Lennart Brodin

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.

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 endophilins 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.

Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton

Actin is an abundant component of nerve terminals that has been implicated at multiple steps of the synaptic vesicle cycle, including reversible anchoring, exocytosis, and recycling of synaptic vesicles. In the present study we used the lamprey reticulospinal synapse to examine the role of actin at the site of synaptic vesicle recycling, the endocytic zone. Compounds interfering with actin function, including phalloidin, the catalytic subunit of Clostridium botulinum C2 toxin, and N-ethylmaleimide-treated myosin S1 fragments were microinjected into the axon. In unstimulated, phalloidin-injected axons actin filaments formed a thin cytomatrix adjacent to the plasma membrane around the synaptic vesicle cluster. The filaments proliferated after stimulation and extended toward the vesicle cluster. Synaptic vesicles were tethered along the filaments. Injection of N-ethylmaleimide-treated myosin S1 fragments caused accumulation of aggregates of synaptic vesicles between the endocytic zone and the vesicle cluster, suggesting that vesicle transport was inhibited. Phalloidin, as well as C2 toxin, also caused changes in the structure of clathrin-coated pits in stimulated synapses. Our data provide evidence for a critical role of actin in recycling of synaptic vesicles, which seems to involve functions both in endocytosis and in the transport of recycled vesicles to the synaptic vesicle cluster.

N-methyl-d-aspartate (NMDA), kainate and quisqualate receptors and the generation of fictive locomotion in the lamprey spinal cord

The motor pattern underlying swimming can be elicited in an in vitro preparation of the lamprey spinal cord by applying excitatory amino acids in the bath activating N-methyl-D-aspartate (NMDA) receptors and kainate receptors, but not quisqualate receptors. L-DOPA exerts a weak rythmogenic effect due to an action on kainate receptors. The kainate-induced rhythm is unchanged when a NMDA receptor antagonist is applied (2APV) and the N-methyl-aspartate-induced fictive locomotion can occur when kainate receptors are blocked (PDA). The burst frequency of the NMA-induced activity (dose range 30-5000 microM) is wide and ranges from 0.05-0.1 Hz up to 2.5-4 Hz, while the kainate-induced activity (dose range 7-30 microM) ranges from 0.5-1 Hz up to 4-8 Hz. This frequency range overlaps largely with that of the intact swimming animal. The findings further consolidate that NMDA receptors are efficient and demonstrates that kainate can also be effective in inducing fictive locomotion, and also that activation of either receptor type is sufficient. It has previously been shown that fictive locomotion elicited via sensory stimuli is depressed by NMDA and kainate receptor antagonists. It is suggested that these effects, presumably via aspartate and/or glutamate actions, are exerted on the input stage of interneuronal network.

A computer based model for realistic simulations of neural networks

The use of computer simulations as a neurophysiological tool creates new possibilities to understand complex systems and to test whether a given model can explain experimental findings. Simulations, however, require a detailed specification of the model, including the nerve cell action potential and synaptic transmission. We describe a neuron model of intermediate complexity, with a small number of compartments representing the soma and the dendritic tree, and equipped with Na+, K+, Ca2+, and Ca2+ dependent K+ channels. Conductance changes in the different compartments are used to model conventional excitatory and inhibitory synaptic interactions. Voltage dependent NMDA-receptor channels are also included, and influence both the electrical conductance and the inflow of Ca2+ ions. This neuron model has been designed for the analysis of neural networks and specifically for the simulation of the network generating locomotion in a simple vertebrate, the lamprey. By assigning experimentally established properties to the simulated cells and their synapses, it has been possible to verify the sufficiency of these properties to account for a number of experimental findings of the network in operation. The model is, however, sufficiently general to be useful for realistic simulation also of other neural systems.

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.

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.

Transmitters, membrane properties and network circuitry in the control of locomotion in lamprey

Abstract The lamprey brainstem and spinal cord can be maintained in vitro . It is a simple vertebrate preparation with comparatively few neurones. The neural correlates of different patterns of behaviour can be elicited in this in-vitro preparation. The subject of this review is the neuronal organization underlying locomotion, and, in particular, the role of different types of interneurones and their transmitters and mode of synaptic interaction. Excitatory amino acids, glycine, GABA, 5-HT, tachykinins and CCK have been implied as putative transmitters. The activation of one type of excitatory amino acid receptor, the NMDA receptor, can elicit TTX-resistant pacemaker-like membrane, potential oscillations. 5-HT can exert indirectly a potentiating effect via a depression of the postspike after-hyperpolarization (Ca 2+ -dependent potassium channels).

Phasic modulation of reticulospinal neurones during fictive locomotion and other types of spinal motor activity in lamprey

The intracellular activity of different types of reticulospinal neurones was studied during fictive locomotion and other types of spinal motor activity in an in vitro preparation of the lamprey brainstem-spinal cord. The examined neurones included large Müller cells of the rhombencephalic and mesencephalic reticular formation, the Mauthner cell, and neurones in the posterior rhombencephalic reticular nucleus with different sizes and conduction velocities. During bouts of fictive swimming initiated spontaneously or by stimulation of the trigeminal nerve or spinal cord, the Müller cells were depolarized and fired action potentials. Bulbar Müller cells in addition showed a phasic modulation of membrane potential with excitation in phase with ipsilateral motoneurones of the rostral spinal cord. The Mauthner cell was depolarized in phase with contralateral motoneurones. Many neurones in the posterior rhombencephalic reticular nucleus showed modulation in phase with ipsilateral motoneurones during fictive swimming. Such oscillations were observed in both fast-conducting neurones, located mainly in the medial part of the nucleus, and slower conducting cells with a more lateral distribution. All examined reticulospinal neurones showed a strong coupling also with other types of spinal motor activity, such as slow alternating bursting and synchronous bilateral ventral root bursts, but the reticulospinal activity had no correlation with respiratory activity recorded from the Xth nerve. The consequences of a phasic reticulospinal activity during locomotion are discussed.

The role of putative excitatory amino acid neurotransmitters in the initiation of locomotion in the lamprey spinal cord. I. The effects of excitatory amino acid antagonists

The activation of N-methyl-D-aspartate (NMDA) and kainate receptors will evoke fictive locomotion in the appropriate motor pattern for locomotion in the isolated lamprey spinal cord, but not a selective activation of quisqualate receptors. The present experiments test whether the initiation of locomotion in response to sensory stimulation depends on these types of receptors. An in vitro preparation of the lamprey spinal cord with part of its tailfin left innervated has been used. In this preparation a sequence of fictive locomotion (i.e. alternating bursts in the segmental ventral roots with a rostrocaudal phase lag) can be elicited by continual sensory stimulation of the tailfin. The effects of excitatory amino acid antagonists were studied by recordings from ventral roots (extracellularly) and motoneurones (intracellularly). It was found that the strong initial bursts of each swimming sequence induced by sensory stimulation were depressed by combined NMDA/kainate antagonists (cis-2,3-piperidine dicarboxylate (PDA) and gamma-D-glutamylglycine (gamma-DGG] whereas the less intense burst activity, occurring particularly towards the end of each swimming sequence, was depressed by a selective NMDA antagonist, 2-amino-5-phosphonovalerate (2-APV). This condition could be mimicked in an isolated spinal cord preparation by an application of L-glutamate; the low-level fictive locomotion induced by low doses of L-Glu (less than 100 microM) was depressed by a NMDA antagonist (2-APV), and, if higher doses were applied, the activity was only depressed by PDA/gamma-DGG. The mode and time course of the depression (by excitatory amino acid antagonists) of fictive locomotion, induced by sensory stimulation, shows that the putative excitatory amino acid neurotransmitter directly or indirectly acts at the pattern generating circuitry within the spinal cord.