Lab techniques are often shrouded in a veil of perceived voodoo and superstition – almost everything and anything a person does in lab can have an associated dance, prayer, or ritual to ensure their success. This is especially true for Drosophila electrophysiology. From lab to lab there are different prescriptions for electrode resistance, dissection technique, and temperature control of the rig. But perhaps the most variable and contested opinions are about proper recording solution. In this article I help you understand the reasoning driving some lab’s choice of Drosophila electrophysiology recording solutions.
Broadly, there are three classes of Drosophila electrophysiology recording solutions:
Class 1: Standard & Class 2: Modified standard
The whole point of electrophysiology is to measure a physiological process and for that we need to keep the preps alive and cells firing! Standard and modified standard are variations on invertebrate-specific recording solutions first used by Jan and Jan in the 1970’s. They contain just about everything you’d think they would for mimicking the ionic gradients of the invertebrate extracellular matrix space and keeping cells alive: NaCl, KCl, MgCl2, CaCl2, sucrose, HEPES/BES, and trehalose.
Class 3: Hemolymph-like (HL)
The hemolymph-like (HL) solutions started popping up in the 1990’s in response to a paper that reported ionic concentrations measured in Drosophila in vivo (Stewart et al 1994). Today, the most common solutions used and reported are HL3, HL3.1, and HL6. In the annuls of Drosophila texts, one can also find recipes for an HL4, HL5, and variations on those. But since those are less commonly used today, I will not include them here. But, if you use them and they are successful for you, mazel! Sometimes the old ways are the best ways.
HL3 is the granddaddy of HL recording solutions. It represents the first attempt at mimicking the ionic concentrations reported from Drosophila in vivo measurements. I use HL3 for recording membrane potentials, excitatory junctional potentials, and miniature end plate potentials from the neuromuscular junction of third instar larvae, and it works pretty well.
In short HL3 contains (in mM): 70 NaCl, 5 KCl, 20MgCl2, 5 trehalose, 115 sucrose, 5 HEPES, and a pH of 7.2, using NaHCO3. But where may you ask, is the CaCl2? Calcium tends to make tissues a bit sticky, so I dissect my preparations in HL3 without CaCl2, and then replace the solution with HL3 containing my desired concentration of CaCl2, but more about this below. HL3 can also be used in adult recordings.
HL3.3 is a slightly modified version of HL3. The primary difference is a reduction in the concentration of MgCl2 used. HL3.3 contains 0.5 mM MgCl2. Why? Well we need to go back to basic biology: Magnesium atom looks very much like the calcium ion. And magnesium ions can, in the case of myosin, compete for calcium binding sites. But magnesium has a longer dwelling period at the binding site, which can cause your prep to loose some movement. I use HL3.1 for extracellular recordings. I find the prep stays fresher longer than it does in HL3.
HL6 is another modified version of HL3. HL6 is used primarily for calcium imaging, either using a fluorescent indicator (like Oregon Green) or a genetic indicator (like GCaMP). And unlike HL3.1, HL6 contains many more modifications in the form of amino acids and sugars (in mM): 15.0 MgCl2, 24.8 KCl, 23.7 NaCl, 20.0 isenthionic acid, 5.0 BES, 80 trehalose, 5.7 L-alanine, 2.0 L-arginine, 14.5 glycine, 11.0 L- histidine, 1.7 L- methionine, 13.0 L-proline, 2.3 L-serine, 2.5 L-threonine, 1.4 L-tyrosine, 1.0 L-valine, and a pH of 7.2. TPEN (0.0001 and Trolox (1.0) can also be added to aid in optophysiology.
You want to keep the cells as fresh and happy as possible for the longest period of time, and you want to make the prep and noise-free as possible with very little movement. HL6 works nicely for this. I tried it for regular EJP and mEPP recordings and found it did not work nearly as nicely as HL3.
Let’s Talk Calcium
Finally, what about the calcium? You will notice I did not report the calcium concentrations used in the recording solutions. A whole article could be devoted to the minutia of which calcium concentration to use for detecting which phenomenon. That being said, here are some numbers for:
1.0 mM CaCl2 is a standard concentration used to record changes in potential at the neuromuscular juntion. But it is not uncommon to see a range of concentrations from 0.3 mM to 2.0 mM. I regularly use 0.5 mM and 1.0 mM, but if you are looking specifically at voltage gated calcium channels, start with 0.2 mM and work up from there.
Extracellular and whole cell patching recordings
Literature values for these studies hover around 1.8mM- 2.0 mM. I have good luck with both.
Lower values around 0.5 mM seem common. If working with voltage gated calcium channel hypomorphs, hypermorphs, or mutants, calcium concentrations can vary from the extremely low 0.2-0.3 mM to the higher 2.0 mM.
The moral of the story is, like every lab is different, every experiment will depend upon solution and calcium tweaks to get the best possible set-up for your experiments. For further, more detailed reading, check out Drosophila Neurobiology: A Laboratory Manual edited Bing Zhang, Marc R. Freeman, and Scott Waddell and Drosophila Protocols edited by William Sullivan, Michael Ashburner, an R. Scott Hawley.