Patch-Clamping Drosophila Sensory Neurons
Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Dros
- PDF / 676,620 Bytes
- 13 Pages / 504.57 x 720 pts Page_size
- 5 Downloads / 182 Views
1
Introduction Ion channels are integral membrane proteins that form pores through the lipid bilayer to facilitate ion permeation. This function makes channel proteins important regulators of a variety of biological processes. For instance, they play a vital role in neuronal signal transduction, neurotransmitter release, muscle contraction, cell secretion, and gene transcription. The patch clamp technique allows direct measurement of ion channel function and can provide essential clues about ion channel expression, regulation, and physiological function. However, probing ion
Nikita Gamper (ed.), Ion Channels: Methods and Protocols, Methods in Molecular Biology, vol. 998, DOI 10.1007/978-1-62703-351-0_30, © Springer Science+Business Media, LLC 2013
385
386
Volodymyr Kucher et al.
channels in native cells and tissues often can be challenging and experimentally limiting. Drosophila melanogaster is a powerful model organism for the study of physiology, including physiological functions of ion channels. The reasons for this are (a) the genetic tractability of this model organism as its complete genome has been decoded and sequenced; (b) many tools are available to modify the genetics of these animals; (c) the structure of the Drosophila nervous system is well defined and understood; and (d) the short life span, small size, and simple diet of these animals keep husbandry inexpensive. However it has been impossible, until recently, to take full advantage of the utility of the Drosophila model organism for studying ion channels as techniques for recording native ion channels from Drosophila neurons were missing. Recent advances in Drosophila cell culture and electrophysiology techniques, as described here, have overcome many of these limitations increasing the utility of Drosophila as a model organism for the study of ion channel biology and physiology. Preparation of primary cell cultures from Drosophila embryos was first developed in the laboratory of Seecof and colleagues in 1968 (1). Several variations in this technique are available for culturing neuronal as well as nonneuronal cell types (2–4). These cultures are suitable for probing ion channel activity, regulation, and function with the patch clamp method (5, 6). Moreover, neurons in primary neuronal cultures from Drosophila retain their native physiological properties: they form functional synaptic connections and exhibit electrical excitability (5, 6). Here we describe the preparation of a primary neuronal cell culture from midgastrula-stage Drosophila embryos and detail patch-clamp analysis of class IV multidendritic (md) sensory neurons in this culture to demonstrate their utility for investigating ion channel function and regulation.
2
Materials
2.1 Preparation of Primary Neuronal Cell Culture from Drosophila Embryos
1. The appropriate Drosophila melanogaster line preferably with the cell type of interest labeled in some manner (see Note 1). 2. Agar, Granulated (Genesee Scientific; Seattle, WA). 3. Sucrose (Sigma, St. Louis, MO). 4. Apple juice, 100
Data Loading...
