Acute Dynamin Inhibition Dissects Synaptic Vesicle Recycling Pathways That Drive Spontaneous and Evoked Neurotransmission

Departments of Neuroscience, Pharmacology, and Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 01/2010; 30(4):1363-76. DOI: 10.1523/JNEUROSCI.3427-09.2010
Source: PubMed


Synapses maintain synchronous, asynchronous, and spontaneous forms of neurotransmission that are distinguished by their Ca(2+) dependence and time course. Despite recent advances in our understanding of the mechanisms that underlie these three forms of release, it remains unclear whether they originate from the same vesicle population or arise from distinct vesicle pools with diverse propensities for release. Here, we used a reversible inhibitor of dynamin, dynasore, to dissect the vesicle pool dynamics underlying the three forms of neurotransmitter release in hippocampal GABAergic inhibitory synapses. In dynasore, evoked synchronous release and asynchronous neurotransmission detected after activity showed marked and unrecoverable depression within seconds. In contrast, spontaneous release remained intact after intense stimulation in dynasore or during prolonged (approximately 1 h) application of dynasore at rest, suggesting that separate recycling pathways maintain evoked and spontaneous synaptic vesicle trafficking. In addition, simultaneous imaging of spectrally separable styryl dyes revealed that, in a given synapse, vesicles that recycle spontaneously and in response to activity do not mix. These findings suggest that evoked synchronous and asynchronous release originate from the same vesicle pool that recycles rapidly in a dynamin-dependent manner, whereas a distinct vesicle pool sustains spontaneous release independent of dynamin activation. This result lends additional support to the notion that synapses harbor distinct vesicle populations with divergent release properties that maintain independent forms of neurotransmission.

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Available from: Ege T Kavalali, Aug 14, 2014
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    • "at the synaptic vesicles that are responsible for the spontaneous release of single quanta belong to the same pool as those that mediate synchronous , or AP - driven release , there is now overwhelming evidence that two different pools of vesicles , whose release is controlled by quite different mechanisms , are involved ( Hablitz et al . , 2009 ; Chung et al . , 2010 ) . A third independent pool responsible for asynchronous release has also been proposed ( Smith et al . , 2012 ) . The facility for AP - driven release does not appear to be dependent upon the time since synapse formation , but rather on the maturity of the presynaptic neuron . Synapse - like contacts between MSNs ( E17 cultures , 15 D"
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    ABSTRACT: Basal ganglia play an essential role in motor coordination and cognitive functions. The GABAergic medium spiny neurons (MSNs) account for ~95% of all the neurons in this brain region. Central to the normal functioning of MSNs is integration of synaptic activity arriving from the glutamatergic corticostriatal and thalamostriatal afferents, with synaptic inhibition mediated by local interneurons and MSN axon collaterals. In this study we have investigated how the specific types of GABAergic synapses between the MSNs develop over time, and how the activity of GABAA receptors (GABAARs) influences this development. Isolated embryonic (E17) MSNs form a homogenous population in vitro and display spontaneous synaptic activity and functional properties similar to their in vivo counterparts. In dual whole-cell recordings of synaptically connected pairs of MSNs, action potential (AP)-activated synaptic events were detected between 7 and 14 days in vitro (DIV), which coincided with the shift in GABAAR operation from depolarization to hyperpolarization, as detected indirectly by intracellular calcium imaging. In parallel, the predominant subtypes of inhibitory synapses, which innervate dendrites of MSNs and contain GABAAR α1 or α2 subunits, underwent distinct changes in the size of postsynaptic clusters, with α1 becoming smaller and α2 larger over time, while both the percentage and the size of mixed α1/α2-postsynaptic clusters were increased. When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged. In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters. Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.
    Frontiers in Cellular Neuroscience 08/2015; 9:290. DOI:10.3389/fncel.2015.00290 · 4.29 Impact Factor
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    • "In a first series of experiments, we checked whether memory is affected by dynasore, a synthetic non-peptidic inhibitor of dynamins [13]. The compound blocks both dynamin-dependent endocytosis [4] and dynamin-dependent evoked release [7] while it generates branched tubular membrane networks, capped by clathrin-coated pits upon intense exocytosis [14], similar to those observed in the neurons of dynamin 1-null mice [5]. We analyzed the effects of dynasore on contextual fear memory [15], a form of associative, hippocampal-dependent memory in which mice have to associate the environment they are exposed to with the occurrence of an aversive stimulus that is delivered during the training phase. "
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    ABSTRACT: Dynamin 1-3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity.
    PLoS ONE 03/2014; 9(3):e91954. DOI:10.1371/journal.pone.0091954 · 3.23 Impact Factor
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    • "Reinforcing this hypothesis, recent work in central synapses showed that vesicles that recycle spontaneously and under depolarizing stimuli do not mix and are segregated in different vesicular pools (Fredj & Burrone, 2009; Chung et al., 2010). Because our data show that MbCD induces spontaneous vesicle release without stimulating FM1-43 uptake, we looked at ultrathin sections of neuromuscular preparations in control conditions or in muscle exposed to MbCD (10 mM) to visualize the distribution of synaptic vesicles in nerve terminals, especially in the vicinity of the active zones which is the location of the RRP. "
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    ABSTRACT: We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-β-cyclodextrin (MβCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MβCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MβCD were larger than those from control. In addition, treatment with MβCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MβCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.
    European Journal of Neuroscience 07/2013; 38(7). DOI:10.1111/ejn.12300 · 3.18 Impact Factor
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