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Drosophila cholinergic neurons and processes visualized with Gal4/UAS–GFP

https://doi.org/10.1016/S1567-133X(01)00011-4Get rights and content

Abstract

Using 7.4 kb of 5′ flanking DNA from the Drosophila cholinergic gene locus to drive Gal4 expression we can visualize essentially all cholinergic neurons and neuropiles after genetic recombination with a UAS–GFP (S65T) reporter gene. In contrast to previous methods somata and neuropiles can be observed in the same samples. Fluorescence intensity is strong enough to allow observations in live animals at all developmental stages. Three-dimensional reconstructions made from confocal sections of whole-mount preparations reveal the extensive cholinergic connections among various regions of the nervous system.

Introduction

Neurotransmitter phenotype is a functionally important characteristic of neurons that depends on the expression of specific neurotransmitter processing genes. In neurons that use acetylcholine as a neurotransmitter, the ‘cholinergic’ gene locus, which contains genetic functions for both choline acetyltransferase (Cha) and the vesicular acetylcholine transporter (Vacht) [1], [11], [19] is specifically transcribed and translated. The distribution of cholinergic elements has been partially documented in Drosophila (see [42]), however, no single technique has allowed simultaneous visualization of both somata and associated nerve fibers and endings [6], [12], [13], [39]. Immunocytochemistry, using anti-choline acetyltransferase (ChAT) antibodies reveals intensely stained neuropile regions but only rarely cell somata [12], [40]. Localization of somata has primarily been inferred from low-resolution in situ hybridization with antisense RNA probes for Cha [2], [42].

Analysis of transgenic reporter genes in Drosophila has revealed that transcriptional regulatory activity of the cholinergic locus resides in the 5′ flanking DNA [16], [17], [18], [39]. Here we describe the characterization of transgenic Drosophila using the Gal4–UAS system [3] recombined with a UAS–GFP fluorescent reporter gene. The main advantages of this approach are the ability to easily reconstruct the complicated relationships of cholinergic cell somata and neuropiles from confocal sections and the ability to view cholinergic elements in live intact animals.

Section snippets

Transgenes and transgenic lines

Gal4, excised from pGaTB [3] as a BamHI/NotI fragment, was inserted into pCaSpeR2 [24] and then the 7.4 kb of 5′ flanking DNA from the Drosophila cholinergic locus, as a BamHI fragment, was inserted to generate pCaSpeR2–Cha–Gal4. The P-element from one transgenic lines (prepared by R. Greenspan) was mobilized [27] resulting in a homozygous second chromosome viable line, 19B, described in this study. Several other lines have similar fluorescence patterns. For further details see Ref. [15].

Results

Fluorescent cells and processes could be observed in many parts of the central and peripheral nervous system at all developmental stages examined. Fluorescence was specific to transgenic lines containing both the cholinergic regulatory DNA–Gal4 transgene and UAS–GFP transgene since lines containing only driver or responder genes showed no specific fluorescent signal.

Acknowledgements

This work was supported by a grant from NINDS NS19482. We thank Dr. Banghua Sun and Ms. Elvia Guiterrez for preparation of dissected nervous system samples used in the early parts of this study as well as their generous help and advice. We also thank Dr. R. Greenspan for making the original Gal4 lines and our institutional colleagues for many useful discussions and help. In particular, K. Ikeda and R. Williamson offered valuable advice and expertise.

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