Biomedical subjects

L Brodin

Publications and source records attributed to L Brodin.

At least 19 recordsLinked to original sources

PubMed2006

Differential efficiency of the endocytic machinery in tonic and phasic synapses.

Efficient synaptic vesicle membrane recycling is one of the key factors required to sustain neurotransmission. We investigated potential differences in the compensatory endocytic machineries in two glutamatergic synapses with phasic and tonic patterns of activity in the lamprey spinal cord. Post-embedding immunocytochemistry demonstrated that proteins involved in synaptic vesicle recycling, including dynamin, intersectin, and synapsin, occur at higher levels (labeling per vesicle) in tonic dorsal column synapses than in phasic reticulospinal synapses. Synaptic vesicle protein 2 occurred at similar levels in the two types of synapse. After challenging the synapses with high potassium stimulation for 30 min the vesicle pool in the tonic synapse was maintained at a normal level, while that in the phasic synapse was partly depleted along with expansion of the plasma membrane and accumulation of clathrin-coated intermediates at the periactive zone. Thus, our results indicate that an increased efficiency of the endocytic machinery in a synapse may be one of the factors underlying the ability to sustain neurotransmission at high rates.

Animals↗

PubMed2001

Measurement of the dynamics of exocytosis and vesicle retrieval at cell populations using a quartz crystal microbalance.

The quartz crystal microbalance-dissipation technique (QCM-D) is used in two different measurement strategies to monitor the mass change and rigidity of populations of excitable cells during exocytosis and subsequent retrieval of dense-core vesicles. Two cell lines, NG 108-15 and PC 12, were grown to confluence on piezoelectric quartz crystals and were examined separately to demonstrate differences in release and retrieval with cells of different morphology, size, and number of dense-core vesicles. Stimulating the cells to exocytosis with media containing an elevated potassium concentration resulted in an increase in the frequency response corresponding to loss of mass from the cells owing to release of vesicles. In Ca2+-free media, the response was completely abolished. The amplitude and peak area in the frequency response corresponding to mass change with stimulated release was larger for PC 12 cells than for NG 108-15 cells, whereas the initial rate constants for the frequency responses were similar. The data suggest (1) that a greater number and larger size of vesicles in PC 12 cells results in a greater amount of release from these cells vs NG 108-15 cells, (2) the recycling of vesicles utilizes similar fusion/retrieval mechanisms in both cell types, (3) that the control of excess retrieval might be related to the number and size of released vesicles, and (4) that measured retrieval has a rapid onset, masking exocytosis and implying a rapid retrieval mechanism in the early stages of release. These results demonstrate that measurements of complex dynamic processes relating to dense-core vesicle release and retrieval can be simultaneously accomplished using the QCM-D technique.

Animals↗

PubMed2000

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Sequential steps in clathrin-mediated synaptic vesicle endocytosis.

Synaptic vesicles are recycled with remarkable speed and precision in nerve terminals. A major recycling pathway involves clathrin-mediated endocytosis at endocytic zones located around sites of release. Different 'accessory' proteins linked to this pathway have been shown to alter the shape and composition of lipid membranes, to modify membrane-coat protein interactions, and to influence actin polymerization. These include the GTPase dynamin, the lysophosphatidic acid acyl transferase endophilin, and the phosphoinositide phosphatase synaptojanin. Protein perturbation studies in living nerve terminals are now beginning to link the actions of these proteins with morphologically defined steps of endocytosis.

Animals↗

PubMed2000

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster.

Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed1999

Presynaptic mitochondria and the temporal pattern of neurotransmitter release.

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.

Adenosine Triphosphate↗

PubMed1999

Immunohistochemical demonstration of exocytosis-regulating proteins within rat molar dentinal tubules.

No morphologically defined synaptic structures have so far been detected between nerve terminals and the dentine-producing odontoblasts. Recent studies of the molecular mechanisms in neuronal exocytosis have identified several proteins that participate in synaptic-vesicle exocytosis. By localizing these proteins with immunohistochemical methods, information about the capacity for synaptic exocytosis should be obtained. Here, antibodies directed against some of the exocytosis-related proteins were used to investigate whether they are present in nerve fibers within the dentinal tubules in rat molars. Antibodies against synaptosome-associated protein of 25 kDa, Rab 3, synaptotagmin and synapsin all produced a punctuate staining pattern, suggesting that the proteins are accumulated in bouton-like elements. The results demonstrate that a set of exocytosis-related proteins is accumulated in the dentinal tubules, most probably within the intradentinal nerves. This finding is consistent with the hypothesis that intradentinal nerves can mediate efferent signals.

Animals↗

PubMed1999

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

Animals↗

PubMed1999

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector.

We have examined whether the Semliki Forest virus (SFV) expression vector can be used to manipulate the exocytotic machinery in cultured hippocampal neurons. Autaptic responses were recorded in individually identified neurons which overexpressed either a non-synaptic protein, the transferrin receptor, or the synaptic SNARE protein SNAP-25 (synaptosomal-associated protein of 25 kDA). In neurons overexpressing the transferrin receptor, autaptic responses occurred in a similar proportion and had similar amplitudes (12-18 h postinfection) as in uninfected control neurons. With increasing time after the infection, an increasing proportion of the transferrin receptor-overexpressing neurons showed changes in the shape of the cell body, but the autaptic responses appeared normal as long as recordings could be performed (up to 30 h postinfection). In contrast, in SNAP-25-overexpressing neurons, the proportion of responding cells was reduced 12-18 h after the infection, and the amplitude of the autaptic current in responding neurons was also reduced. The sensitivity to exogenously applied glutamate was, however, unchanged. Biochemical analysis showed that 50% of the overexpressed SNAP-25 was palmitoylated. The levels of two other SNAREs, syntaxin and synaptobrevin (also called vesicle-associated membrane protein), were not affected. Our results indicate that the SFV vector can provide an effective tool to study the function of proteins participating in neurotransmitter release.

Animals↗

PubMed1998

Dissociation between Ca2+-triggered synaptic vesicle exocytosis and clathrin-mediated endocytosis at a central synapse.

We have tested whether action potential-evoked Ca2+ influx is required to initiate clathrin-mediated synaptic vesicle endocytosis in the lamprey reticulospinal synapse. Exo- and endocytosis were temporally separated by a procedure involving tonic action potential stimulation and subsequent removal of extracellular Ca2+ (Ca2+e). A low concentration of Ca2+ ([Ca2+]e of 11 microM) was found to be required for the induction of early stages of endocytosis. However, the entire endocytic process, from the formation of clathrin-coated membrane invaginations to the generation of synaptic vesicles, proceeded in the absence of action potential-mediated Ca2+ entry. Our results indicate that the membrane of synaptic vesicles newly incorporated in the plasma membrane is a sufficient trigger of clathrin-mediated synaptic vesicle endocytosis.

Animals↗

PubMed1998

Co-localized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission.

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA(B) receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (-ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA(B) agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA(B) antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.

Animals↗

PubMed1997

Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions.

The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain-containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin's SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.

Amino Acid Sequence↗

PubMed1997

Glial and neuronal glutamine pools at glutamatergic synapses with distinct properties.

The main pathway for transmitter glutamate turnover in excitatory synapses is thought to involve an uptake in glial processes, a conversion into glutamine, which recycles to the presynaptic terminal to serve as the main precursor for new synthesis of glutamate. To investigate whether the mechanisms of glutamine and glutamate turnover are linked with the properties of different glutamate synapses, the distribution of glutamine was studied in two types of glutamate synapse in the lamprey spinal cord using immunogold post-embedding electron microscopy. The synapses examined are formed by primary afferent axons (dorsal column axons), which predominantly exhibit a tonic firing pattern, and by giant reticulospinal axons, which primarily fire in brief bursts. Glial cell processes and postsynaptic dendrites displayed the highest density of glutamine labeling in both types of synapse. The level of glutamine was significantly higher in the glial cell processes surrounding the tonic dorsal column synapses, as compared to those surrounding the reticulospinal synapses. The axoplasmic matrix and presynaptic mitochondria, as well as postsynaptic dendrites, contained similar levels of glutamine labeling in both cases. The glutamate labeling in glial processes was also similar at the two types of synapse, while axoplasmic matrix and presynaptic mitochondria displayed four to six times higher levels in the tonic axons. In conjunction with our previous results, showing a different transport activity in glial processes of the two types of excitatory synapse, the results of the present study suggest that the glial pool of neurotransmitter precursor is linked to the rate of transmitter synthesis and release in adjacent synapses.

Animals↗

PubMed1997

Distinct effects of clostridial toxins on activity-dependent modulation of autaptic responses in cultured hippocampal neurons.

Clostridial neurotoxins proteolyse specific proteins implicated in synaptic vesicle exocytosis, but their actions on the release machinery in functional synapses is not well understood. Here we examine the effects of botulinum toxin A (BoNT/A) and tetanus toxin (TeTx) on autaptic transmission in cultured rat hippocampal neurons using whole-cell voltage clamp recordings. The proportion of cells responding to stimulation with an excitatory postsynaptic current (EPSC) and the magnitude of the remaining responses decreased gradually with increasing concentration of either toxin. However, the activity-dependent modulation (5 Hz repetitive stimulation) of EPSCs remaining after toxin inhibition differed markedly between the two toxins. The TeTx inhibition was associated with a persistent activity-dependent depression similar to that in control cells. In contrast, the BoNT/A inhibition was accompanied by a reversal of the modulation into facilitation, resembling that induced by lowering of the calcium concentration. These results demonstrate a difference between BoNT/A and TeTx in their mode of inhibition of synaptic vesicle exocytosis, which suggests that they exert their preferential actions at distinct steps of the release process.

Animals↗

PubMed1997

Sustained neurotransmitter release: new molecular clues.

Chemical synapses convey impulses at high frequency by exocytosis of synaptic vesicles. To avoid failure of synaptic transmission, rapid replenishment of synaptic vesicles must occur. Recent molecular perturbation studies have confirmed that the recycling of synaptic vesicles involves clathrin-mediated endocytosis. The rate of exocytosis would thus be limited by the capacity of the synaptic clathrin machinery unless vesicles could be drawn from existing pools. The mobilization of vesicles from the pool clustered at the release sites appears to provide a mechanism by which the rate of exocytosis can intermittently exceed the rate of recycling. Perturbation of synapsins causes disruption of vesicle clusters and impairment of synaptic transmission at high but not at low frequencies. Both clathrin-mediated recycling and mobilization of vesicles from the reserve pool are thus important in the replenishment of synaptic vesicles. The efficacy of each mechanism appears to differ between synapses which operate with different patterns of activity.

Animals↗

PubMed1996

Synaptic and nonsynaptic monoaminergic neuron systems in the lamprey spinal cord.

In the lamprey spinal cord, dopamine- (DA) and 5-hydroxytryptamine-(5-HT) containing cells appear to play an important role in controlling the firing properties of motoneurons and interneurons and, thereby, in modulating the efferent motor pattern. To determine the detailed morphology and synaptic connectivity of the intraspinal DA and 5-HT systems in Lampetra fluviatilis and Ichthyomyzon unicuspis, DA and 5-HT antisera were used in light and electron microscopic immunocytochemical experiments. Two main groups of labeled cells were distinguished: DA-containing liquor-contacting (LC) cells distributed along the central canal, and 5-HT+DA-containing multipolar cells located near the midline ventral to the central canal. Both types were synaptically connected with other neuronal elements. The DA-immunoreactive LC cells, which extended a ciliated process into the central canal, received symmetrical synapses from unlabeled terminals containing small synaptic vesicles. The distal process of the LC cells could be traced to the lateral cell column, to the ventral aspect of the dorsal column, or to the ventromedial area. Ultrastructural analysis of DA fibers in these regions showed the presence of labeled terminals containing numerous small synaptic vesicles and a few dense-core vesicles. These terminals formed symmetrical synapses with unlabeled cell bodies and dendrites, with GABA-immunopositive LC cells, and with the multipolar DA+5-HT cells. The multipolar DA+5-HT cells also received input from unlabeled synapses. Intracellular recording from these cells showed that they received excitatory postsynaptic potentials in response to stimulation of fibers in the ventromedial tracts and dorsal roots. The terminals of the multipolar DA+5-HT neurons in the ventromedial spinal cord contained numerous dense-core vesicles and small synaptic vesicles, but no synaptic specializations could be detected. In addition, a small number of larger DA-immunoreactive cells were observed in the lateral cell column at rostral levels. The lamprey spinal cord thus contains distinct populations of synaptically interconnected monoaminergic neurons. Dopamine-containing LC cells synapse onto DA+5-HT-containing multipolar cells, in addition to GABAergic LC cells and unidentified spinal neurons. In contrast, the multipolar cells appear to exert their influence by nonsynaptic mechanisms.

Amines↗