Current methods for in vivo patch-clamp electrophysiology and optical imaging of calcium transients in C. elegans neurons rely on veterinary-grade cyanoacrylate glue for immobilizing animals (Goodman et al., 1998; Lockery and Goodman, 1998; Kerr, et al., 2000). Just about ten years have passed since the first studies measuring cellular responses in individual neurons were published, all of them using Nexaband S/C (WPI, Sarasota, FL) to immobilize worms on thin, 2% agarose pads. Recently and tragically, Abbott discontinued the manufacture of Nexaband S/C. HistoacrylBlue has been used to immobilize animals during recordings from the C. elegans neuromuscular junction (Richmond and Jorgensen, 1999). Unfortunately, the dye added to this adhesive is fluorescent when viewed under a GFP filter set, obscuring GFP-tagged neurons.
Alternative methods for immobilization have emerged from the recent explosion in the design and fabrication of microfluidics devices for worms (Ben-Yakar et al., 2009). These devices have been used for long- and short-term imaging work and appear to be significantly less toxic than even the best cyanoacrylate adhesives. However, the need for physical access to the animals during an electrophysiology experiment precludes the use of existing traps for in vivo recording. Thus, despite the great advances provided by microfluidics devices, there is a continuing need for a minimally toxic, cyanoacrylate adhesive.
We are pleased to report a suitable alternative, custom-formulation from GluStitch Inc. (http://www.glustitch.com, contact: Steven Blacklock, firstname.lastname@example.org): 80% octyl-20% butyl cyanoacrylate (r = 5.75 centiPoise). The viscosity and polymerization rate of this glue is such that it can be applied in a targeted way to one side of a worm without coating the entire animal. With this formulation, it is possible to see the GFP label and also to expose GFP-tagged neurons of interest, the first step needed for electrophysiological recordings. Additionally, worms appear healthy hours after being glued. Other glues and adhesion methods tested were deemed unsuitable. These include: Nexcare (3M), other formulations from GluStitch, Inc. (high viscosity (75 cP) 100% butyl cyanoacrylate, 50/50 octyl/butyl cyanoacrylate, 100% butyl cyanoacrylate (3.7 cP)) and Gluture (Abbott Laboratories). Good luck and good patching.
Ben-Yakar A, Chronis N, and Lu H. (2009). Microfluidics for the analysis of behavior, nerve regeneration and neural cell biology in C. elegans. Curr. Opin. Neurobiol. [Epub ahead of print].
Goodman MB, Hall DH, Avery L, and Lockery SR. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron 20, 763-772.
Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, and Schafer WR. (2000). Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26: 583-594.
Lockery SR and Goodman MB. (1998). Tight-seal whole-cell patch clamping of Caenorhabditis elegans neurons. Meth. Enzymol. 293, 201-217.
Richmond JE and Jorgensen EM. (1999). One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nat. Neurosci. 2, 791-797.
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Verena Wolfram says
In Dr. R. Baines lab (University of Manchester, UK) we are routinely recording from motoneurons in the Drosophila 1st instar larva. For a while now we are experiencing problems with our patch clamp recordings and we have narrowed it down to the glue we are using. They are ranging from recording unusually very long synaptic currents, over silent cells (no synaptic currents at all) to the point of the inability to get cell attached. Most problems we encounter when recordings are done in standard potassium salines (internal and external). The glues we have been using are: Braun – Histoacryl containing n-butyl; 3M Vetbond containing n-butyl; slightly better but still not satisfactory results were obtained with Gluture 60% 2-octyl 40% n-butyl. It seems that some glues get worse with time. We will be trying out the Wormglue in the near future.
For what reasons were the other glues not suitable? Were they able to keep the worms alive and see GFP with low background for imaging?
Andrew Hardaway says
Do you have any suggestions for glue application?