geometric axes: anterior-posterior, dorsal-ventral and left-right. Left-right axis formation, displayed in heart and gut development, is the least understood, even though it has been studied for many years. In Xenopus laevis gastrulae, a fibronectin-rich extracellular matrix is deposited on the basal surface of ectoderm cells over which cardiac and visceral primordia move during development. Here I report experiments in which localized perturbation of a small patch of extracellular matrix by microsurgery was correlated with localized randomization of left-right asymmetries. Global perturbation of the extracellular matrix by microinjection of Arg-Gly-Asp peptides or heparinase into the blastocoel resulted in global randomization of left-right asymmetries. From these observations, I suggest that left-right axial information is contained in the extracellular matrix early in development and is independently transmitted to cardiac and visceral primordia.     

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.     

A dopamine- and cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions

Several mammalian neurotransmitter candidates, for example, serotonin, dopamine and noradrenaline, may exert some of their synaptic effects by regulating protein phosphorylation systems. Comparison of the regional distribution of brain phosphoproteins with neurotransmitter systems may help to identify the specific phosphoproteins involved in the functions of particular neurotransmitters. Here we report the association of one such phosphoprotein with the dopamine pathways in brain. This protein, of apparent molecular weight (MW) 32,000 (32K), seems to be present only in nervous tissue. Its regional distribution within the brain is very similar to the pattern of dopamine-containing nerve terminals; more specifically, the protein appears to be enriched in those dopaminoceptive neurones which possess D-1 receptors (dopamine receptors coupled to adenylate cyclase). The state of phosphorylation of the protein in these dopaminoceptive neurones can be regulated by both dopamine and cyclic AMP. These results suggest that the phosphoprotein may mediate certain of the trans-synaptic effects of dopamine acting on dopaminoceptive neurones.     

Release of endogenous Zn2+ from brain tissue during activity

The role of divalent transition metal ions in neural function is poorly understood. In excess, these ions are associated with neurological disorders such as Wilson's disease, Pick's disease and epileptic seizures. We suggest that zinc ions, which are contained in nerve terminals, are extruded into the extracellular space during neuronal activity. Excessive levels of zinc may be released during intense neuronal activation, and contribute to the paroxysm and toxic damage observed. Zinc ions are contained in high concentrations in mossy fibres of the hippocampal formation, and it is the postsynaptic neurones of these fibres which are most susceptible to the toxic effects of kainic acid, a potent convulsant, or to chronic exposure to organometallic compounds. Here we demonstrate for the first time that Zn2+ is released into the extracellular space during excitation of hippocampal slices.     

Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.     

Oxytocin induces morphological plasticity in the adult hypothalamo-neurohypophysial system

The hypothalamo-neurohypophysial system offers a unique example in the adult mammalian central nervous system (CNS) of a functional and structural plasticity related to a physiological state. During lactation, oxytocin neurones evolve a synchronized electrical activation which permits pulsatile hormone release at milk ejection. At the same time, in the supraoptic (SON) and paraventricular nuclei, glial coverage of neurones diminishes, so that large portions of their surface membrane become directly juxtaposed; synaptic remodelling also associates pairs of neurones through the formation of common presynaptic terminals. These structural changes, reversible after weaning, affect exclusively oxytocinergic neurones and could facilitate their synchronized electrical activity. As several observations suggest that oxytocin itself is released centrally, we have examined the effect of prolonged intracerebroventricular infusions of oxytocin on the structure of the SON of non-lactating animals. We report here that the peptide indeed engenders the structural reorganization characteristic of the oxytocin system when it is physiologically activated. Similar infusion of vasopressin has no effect. Our observations thus demonstrate that a central neuropeptide can induce anatomical changes in the adult CNS, and suggest that oxytocin can regulate its own release by contributing to the dramatic restructuring of the nuclei containing the neurones responsible for its secretion.     

Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.

Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.