In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.
Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.
The following description is not specific for one motoneuron. It can be used with any neuron. In this example, we use the flight motoneuron 5 (MN5) that innervates the two dorsalmost fibers of the dorsolongitudinal wing depressor muscle (DLM). To identify and visualize MN5 we use the UAS/GAL4 system to express GFP in the flight motoneurons (and few other neurons).
1. Dissection of Adult Drosophila to Access the Dorsal Part of the Ventral Nerve Cord (VNC)
2. Visualization of MN5 (See Figures 2 and 3), Head Off
3. Alternative Method for Visualization of MN5
For our application it is important to have the cell labeled, for example with targeted expression of GFP. Alternatively, DiI, a lipophilic dye, can be used to retrogradely label this neuron. In this case dye crystals are inserted into the flight muscle using an insect minutien pin, and the wound is closed using UV curing glue.
4. Preparation of the Enzyme Pipette that is Used for Cleaning
It is important to ensure a steady flow of saline through the recording chamber. Having the bath volume rise and fall has to be avoided as it causes vibration and offset potential changes during experiments. Perfusion and suction have to be set accordingly. This makes solution exchange much easier, which in turn is important for rapid wash out of protease before the experiment and rapid exchange of pharmacological agents during the experiment.
5. Cleaning of GFP-tagged MN5 (Figure 3 and Video)
Note to the reader: Images of MN5 before and after cleaning are difficult to distinguish in Figure 3. This is what you get in real life. It will take some training to learn to distinguish the subtle differences between cleaned and non-cleaned neurons. Please note that these subtle differences can be seen better in moving images. Therefore, the movie provides a better impression than the static images (Figure 3).
6. Electrophysiology
7. Representative Results
After the cleaning procedure both MN5 are ready for in situ whole cell patch clamp (Figure 3). The following section shows representative voltage clamp and current clamp traces as recorded from MN5 soma after using the described cleaning procedure. MN5 expresses a variety of voltage gated ion channels (see also13,14). We show an example of voltage gated calcium currents (Figure 4), voltage gated potassium currents (Figure 5) and action potential traces (Figure 6). Voltage gated calcium and potassium currents were evoked by similar voltage protocols, but the solutions that have been used differ in order to allow only the ion channels under investigation to pass current (for details see13,14). All other major voltage gated ion channels have been blocked (voltage gated sodium channels with 100 nM tetrototoxin – pipetted directly into the bath; the perfusion was halted for two minutes – calcium channels with 500 μM cadmiumchloride, potassium channels with 2 mM 4-aminopyridin (4-AP) and 30 mM tetraethylammoniumchloride extracellularly and 0.5 mM 4-AP, 20 mM tetraethylammoniumbromide and 144 mM cesiumchloride intracellularly via the intracellular patch solution in the patch pipette (for details see13,14). Action potential traces were evoked by current injection into the MN5 soma (Figure 6) without usage of any ion channel blockers12.
Figure 1. The cleaning setup. The fly is pinned down in a sylgard coated lid of a 35 mm Petri dish in which we glued a plastic ring (inner diameter 9 mm, 1.3 mm thick) with petrolatum (A, B). After submerging the fly in saline (C) it is opened along the dorsal midline. Gut and esophagus are removed to expose the ventral nerve cord (D). For better accessibility of the thoracic neuromere the head is removed (E). After mounting the preparation onto an upright epifluorescence microscope, the perfusion system (F, G, saline-in, saline-out, white arrows on the left) as well as the ground wire are brought into position (F, G, white arrow on the right). The enzyme filled cleaning pipette is brought into position after the water immersion objective (40x, NA 0.8) has been lowered into the bath (H). For clarity we show it already before the objective has been lowered (F, G, white arrow on the right). The angle at which the enzyme pipette that is held by a 1-HL-U electrode holder (J) is entering the recording chamber is important as the pipette must not touch both the objective and the plastic ring. This angle varies from setup to setup. Here it is approximately 30° (see drawing in J, arrow in K). The arrangement of the pipette in its holder which is attached to a headstage is shown in (L). The headstage is attached to a motorized micromanipulator. Click here to view larger figure.
Figure 2. MN5 in the ventral nerve cord. MN5 can be readily identified by its location in the Drosophila ventral nerve cord (VNC) when GFP is genetically expressed by the use of motoneuron specific GAL4 drivers (A). The dotted rectangle depicts the thoracic neuromere. To get an idea of how the sizes relate to each other, we show a cleaning patch pipette approaching the left MN5 from the left side of the dorsolongitudinal flight muscle. For better visualization, the patch pipette was filled with a red dye (dextran-tetramethylrhodamine, A). The contralaterally projecting MN5 are located on the dorsal surface of the mesothoracic neuromere of the VNC on each side of the midline (projection view of a confocal image stack in B). The ipsilaterally projecting MN1-4 are located more laterally and anteriorly related to MN5 (arrows in B). The dotted circle between both MN5 depicts the dendrites of the flight motoneurons in the neuropil including MN5 (B). GFP label in (B) was enhanced by immunohistochemical staining with an antibody against GFP. Details of MN5 morphology are made visible by filling the neuron intracellularly (iontophoretically) with the invisible tracer neurobiotin and subsequent confocal image acquisition (C). Staining was made visible with streptavidin coupled to a fluorophore. Scale bar is 30 μm.
Figure 3. MN5 before and after cleaning. Visualization of MN5 is achieved by expression of GFP using the UAS/GAL4 system (A, C, see label). MN5 before (A, B) and after (C, D) the cleaning procedure. Left hand side of each picture represents anterior, right hand side represents posterior. Neurons to record from are cleaned with a protease filled patch pipette with a broken tip. The enzyme pipette is only faintly visible (A, B, see label). Inset in A shows an enlargement of bottom MN5 with the enzyme pipette approaching the cell. MN5 cannot be seen in bright light before the cleaning procedure (B). Trachea need to be removed if they cover the cells under investigation. After the cleaning procedure, the contrast between cell and surrounding tissue is more crisp (C, bottom MN5), and the cell can now be seen in bright light (D). The pipette tip can be seen more clearly when bright light is used. The enlargement depicts the area in the dotted square with MN5 cell body borders encircled with a dotted line for better identification. Visualization in bright light often helps judgment of cleanliness.
Figure 4. Calcium current in MN5. Example recording of whole cell calcium currents as recorded from MN5. At least two different calcium currents are shown, a low voltage activated transient calcium and a sustained high voltage calcium current. Currents were evoked from a holding potential of -90 mV. Voltage steps from -90 mV to +20 mV were applied. Blockers were used to block most other ion channels. Artifacts were omitted for clarity (for characterization of MN5 calcium currents see Ryglewski et al., 2012).
Figure 5. Potassium currents in MN5. Example recording of whole cell potassium currents as recorded from MN5. Currents shown include calcium activated potassium currents as no calcium channel blockers were applied. Currents were evoked from a holding potential of -90 mV for the total potassium current (A) or from a holding potential of -20 mV to inactivate transient potassium currents and show only the sustained potassium current (B). Voltage steps from -90 mV to +20 mV were applied in 10 mV increments. Offline subtraction of potassium currents as evoked from a holding potential of -90 mV (A) and -20 mV (B) reveal pure transient potassium currents (C). Voltage gated sodium channels were blocked. Artifacts were omitted for clarity (for characterization of MN5 voltage and calcium potassium currents see Ryglewski and Duch, 2009).
Figure 6. Firing pattern exhibited by MN5. Example recording of firing patterns as elicited in MN5 by somatic current injections (0.4 nA, black, 0.5 nA, gray) of 200 ms duration. Resting membrane potential was -59 mV. No ion channel blockers were used. (for current clamp analysis of MN5 firing patterns see Duch et al., 2008).
When visualizing the cells with fluorescent proteins like GFP, it is important to not over-expose the preparation to too much light. This may result in photo damage. We use 100W HBO short arc mercury bulbs for illumination, and we also use neutral density (ND) of 0.8 (Chroma ND filters 0.3 and 0.5). To be able to judge on the quality of the cleaning good visibility is crucial. Therefore, ND filters may be removed for short periods of about 20 s for multiple times.
When applying some positive pressure, the cell body “flaps” a little. This helps judging the quality of cleanliness. In the ventral nerve cord contrast is very low, and movement can be extremely helpful to visualize cells. The electrode holder that is being used for the cleaning procedure (and also for patch clamping) needs to be tightly sealed; otherwise controlled pressure application will not be possible. Loosing pressure will disturb successful removal of tissue and debris and will prevent giga-seal formation
In case in situ patch clamp recordings shall be performed after cleaning the cell, be aware that there is not only a sheath surrounding the ventral nerve cord as a whole (which needs to be removed for any patch clamp recording of any central neuron of adult flies), but cells themselves may be surrounded by a sheath as well. MN5, for example, is surrounded by a non-cellular sheath that can be seen only with brief high intensity illumination. In addition, cells may be located underneath the trachea. In case the cell cannot be accessed, the trachea have to be pulled aside if not ripped off with the pipette during the cleaning procedure (after having been pulled aside). The whole cleaning procedure needs to be done without pulling the tissue too much. This procedure will require training. The better the cleaning, the higher will be the frequency of immediate giga-seals (within 1 s after touching the cell and releasing positive pressure).
Depending on the cleaning protocol and the break-in the following scenarios are possible: First, if the cells were not cleaned well enough, a giga-seal is not possible and the experiment must be terminated. Second, in some cases cleaning is good enough for giga-seal formation but a thin, hardly visible remnant of the sheath remains around the soma. This will cause the membrane to rupture and cause leak currents of various amplitudes. A less severe consequence of insufficient cell cleaning is that some remnants of the sheath cause increases in access resistance. Depending on the experimental design different quality criteria for the patch might apply. For monitoring action potential patterns in current clamp the membrane potential of the cell must be healthy (-55 mV or more hyperpolarized in the case of MN5), but access resistance is not such an important issue. However, when evoking action potentials by current injection access resistance becomes an issue. For evoking large amplitude potassium currents which originate close to the recording site the leak should be small (in the case of MN5 input resistance must by higher than 80 MΩ) and access resistance should not be larger than 15 MΩ. For the space clamp that is needed in voltage clamp recordings of small amplitude dendritic calcium currents which originate at quite some distance from the soma no leak can be introduced (for MN5 input resistance must be above 120 MΩ) and access resistance cannot be higher than 12 MΩ. In most recordings we measure calcium currents with access resistances between 8 and 10 MΩ(as read from the dial on the axopatch 200B amplifier after series resistance and whole cell capacitance compensation). For each cell type to be recorded, such quality criteria must be determined individually.
Using the cleaning procedure described here, we can hold the recordings stable for approximately 90 minutes. We do not observe considerable run-down (specifically tested for Cav2 and Cav3 calcium currents and for Shaker, Shal, and delayed rectifier potassium currents). However, we have not attempted to hold the patch longer and therefore cannot state whether longer recordings would be possible.
The authors have nothing to disclose.
Agent/item | Company | Catalog number |
Protease type XIV | Sigma Aldrich USA | P5147 |
Microfil flexible injection needle | World Precision Instruments USA | MF28G-5 |
Borosilicate Glass Capillaries, o.d. 1.5 mm, i.d. 1.0 mm, no filament | World Precision Instruments USA | PG52151-4 |
DiI | Invitrogen USA | D3899 |
Sylgard Elastomer Kit 184 (Dow Corning) | www.ellsworth.com | 184 SIL ELAST KIT |
ND filter set (unmounted) | Chroma | 22000b series |
Electrode holder 1-HL-U | Molecular Devices | 1-HL-U |