Enhancement of the endosomal endocytic pathway increases quantal size

doi:10.1016/j.mcn.2008.10.005

Molecular and Cellular Neuroscience

Volume 40, Issue 2, February 2009, Pages 199-206

https://doi.org/10.1016/j.mcn.2008.10.005 Get rights and content

Abstract

We combined recordings of spontaneous quantal events with electron microscopy analysis of synaptic ultrastructure to demonstrate that the size of a neurosecretory quantum increases following an activation of the endosomal endocytic pathway. We reversibly activated the endosomal endocytic pathway in Drosophila motor boutons by application of high K⁺ solution. This treatment produced the formation of numerous cisternae, vacuoles and enlarged vesicles. Spontaneous quantal events recorded immediately after the cessation of high K⁺ application were significantly enlarged, and this increase in quantal size was reversed after a 10 minute resting period. Actin depolymerization produced by latrunculin B pretreatment inhibited both the formation of endosome-like structures and the increase in quantal size. Loading the preparations with the dye FM1-43 followed by photoconversion of the dye combined with electron microscopy analysis revealed that the observed cisternae are likely to be the product of both bulk membrane retrieval and vesicle fusion.

Introduction

Neurotransmitters are packed in synaptic vesicles and released by the fusion of a vesicle with the presynaptic membrane. A current generated by a postsynaptic cell in response to a single vesicle fusion is termed the quantal current. The efficacy of neuronal transmission can be regulated either via the number of vesicles releasing transmitters or via the magnitude of the quantal current (the quantal size).

Regulation of quantal size may underlie various forms of synaptic plasticity (Edwards, 2007) and can be determined by postsynaptic (Malinow and Malenka, 2002) or presynaptic factors (Liu, 2003). Presynaptic mechanisms that may determine the quantal size are the vesicular transmitter concentration (Wilson et al., 2005, Wu and Wu, 2007), fusion pore opening time (Lindau and varez de, 2003), or synaptic vesicle size (Zhang et al., 1998, Karunanithi et al., 2002).

After releasing their content into the synaptic gap, vesicles are internalized and recycled. It has been demonstrated that vesicles are formed via clathrin-mediated endocytosis (De and Takei, 1996), which involves formation of a clathrin coat, pinching off from the plasma membrane and clathrin removal. The regulation of vesicle size by a clathrin adaptor (Zhang et al., 1998) suggested that vesicle sizes are “tailored” by the clathrin coat assembly (Zhang et al., 1999).

Vesicles may be reused directly after their reuptake (Koenig and Ikeda, 1996, Murthy and Stevens, 1998) or regenerated from endosomal intermediates or cisternae (Heuser and Reese, 1973, Takei et al., 1996, Leenders et al., 2002, de Lange et al., 2003). The question of whether the size of a vesicle and a neurosecretory quantum might be affected by the vesicle recycling pathway has not yet been explored. In the present study we investigated how the quantal size is affected at Drosophila motor boutons when the endosomal recycling pathway is activated.

Section snippets

Results

It has been demonstrated previously that endosomal recycling pathway can be activated by maintaining a prolonged membrane depolarization by employing either continuous stimulation by action potentials (Heuser and Reese, 1973, Teng and Wilkinson, 2000) or high K⁺ applications (Marxen et al., 1999, Holt et al., 2003, de Lange et al., 2003, Coggins et al., 2007). To test whether this can be achieved in Drosophila motor boutons, we stimulated preparations by 5 minute application of 90 mM KCl and

Discussion

The main finding of our study is that quantal size may depend on the vesicle recycling pathway. We have demonstrated this by activation of the endosomal endocytic pathway employing high K⁺ applications. Subsequent recordings of spontaneous synaptic activity demonstrated that the quantal size was reversibly increased. The conclusion about the dependence of quantal size on the endocytic pathway was further strengthened by our experiments demonstrating that pretreatment with the actin

Preparations and chemicals

Canton S strain of Drosophila melanogaster was used in this study. Experiments were performed on Ib boutons (Lnenicka and Keshishian, 2000, Dasari and Cooper, 2004) of the muscles 6 and 7 of abdominal segments 2, 3 or 4 of the third instar larvae. Preparations were dissected in physiological solution containing (in mM) 130 NaCl, 36 sucrose, 5 KCl, 2 CaCl2, 2 MgCl2, and 5 HEPES, pH 7.3. (Jan and Jan 1976), pinned to sylgard, cut open along the dorsal midline, and internal organs were removed to

Acknowledgment

The authors acknowledge the support from NIH grant R01 MH61059.

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Enhancement of the endosomal endocytic pathway increases quantal size

Abstract

Introduction

Section snippets

Results

Discussion

Preparations and chemicals

Acknowledgment

J. Neurosci. Methods

Neurosci. Res.

Neuron

Brain Res.

Curr. Biol.

Neuron

Neuroscience

Biochim. Biophys. Acta

Curr. Opin. Neurobiol.

Neuroscience

Neuron

Neuron

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

J. Neurobiol.

Quantal analysis of EPSCs recorded from small numbers of synapses in hippocampal cultures

J. Neurophysiol.

Origin of variability in quantal size in cultured hippocampal neurons and hippocampal slices

Proc. Natl. Acad. Sci. U. S. A.

Stimulated exocytosis of endosomes in goldfish retinal bipolar neurons

J. Physiol.

Two modes of vesicle recycling in the rat calyx of Held

J. Neurosci.

Molecular mechanisms in synaptic vesicle endocytosis and recycling

Neuron