Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

The protocol described above allows one to patch clamp record from the M-cells and its homologs. The technique is challenging and care should be taken in several key areas throughout the procedure. We will now discuss some of these areas and will offer helpful hints to ensure success.

Construction of the dissecting/recording chamber is a critical aspect of the experiment. The M-cells are difficult to see unless differential interference contrast is used. Nomarski DIC works best with clean glass coverslips, but we have found that a very thin layer of Sylgard (~1-2 mm) will not distort the DIC to such an extent that it interferes with identification of the M-cells. Therefore, we use a recording chamber that has a thin glass coverslip on the bottom upon which is placed a small plastic washer (~5 mm diameter). We mix Sylgard 184 in a petri dish and carefully add the liquid Sylgard to the inside of the washer with a Pasteur pipette. The Sylgard can be cured at room temperature over a couple of days or can be heated for rapid curing. The washer may be removed after the Sylgard has cured, although this is not necessary. Once the dissection/recording chamber has been made, it can be used for several weeks before a fresh Sylgard-lined glass slide is needed.

The next step in the procedure is to make up all of the relevant solutions on the day of the experiment (Table 3). Intracellular solutions containing everything but ATP and GTP are made in advance and stored in 5 ml aliquots at -20 °C. On the day of recording an aliquot is thawed to room temperature and fresh ATP and GTP is added. The solution is adjusted to 280 mOsm and pH 7.4. In our hands, we find that addition of fresh ATP and GTP on a daily basis results in good recordings. All solutions need to be kept sterile for the best chance of success.

Intracellular solutions must be filtered through a 0.22 μm filter (Sigma) to remove small particles and debris that might affect the recording. To do this, we first draw the intracellular solution into a 1 ml syringe, affix the filter to the end of the syringe and add a plastic pipette tip that was heated and pulled to fine tip, to the end of the filter. This allows us to apply the filtered intracellular solution directly to the inside of the glass patch pipettes.

Once the solutions are prepared, the embryo can be anesthetized and dissected. Prior to the dissection, care should be taken to adequately assess the age of the organism. Each embryo within a clutch ages at a slightly different rate. Therefore when assessing the age of the organism it is important to do so according to established procedures ^10,13, by using phenotypical cues such as the number of somites, the overall shape of the body and the time of egg fertilization. All dissections are performed with a Dumont #5 fine pair of forceps. These need to be sharp and in good working condition otherwise it will be very difficult to remove the skin overlaying the hindbrain and any connective tissue on the ventral surface of the hindbrain. We often have to sharpen the forceps after a few weeks of use, and do this ourselves with a sharpening stone.

To pin the fish to the thin layer of sylgard, we use pins fashioned from fine tungsten wire 0.001 inches in diameter. The pins are cut with an old, micro-dissection scissors under a dissecting microscope, and are small enough to fit right through the notochord. Pinning the embryos at any other point does not immobilize them and makes dissections almost impossible to perform. When first learning to perform the recordings, it can be difficult to identity the M-cell from its homologs (particularly MiD2 cm which is located in the neighboring rhombomere – rhombomere 5). In some embryos these two pairs of cells appear similar in size. The otoliths provide a convenient landmark to aid in the identification of M-cells. At 48 hpf the M-cells are located right next to the otoliths, and even though the otoliths are removed during the dissection, they leave behind a slightly damaged lateral region of the hindbrain, which serves as a marker for their original position. Transgenic fish that express GFP or some other fluorescent marker in the M-cell, provide excellent preparations for new experimenters. In our hands the M-cells have a membrane capacitance of approximately 18-24 pF at 48 hpf, while the smaller homologs are closer to 12-14 pF. Cells with greater surface area have larger membrane capacitance values (measured in pico Farads (pF)) compared with smaller cells. Thus, membrane capacitance can be used as a rough indicator of cell size, where larger cells have larger capacitance values. This electrophysiological characteristic serves as another property by which the M-cell can be identified from its homologs. Finally, we often use Lucifer yellow in our recording (intracellular) solutions, which allows us to conduct a firm identification of the cell type under investigation using fluorescence imaging once recordings are complete.

Pipette tip resistances in the range of 3.5-4.5 MΩ are the best compromise between tip size and low access resistance, which typically ranges from 8 to 15 MΩ. This is particularly important for events with fast kinetics; ~0.1 msec (20-80%) rise times and ~0.5 msec exponential decays ^12,14,15 (Figures 3A and 3B). Data is acquired at a digitization rate of 50-100 kHz and filtered with a low pass frequency of 5-10 KHz. With regard to the patch pipettes, we have found that they do not need to be fire-polished to obtain a recording, but anecdotally, it appears to offer slightly better chance of obtaining good seals compared with unpolished pipettes. Since the pipette tips are relatively large, we do not add very much positive pressure to the pipette before entering the solution. It takes practice deciding upon the appropriate amount of positive pressure to apply as an excess of pressure will move the cell too much, and will prevent seal formation. It may also damage synaptic contacts as the cell is gently displaced from its original position. On the other hand, too little pressure results in dirty tips and does not clean the surface of the cell well enough to form good seals. A happy medium is only attained by significant practice and experience. Seal formation can be enhanced with applications of negative voltages to the pipette. We typically have seals of 1.2-2 GΩ, which are excellent for breakthroughs into the whole cell mode.

When pharmacological agents need to be applied to the cell cytoplasm, we include them in the intracellular solution prior to filling the pipette. Care should be taken when preparing the pipette solutions to avoid loss of the agents by having it bind to the 0.22 μm filter. Therefore, we filter the intracellular medium first, and then carefully add the pharmacological agent to the filtered solution. Application of compounds to the intracellular compartment has its own unique set of challenges. For instance, addition of drugs to the intracellular medium usually reduces the chances of forming a GΩ seal, but not to the point where it is a major hindrance to the experiment.

One should realize that application of pharmacological agents in this manner does not allow the experimenter to record the most appropriate control since the agent has immediate access to the cell from the start of the whole cell configuration. Therefore, while we record data throughout this type of experiment, one must be aware that the next best control is the intracellular application of an inactive form of the agent. In many instances this takes the form of a heat-inactivated compound, a scrambled peptide or an inactive isomer obtained from the supplier.

We acquire data in the form of synaptic currents (mPSCs), voltage-gated currents and action potentials. Miniature currents may be analyzed by a few excellent programs (pClamp, Axograph, etc.), although we prefer to use Axograph X (John Clements). Axograph X runs on both Windows and Macintosh platforms and can be used for both the acquisition and analysis of electrophysiological data. mEPCs are acquired with the aid of a template of the experimenter's choice, obtained from the recording itself. Individual events can be analyzed for properties such as rise time, amplitude, half width and decay time, etc. We export the spreadsheet of data to Kaleidagraph (Synergy Software)where we can produce figures, including copies of event traces from Axograph X.

One of the more difficult aspects of the experiment is determining whether the recording was performed well enough to provide clean, reliable data. For instance, space-clamping issues may arise when recording mPSCs from M-cells that have large axons and extensive dendritic processes. When voltage clamping, one can generally control the voltage at the tip of the pipette reasonably well (especially if the access resistance (R_a) is low), but may not have proper control of the membrane potential in dendritic processes or axons that are far away from the pipette tip. One method of determining if the cell was properly space clamped is to produce a scatter plot of the rise time vs the amplitude of the mPSCs that were recorded. A correlation between the data is suggestive of inadequate space clamping. In other words, if the space clamp is poor then mPSCs that occur close to the pipette will have quicker rise times and greater amplitudes than events occurring some distance away from the pipette. If a correlation does exist, then the data is problematic and cannot be used.

Another aspect of electrophysiological recordings that needs to be addressed is that of leak currents. This is especially true when voltage clamping a cell in order to record voltage-gated currents such as Na⁺ or K⁺ currents. When the membrane potential is deviated from rest, leak currents can be recorded along with voltage-gated activity. Leak currents (I_L), a combination of potassium (I_K), sodium (I_Na) and chloride (I_Cl) current through non-voltage-gated channels will obscure the activity of interest and must be subtracted from the recording. The amplifier is capable of performing this function online during the recording by running what is typically called a P/N protocol. During a P/N protocol the amplifier will run several small depolarizations (or hyperpolarizations) from rest and will record the resulting leak current that occurs. It is important to use small enough pulses that do not activate voltage-gated channels. The currents that occur during these pulses are recorded and averaged so that these leak currents can be subtracted from the voltage-gated channel activity.

Lastly, one needs to take into account the junction potential, which occurs at the tip of the electrode between the intracellular and extracellular solutions. Intracellular and extracellular solutions are typically composed of different ions, with differing activities and mobilities. Larger ions with lower mobilities will offer a greater resistivity to ion movement than smaller ones and this can result in a small but significant potential difference at the junction of the solutions. This junction potential must be taken into account when determining the appropriate voltages experienced by the cell.

The technique presented here is one used by our lab for many years. However, there are alternative methods of recording from the M-cell. Most notably, Joseph Fetcho and colleagues have developed an excellent preparation in which one can record from the M-cells of older larvae (4-5 days old) in a less invasive manner than presented here, where the M-cell is approached from the dorsal surface¹⁶. We had tried a similar technique but found it easier to approach the M-cell from the ventral side of the hindbrain, where the cell is closer to the surface of the brain, particularly in young fish (i.e. 24 hpf to 72 hpf). Furthermore, an approach from the dorsal surface usually requires the use of a transgenic line that expresses a fluorescent tag in the M-cell, which has the potential to introduce additional variables into the recording. This issue is not present when using wild type fish. However, with the technique presented here, we are limited to studying younger animals (24 hpf to 72 hpf). Thus, each approach has its benefits and limitations.

Student's t-test can be used to compare two groups of data while an analysis of variance (ANOVA) can be used to compare multiple groups. Care must be taken to choose the appropriate post-hoc test for parametric vs non-parametric data.