Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses

Abstract

The neuromuscular system of Drosophila has been widely used in studies on synaptic development. In the embryo, the cellular components of this model system are well established, with uniquely identified motoneurons displaying specific connectivity with distinct muscles. Such knowledge is essential to analyzing axon guidance and synaptic matching mechanisms with single-cell resolution. In contrast, to date the cellular identities of the larval neuromuscular synapses are hardly established. It is not known whether synaptic connections seen in the embryo persist, nor is it known how individual motor endings may differentiate through the larval stages. In this study, we combine single-cell dye labeling of individual synaptic boutons and counterstaining of the entire nervous system to characterize the synaptic partners and bouton differentiation of the 30 motoneuron axons from four nerve branches (ISN, SNa, SNb, and SNd). We also show the cell body locations of 4 larval motoneurons (RP3, RP5, V, and MN13-Ib) and the types of innervation they develop. Our observations support the following: (1) Only 1 motoneuron axon of a given bouton type innervates a single muscle, while up to 4 motoneuron axons of different bouton types can innervate the same muscle. (2) The type of boutons which each motoneuron axon forms is likely influenced by cell-autonomous factors. The data offer a basis for studying the properties of synaptic differentiation, maintenance, and plasticity with a high cellular resolution.

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila

“…We found abnormal formation of synapses on a small subset of dorsal muscles. As shown in Figure 4A & A1, the ISN nerve traveled towards dorsal muscles 3, 2, and 1 and formed a typical âbeads-on-a-stringâ pattern of synapses extending along the surface of these muscles (Hoang and Chiba, 2001). However, approximately 90% of the synapses found onto muscle 2, and to a lesser extent, onto muscle 3, in fmi E59 /Df larvae differed dramatically from the control synapses.…”

“…To this end, we have investigated the larval NMJ of the flamingo (fmi) mutant. The Drosophila NMJ is a well-known model system for studying synaptic formation and growth at the single cell level (Keshishian et al, 1993;Budnik and Gramates, 1999;Hoang and Chiba, 2001). The larval bodywall muscle-synapse is segmented and readily recognized anatomically.…”

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…A key prerequisite for detecting and working with these phenotypes is the existence of enormously detailed descriptions of all embryonic motor and sensory neurons including their axonal and dendritic morphology (Landgraf, Bossing, Technau, & Bate, 1997;Merritt & Whitington, 1995;Rickert, Kunz, Harris, Whitington, & Technau, 2011). For functional analyses, they can either be stained with antibodies [all neurons (anti-HRP), (subsets of ) sensory (22C10, Mab49C4) or motor axons (anti-FasII, anti-FasIII)] or using GAL4/UAS-based (Chiang et al, 2011) as well as manual single-cell labeling strategies (Hoang & Chiba, 2001;Landgraf et al, 1997). Simple strategies based on anti-HRP-labeled histological landmarks were developed to measure the extent of axon/ nerve growth toward their respective target areas, thus generating quantitative data for mutant or experimental analyses (Bottenberg et al, 2009).…”

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“…Do MNs transmit a temporally similar 28 pattern of activity to the one they received, or do they change the pattern? If the latter, 29 to what extent do MNs contribute to shaping the final timing of motor behavior? hemisegments expressed EKI, while the same identified MNs in other hemisegments did 83 not (Fig.…”

“…The body of a Drosophila larva comprises multiple 114 segments, and in each hemisegment is a repeated set of 30 muscles [29,44]. This 115 organization is mirrored across the midline.…”