A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult

RESEARCH ARTICLE A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult Alexandra L. Strauss, Fumiko Kawasaki, Richard W. Ordway* Department of Biology and Center for Molecular and Cellular Neuroscience, The Pennsylvania State University, University Park, Pennsylvania, United States of America * a11111 OPEN ACCESS Citation: Strauss AL, Kawasaki F, Ordway RW (2015) A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult. PLoS ONE 10(6): e0129957. doi:10.1371/ journal.pone.0129957 Academic Editor: Brian D. McCabe, Columbia University, UNITED STATES Received: November 10, 2014 Accepted: May 14, 2015 Abstract Previous studies of Drosophila flight muscle neuromuscular synapses have revealed their tripartite architecture and established an attractive experimental model for genetic analysis of glial function in synaptic transmission. Here we extend these findings by defining a new Drosophila glial cell type, designated peripheral perisynaptic glia (PPG), which resides in the periphery and interacts specifically with fine motor axon branches forming neuromuscular synapses. Identification and specific labeling of PPG was achieved through cell typespecific RNAi-mediated knockdown (KD) of a glial marker, Glutamine Synthetase 2 (GS2). In addition, comparison among different Drosophila neuromuscular synapse models from adult and larval developmental stages indicated the presence of tripartite synapses on several different muscle types in the adult. In contrast, PPG appear to be absent from larval body wall neuromuscular synapses, which do not exhibit a tripartite architecture but rather are imbedded in the muscle plasma membrane. Evolutionary conservation of tripartite synapse architecture and peripheral perisynaptic glia in vertebrates and Drosophila suggests ancient and conserved roles for glia-synapse interactions in synaptic transmission. Published: June 8, 2015 Copyright: © 2015 Strauss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Institutes of Health 1R01NS065983-01A1. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction A common synaptic architecture in vertebrates involves assembly of presynaptic and postsynaptic elements with glial processes to form tripartite (three-part) synapses. Previous work has established important roles for glia-synapse interactions in synaptic development and function (reviewed in [1–3]). Studies of vertebrate neuromuscular synapses, for example, have shown that specialized peripheral glia called Perisynaptic Schwann Cells (PSCs) contribute to tripartite synapse structure and function [4–6]. Recent work on Drosophila neuromuscular synapses, specifically those of the Dorsal Longitudinal Flight Muscles (DLM) in the adult, established the presence of tripartite synapses and functional glia-synapse interactions in an invertebrate [7, 8]. Thus Drosophila offers a unique experimental model in which powerful genetic approaches may be applied to the study of glutamatergic tripartite synapses which are accessible in the periphery. PLOS ONE | DOI:10.1371/journal.pone.0129957 June 8, 2015 1 / 13 Peripheral Perisynaptic Glia in Drosophila Despite this progress, the origin of glial cell processes which participate in tripartite DLM neuromuscular synapses, and thus the potential for selective genetic manipulation of these glial elements, has not been determined. Among several known types of peripheral glia in Drosophila [9, 10], including those which ensheath peripheral axons, none has been implicated in gliasynapse interactions. In the absence of a cell type-specific marker which labels perisynaptic glial processes, it is not known whether a distinctive type of glial cell contributes to tripartite neuromuscular synapses. Here we have utilized cell type-specific KD of a glial marker, GS2, as a novel approach to identify and specifically label perisynaptic glia. Moreover, electrophysiological studies of GS2 KD synapses extend previous genetic and functional analysis of GS2 [11], a Drosophila enzyme homologous to glial cytoplasmic Glutamine Synthetases implicated in neural function [12]. The present study provides further characterization of Drosophila tripartite neuromuscular synapses by defining a new type of peripheral glial cell which provides synaptic glial processes. In addition, this work establishes that glia-synapse interactions are a common feature of several different neuromuscular synapses of the Drosophila adult. Results The DLM neuromuscular synapse preparation (Fig 1A) includes six DLM muscle fibers innervated by five motor axons [13, 14] which exit the CNS within the Posterior Dorsal Mesothoracic Nerve (PDMN). Main branches of the PDMN project to the surface of the DLMs as indicated by neuronal and glial markers (Fig 1B) and motor axons branch extensively over the muscle surface in close association with glia (Fig 1C–1E). Fine terminal axon branches make synaptic contacts on the muscle (Fig 1C) and interact with glial processes (Fig 1D and 1E) to form tripartite neuromuscular synapses (Fig 1F–1H, S1 Fig and [7]). However, the cellular organization of glia at DLM neuromuscular synapses has not been defined. Although cell typespecific genetic approaches provide the potential for in vivo analysis of glial function, the use of glial molecular markers, such as GS2 [15–17] and the glutamate transporter, dEAAT1 [18], cannot distinguish whether synaptic glial processes are contributed by a distinct cell type. Further analysis took advantage of the GAL4-UAS system [19] to generate a cell type-specific marker which labels perisynaptic glia. In initial studies, different "driver" transgenic lines, which express the yeast GAL4 transcription factor in a cell type-specific manner, were crossed to flies carrying a transgene for expression of membrane-targeted GFP under the control of a GAL4-responsive Upstream Activation Sequence (UAS-mCD8-GFP). A screen of previously established glial GAL4 driver lines identified none that selectively labeled a distinct glial cell type in proximity to synapses. However, several lines which express in peripheral glia failed to mark synaptic glial processes labeled by GS2. One example is moody-GAL4 [20] which drives expression in subperineurial glial cells, a subtype of glia that forms the blood-brain barrier in peripheral nerves [21, 22]. Using this GAL4 driver, GFP expression is not observed in glial processe (...truncated)