Presynaptic Terminal Membrane Research Articles

Trafficking of synaptic vesicles is changed at the hypothalamus by exposure to an 835 MHz radiofrequency electromagnetic field.

With the rapidly increasing use of mobile phones and their close-contact usage to the brain, there are some concerns about the possible neuronal effects induced by exposure to excessive electromagnetic radiation. Exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate (SAR) 5 h/day for 12 weeks) may affect hypothalamic presynaptic neurons in C57BL/6 mice. The number and size of the synaptic vesicles (SVs) in the hypothalamic presynaptic terminals were significantly decreased after RF-EMF exposure. Further, the density (SVs numbers/μm) of docking and fusing SVs in the active zones of the presynaptic terminal membrane was significantly decreased in hypothalamic neurons. The expression levels of synapsin I/II and synaptotagmin 1, two regulators of SV trafficking in neurons, were also significantly decreased in the hypothalamus. In parallel, the expression of calcium channel was significantly decreased. These changes in SVs in the active zones may directly decrease the release of neurotransmitters in hypothalamic presynaptic terminals. Therefore, we further studied the possible changes in hypothalamic function by testing the core body temperature and body weight and performed the buried pellet test. The trafficking of SVs was changed by RF-EMF; however, we could not find any significant phenotypical changes in our experimental condition.

Presynaptic CamKII regulates activity-dependent axon terminal growth.

Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). Here, we identify a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, we find that both total and phosphorylated CamKII (p-CamKII) are enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. We have previously found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, our studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure.

Differential expression of vesicular glutamate transporters 1 and 2 may identify distinct modes of glutamatergic transmission in the macaque visual system

Glutamate is the primary neurotransmitter utilized by the mammalian visual system for excitatory neurotransmission. The sequestration of glutamate into synaptic vesicles, and the subsequent transport of filled vesicles to the presynaptic terminal membrane, is regulated by a family of proteins known as vesicular glutamate transporters (VGLUTs). Two VGLUT proteins, VGLUT1 and VGLUT2, characterize distinct sets of glutamatergic projections between visual structures in rodents and prosimian primates, yet little is known about their distributions in the visual system of anthropoid primates. We have examined the mRNA and protein expression patterns of VGLUT1 and VGLUT2 in the visual system of macaque monkeys, an Old World anthropoid primate, in order to determine their relative distributions in the superior colliculus, lateral geniculate nucleus, pulvinar complex, V1 and V2. Distinct expression patterns for both VGLUT1 and VGLUT2 identified architectonic boundaries in all structures, as well as anatomical subdivisions of the superior colliculus, pulvinar complex, and V1. These results suggest that VGLUT1 and VGLUT2 clearly identify regions of glutamatergic input in visual structures, and may identify common architectonic features of visual areas and nuclei across the primate radiation. Additionally, we find that VGLUT1 and VGLUT2 characterize distinct subsets of glutamatergic projections in the macaque visual system; VGLUT2 predominates in driving or feedforward projections from lower order to higher order visual structures while VGLUT1 predominates in modulatory or feedback projections from higher order to lower order visual structures. The distribution of these two proteins suggests that VGLUT1 and VGLUT2 may identify class 1 and class 2 type glutamatergic projections within the primate visual system (Sherman and Guillery, 2006).

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis

CaV2.2 voltage-gated calcium ­channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with CaV2.2 in purified presynaptic terminal membranes. A number of known and new CaV2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-CaV2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct CaV2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of CaV2.2 was beyond reasonable doubt. The identification of the long-splice CaV2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.

Molecular architecture of glycinergic synapses

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.

Munc18: A Presynaptic Transmitter Release Site N type (CaV2.2) Calcium Channel Interacting Protein

Munc18 is a presynaptic protein that is essential for transmitter release. Recent studies have indicated that this protein is involved in secretory vesicle docking but its binding partners in this role remain a mystery. We demonstrate using the isolated calyx-type presynaptic terminal of the chick ciliary ganglion that staining for Munc18 co-localizes and covaries with that for transmitter release site N type calcium channels (CaV2.2), consistent with elements of a common release site complex. Biochemical analysis demonstrated that the protein co-precipitates with CaV2.2 from lysates of rat or chick brain, including the synaptic, long-splice variant; presynaptic terminal surface membrane proteins, and a cell line co-expressing Munc18 and CaV2.2. Munc18 bound with high affinity to the CaV2.2 II-III intracellular loop, low affinity to the I-II loop but not to other channel intracellular regions. Over-expression of Munc18 in dorsal root ganglion neurons did not affect CaV2.2 current amplitude or fast kinetics but siRNA-knockdown resulted in a negative shift in the steady state inactivation curve, an effect attributed to an indirect action via syntaxin 1. Recombinant Munc18 also co-precipitated strongly with the v-SNARE synaptotagmin but only weakly with other SNAREs. Thus, the calcium channel may serve as a surface membrane platform anchoring a Munc18-containing bridge to synaptotagmin and the synaptic vesicle.

A syntaxin 1, Galpha(o), and N-type calcium channel complex at a presynaptic nerve terminal: analysis by quantitative immunocolocalization.

Presynaptic Ca(V)2.2 (N-type) calcium channels are subject to modulation by interaction with syntaxin 1 and by a syntaxin 1-sensitive Galpha(O) G-protein pathway. We used biochemical analysis of neuronal tissue lysates and a new quantitative test of colocalization by intensity correlation analysis at the giant calyx-type presynaptic terminal of the chick ciliary ganglion to explore the association of Ca(V)2.2 with syntaxin 1 and Galpha(O). Ca(V)2.2 could be localized by immunocytochemistry (antibody Ab571) in puncta on the release site aspect of the presynaptic terminal and close to synaptic vesicle clouds. Syntaxin 1 coimmunoprecipitated with Ca(V)2.2 from chick brain and chick ciliary ganglia and was widely distributed on the presynaptic terminal membrane. A fraction of the total syntaxin 1 colocalized with the Ca(V)2.2 puncta, whereas the bulk colocalized with MUNC18-1. Galpha(O,) whether in its trimeric or monomeric state, did not coimmunoprecipitate with Ca(V)2.2, MUNC18-1, or syntaxin 1. However, the G-protein exhibited a punctate staining on the calyx membrane with an intensity that varied in synchrony with that for both Ca channels and syntaxin 1 but only weakly with MUNC18-1. Thus, syntaxin 1 appears to be a component of two separate complexes at the presynaptic terminal, a minor one at the transmitter release site with Ca(V)2.2 and Galpha(O), as well as in large clusters remote from the release site with MUNC18-1. These syntaxin 1 protein complexes may play distinct roles in presynaptic biology.

Receptors with opposing functions are in postsynaptic microdomains under one presynaptic terminal.

Fast excitatory synaptic transmission through vertebrate autonomic ganglia is mediated by postsynaptic nicotinic acetylcholine receptors (nAChRs). We demonstrate a unique postsynaptic receptor microheterogeneity on chick parasympathetic ciliary ganglion neurons-under one presynaptic terminal, nAChRs and glycine receptors formed separate but proximal clusters. Terminals were loaded with [3H]glycine via the glycine transporter-1 (GlyT-1), which localized to the cholinergic presynaptic terminal membrane; depolarization evoked [3H]glycine release that was calcium independent and blocked by the GlyT-1 inhibitor sarcosine. Ganglionic synaptic transmission mediated by nAChRs was attenuated by glycine. Coexistence of separate clusters of receptors with opposing functions under one terminal contradicts Dale's principle and provides a new mechanism for modulating synaptic activity in vivo.

Phospholipaise A2 and arachidonic acid-mediated mechanism of neuroexocytosis: a possible target of botidinum neurotoxin A other then SNAP-25.

The vesicular neuroexocytosis process consists of two important steps: fusion of transmitter-loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into the synaptic cleft. We previously reported that in nerve growth factor (NGF)-differentiated PC12 cells, arachidonic acid (AA) release is associated with acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits both processes and AA itself or a phospholipase A(2) (PLA(2)) activator can cause ACh release in BoNT/A-poisoned cells in which SNAP-25 has supposedly been hydrolyzed. In the present study, we examined the roles of two endogenous intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP-25. A PLA(2) activator, mastoparan, was used to induce the release of AA and ACh from NGF-differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca(2+) influx via the neuronal-type voltage-sensitive Ca(2+) channels. Release of ACh followed a rise in intracellular free Ca(2+) concentration; the increased Ca(2+) activated PLA(2) and, thereby, increased the AA level. Scanning and transmission electron microscopy confirmed that mastoparan-induced ACh and AA release were not due to simple diffusion through damaged plasma membranes. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K(+) induced ACh exocytosis. The results support the conclusion that PLA(2) and AA have important roles in neuroexocytosis that are independent of SNAP-25. Both PLA(2) and AA have been shown to be involved in actin cytoskeletal organization related to vesicle fusion and exocytosis. This mechanism may be an alternative target of BoNT/A other than SNAP-25.

Temperature-Sensitive Neuromuscular Transmission in Kv1.1 Null Mice: Role of Potassium Channels under the Myelin Sheath in Young Nerves

In mammalian myelinated nerves, the internodal axon that is normally concealed by the myelin sheath expresses a rich repertoire of K channel subtypes thought to be important in modulating action potential propagation. The function of myelin-covered K channels at transition zones, however, has remained unexplored. Here we show that deleting the voltage-sensitive potassium channel Kv1.1 from mice confers a marked temperature-sensitivity to neuromuscular transmission in postnatal day 14 (P14)-P21 mice. Using immunofluorescence and electrophysiology, we examined contributions of four regions of the peripheral nervous system to the mutant phenotype: the nerve trunk, the myelinated segment preceding the terminal, the presynaptic terminal membrane itself, and the muscle. We conclude that the temperature-sensitive neuromuscular transmission is accounted for solely by a deficiency in Kv1.1 normally concealed in the myelinated segments just preceding the terminal. This paper demonstrates that under certain situations of physiological stress, the functional role of myelin-covered K channels is dramatically enhanced as the transition zone at the neuromuscular junction is approached.

Innervation and Target Tissue Interactions Induce Rab-GDP Dissociation Inhibitor (GDI) Expression during Peripheral Synapse Formation in Developing Chick Ciliary Ganglion NeuronsIn Situ

Regulated exocytosis of neurotransmitter from synaptic vesicles involves the function of a small GTP-binding protein, Rab3A. Rab-GDP dissociation inhibitor (GDI) is an important modulator of Rab function and subcellular distribution. We have characterized the respective roles of innervation and target tissue interactions in regulating GDI expression during synapse formation in chick ciliary ganglion (CG) neurons developing in situ. Here we report the first full-length chick GDI cDNA sequence. It is highly homologous to mammalian GDI isoforms and includes all of the sequence-conserved regions critical for Rab3A binding. This chick GDI mRNA is predominantly expressed in neurons as judged by Northern blot analysis of tissue distribution and by in situ hybridization of CG sections. Developmental increases in CG GDI mRNA levels occur in two phases as determined by reverse transcription (RT)-PCR and by Northern analysis of both normal-developing and input- or target tissue-deprived ganglia. The initial phase appears to be independent of cell-cell interactions. In contrast, the second, larger increase is induced by both presynaptic inputs and postganglionic target tissues but does not occur until target tissue innervation. Synaptic interaction with the target seems necessary for the regulatory response to both inputs and target tissues. GDI protein levels show similar changes. The developmentally delayed ability of inputs and targets to influence GDI levels differs from the regulation of neurotransmitter receptor expression in CG neurons. These results suggest that distinct extrinsic regulatory signals influence the expression of synapse-related components at the presynaptic axon terminal versus postsynaptic membrane in an individual neuron.

The formation of neuromuscular synapses.

The formation of synapses requires a series of steps including the generation of neurons and their target cells, the guidance of axons to their targets, and the induction of a specialized presynaptic terminal and postsynaptic membrane. This review is primarily concerned with the inductive events that lead to postsynaptic differentiation. Because this phase of synapse formation begins only after motor neurons have navigated to their target muscle cells, the first section of this review summarizes our current understanding of the principles that govern the guidance of motor axons to skeletal muscle cells. The second section will review our current knowledge of signaling molecules and mechanisms that are critical for inducing postsynaptic differentiation. Although the principles and mechanisms of synapse formation are likely to be similar in the PNS and the CNS, much of our understanding about the mechanisms of synapse formation arises from studies of the vertebrate neuromuscular synapse.

Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex.

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments in vitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 microM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 microM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction in vitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.

Dynamic responses of presynaptic terminal membrane pools following KCl and sucrose stimulation

The cholinergic presynaptic terminals of Torpedo electric organ have been examined morphometrically following stimulation by KCl and sucrose. The objective was to confirm correlations predicted by the vesicle hypothesis between miniature end-plate potentials (MEPPs) and morphometric changes in terminal ultrastructure. Both secretegogues generated high frequencies of MEPPs and also distinctive though differing ultrastructural changes. The synaptic vesicles show classes of 68 and 90 nm diameters and both store acetylcholine (ACh). KCl stimulation depleted the 90 nm class first whereas sucrose reversed the order of depletion. Very few instances of actual vesicle fusion were seen. Dose-response correlations between vesicle density and secretegogue strength (mM) and duration were higher with sucrose. Both secretegogues produced declines in vesicle numbers and densities and yielded multimodal distributions of large vesicles with an average 160 nm mean diameter. No meaningful correlations were detected between numbers of MEPPs and vesicles and little evidence was found to indicate that vesicles were fusing to terminal plasma membrane in numbers approximating MEPP release. Linear regression analysis was used to quantitatively examine relationships between the vesicle membrane pool and other pools of the putative exo/endocytotic pathway. Correlation coefficients between vesicle and terminal plasma membrane pools were non-significant and of positive sign, indicating independent, similar responses. Non-significant, negative coefficients were obtained when vacuole and 160 nm vesicle membrane values were included. These tests further argue against claims that vesicles are actively fusing with the plasma membrane. These conflicting findings for both secretegogues preclude meaningful correlations between vesicle changes and numbers of MEPPs generated and again emphasize the difficulty of validating the vesicle hypothesis by ultrastructural means. On the other hand, the study shows that vesicular, vacuolar and terminal membrane pools are dynamically changing during transmitter release, presumably interacting with cytosolic membrane constituents. A dynamical release process therefore has been proposed to account for the two classes of MEPPs, the rapid changes in class ratio and the mutable characteristics of the bell-MEPP that presently challenge the quantal-vesicular claims of prepackaged, immutable, exocytotically released packets of transmitter. This model features a state for each MEPP class with class and size determined at moment of release. For example, a single flicker of a channel would generate the sub-MEPP (defined subunit of an MEPP) and 7–20 flickering channels would generate the bell-MEPP.

Calcium-independent activation of the secretory apparatus by ruthenium red in hippocampal neurons: a new tool to assess modulation of presynaptic function

The functional plasticity of the nervous system may result in part from the direct modulation of the effectiveness of the release machinery of synaptic terminals. To date, direct modulation of secretion in neurons has proven difficult to study because of the lack of a suitable tool to probe the release machinery independently of calcium influx. We report that the polyvalent cation ruthenium red (RR) directly evokes rapid and reversible calcium-independent quantal secretion in hippocampal neurons by binding to external sites on the presynaptic terminal membrane. This binding can be displaced by heparin and is not associated with ultrastructural damage to the synaptic terminals. The use of RR-evoked release as a tool has allowed us to detect a direct modulation of the secretory apparatus after activation of A1 adenosine receptors on hippocampal neurons.

Identification of a 140 kDa protein of rat presynaptic terminal membranes encompassing the active zones

A polyclonal antiserum raised against the car☐y-terminal 17 amino acids of the rat p185 c-neu (anct) reacted with a 140 kDa polypeptide in membranes of synaptosome fractions from neocortex and hippocampus of 11-day-old and adult rats. The same antiserum reacted with a 185 kDa polypeptide in microsome membranes from rat pheochromocytoma cells (PC12). By light microscopic immunocytochemistry, the anct antibodies against the 140 kDa protein were localized in the neuropile of brain, cerebellum and spinal cord of 11-day-old and adult rats. Especially prominent staining was obtained in the CA2-CA3 zones of the hippocampus, and in the substantia gelatinosa in the spinal cord. The finely granular and diffuse pattern of the immunostatin was consistent with synaptic localizations. Interestingly, antibodies against the entire endodomain of p185 c-neu (a-Bacneu) were localized in granular structures, probably representing axo-somatic and axo-dendritic synapses, on a subset of pyramidal neurons of the CA3 zone. By immunoelectron microscopy, conducted in the hippocampus of adult rats, the anct antibodies were localized at the cytoplasmic site of presynaptic terminals in the giant mossy fiber type in the CA3 and CA4 regions. The immunolocalization of the anct antibodies was restricted in segments of the presynaptic membrane facing the synaptic cleft which include the active zone. The identity and function of the 140 kDa membrane protein of rat brain presynaptic terminals, detected by the anct antibodies, is unknown. The 140 kDa protein may be related to p185 c-neu, a tyrosine kinase, or to other known or unknown kinases.

Mathematic modelling of the enteric nervous network. 5. Excitation propagation in a planar neural network

A mathematical model of the enteric nervous system (Auerbach's plexus) as a planar neural network has been developed, based on the actual morphological data of its organisation. The network is composed of excitatory (cholinergic) and inhibitory (adrenergic) neurones interconnected by polysynaptic channels, formed of the geometrically non-uniform unmyelinated nerve axons. The synaptic zones are modelled as a three-compartment open pharmacokinetic system, i.e., presynaptic terminal, synaptic cleft and postsynaptic membrane where the pharmacokinetic mechanisms of electrochemical coupling are considered. All the chemical reactions of transformation of acetylcholine and adrenaline within them are described by first order Michaelis-Menten kinetics. The propagation of the electrical impulse along the pathways and in the vicinity of the nerve terminal is described by the modified Hodgkin-Huxley equations. The results of numerical simulation of the propagation of excitation within the neuronal chain, inhibitory feedback circuit, and a planar neuronal network under normal physiological conditions and after treatment with cholinergic/adrenergic agonists and antagonists are presented. The model predicts the dose-dependent influence of pharmacological agents on the neural network function.

Membrane fusion of erythrocytes as a model study of the effects of muscle relaxants on presynaptic exocytosis.

The exact mechanism by which synaptic vesicles fuse with the presynaptic nerve terminal membrane has not been determined. We therefore investigated the effects of muscle relaxants on membrane fusion using the human red blood cell as a model membrane. The kinetics of calcium-phosphate-induced membrane fusion were monitored continuously by noting the relief of fluorescence self-quenching of octadecyl-rhodamine B chloride (R18)-labeled and unlabeled membranes. The nondepolarizing muscle relaxants, vecuronium and pancuronium, inhibited membrane fusion of ghosts, inside-out vesicles (IOVs), and phosphatidylserine (PS)-loaded IOVs, whereas succinylcholine did not affect membrane fusion. The inhibitory effect of nondepolarizing muscle relaxants on membrane fusion suggests the prejunctional involvement of nondepolarizing muscle relaxants.

Selective Alterations in Presynaptic Cytomatrix Protein Organization Induced by Calcium and Other Divalent Cations That Modulate Exocytosis

Rises in intracellular calcium cause several events of physiological significance, including the regulated release of neuronal transmitters. In this study, the effects of divalent cations on the structural organization of cytomatrix in presynaptic terminals was examined. [35S]Methionine-radiolabeled guinea pig retinal ganglion cell cytomatrix proteins were axonally transported [in slow component b (SCb) of axonal transport] to the neuron terminals in the superior colliculus. When the peak of radiolabeled cytomatrix proteins reached the terminals, synaptosomes containing the radiolabeled cytomatrix proteins were prepared. Approximately 40% of each SCb protein was soluble after hypoosmotic lysis of the radiolabeled synaptosomes in the presence of divalent cation chelators. Lysis of synaptosomes in the presence of calcium ions over a range of concentrations, however, caused a dramatic decrease in solubility of the presynaptic SCb proteins. The cytoplasmic effects may result from a calcium-dependent condensation of cytoplasm around presynaptic terminal membrane systems. There are two major presynaptic SCb proteins (at 60 and 35 kDa), that exhibited exceptional behavior: they remained as soluble in the presence of calcium as under control conditions, suggesting that they were relatively unaffected by the mechanism causing the decrease in SCb protein solubility. Also examined were the effects of other alkaline earth and transition metal divalent cations on the presynaptic SCb proteins.

Differing actions of convulsant and nonconvulsant barbiturates: An electrophysiological study in the isolated spinal cord of the rat

The effects of various pairs of convulsant and nonconvulsant barbiturates on mono-and polysynaptic activity were studied in the isolated spinal cord of the immature rat, using extracellular recording. The convulsant barbiturates, 5-ethyl-5-(3-methylbut-2'-enyl) barbituric acid (3M2B), 5-ethyl-5-(1,3-dimethylbut-1'-enyl) barbituric acid (1,3M1B) and (+)-5-(1,3-dimethylbutyl)-5-ethyl barbituric acid [(+) DMBB] all increased the monosynaptic reflex at concentrations between 5 and 50 μM with no change in polysynaptic activity. When the concentration was raised to between 100 and 300 μM, however, the convulsants all reduced the monosynaptic reflex, thus producing a biphasic dose-response relationship. The nonconvulsant barbiturates phenobarbital, 5-ethyl-5-(3-methylbut-1'-enyl) barbituric acid (3M1B), amylobarbital (3MB) and (−)-5-(1,3-dimethylbutyl)-5-ethyl barbituric acid [(−)DMBB] produced only a decrease in mono- and polysynaptic reflexes. At concentrations which enhanced the monosynaptic reflex, the responses of motoneurones to glycine and eledoisin-related peptide (an analogue of substance P) were reduced by (+)DMBB, while 1,3M1B and 3M2B had no significant effects upon any of the neurotransmitters tested. At concentrations which depressed the monosynaptic reflex, the convulsants all reduced the response to glycine whereas the nonconvulsant barbiturates all increased the response to GABA. With the exception of phenobarbital, both convulsant and nonconvulsant barbiturates produced a direct depolarisation of the presynaptic terminal membrane, with only the convulsants producing a depolarisation of the membrane of the motoneurone. Using another convulsant barbiturate, 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB), this direct depolarising action was found to be calcium-dependent.